HK1099361B

HK1099361B - Method and kit for combination detection of hbv pres1 and hbcag

Info

Publication number: HK1099361B
Application number: HK07105485.4A
Authority: HK
Inventors: 葛胜祥; 袁权; 赵昱; 罗文新; 张军; 夏宁邵
Original assignee: 厦门大学; 北京万泰生物药业股份有限公司
Filing date: 2007-05-25
Publication date: 2010-09-24

Description

Method for combined detection of HBV pre-S1 antigen and core antigen and diagnostic kit

Technical Field

The present invention relates to the field of hepatitis B virus detection, and more particularly to a method for diagnosing hepatitis B virus infection by detecting pre-S1 antigen (HBV PreS1) associated with hepatitis B virus in combination with core antigen (HBcAg). The invention also discloses a combined detection kit (especially an enzyme-linked immunosorbent assay and a chemiluminescence assay) for the pre-S1 antigen and the core antigen of the hepatitis B virus. The invention also relates to a detection method of the pre-S1 antigen, a virus splitting method and a related kit.

Background

Hepatitis B virus (Hepatitis B virus) infection is one of the most important public health problems worldwide.

Currently, HBV serum markers (such as HBsAg, HBsAb, HBeAg, HBeAb and HBcAb, which are commonly called as "two-in-one-half detection") are widely used as the conventional detection standards for the current HBV infection and the previous HBV infection. However, the high variability combined with the large base of HBV carriers leads to a high incidence of false negatives in the conventional "two-and-a-half" test. Moreover, the conventional "two-half and two-half" test cannot quantitatively reflect the replication and infectivity of the virus, and results which are difficult to be understood are often generated, and the test that the individual is not infected with HBV cannot be directly determined from the "two-half and two-half" index.

HBV DNA is a direct indicator of HBV replication, and dot blot detection (or PCR detection) is a gold standard for infection and infectivity judgment of hepatitis B patients and HBV carriers. PCR detection and dot blot detection can be used as direct indicators of HBV infection and infectivity, but are not suitable for large-scale general investigation or conventional use.

Among all the serum marker antigens (HBV PreS1, HBcAg, HBxAg, DNAP, HBV PreS2, etc.) that may be highly related to HBV DNA: HBcAg has been considered as an antigen directly related to HBVDNA, and detection is of diagnostic significance in HBcAg in replication-competent virus quantification and HBsAg-negative HBV infectors and HB patients. However, in view of the difficulties in detecting HBcAg, currently developed or reported HBcAg immunological diagnostic reagents are usually performed by pre-treating the sample before detection (splitting virus, breaking membrane and inactivating hbcabs) or by detecting HBcAg-hbcabs immunocomplexes in a circuitous way, and the former has complicated operation and procedures, is not easy to be accepted by clinical clients, and is not suitable for large-scale donor screening and epidemiological investigation. The latter is difficult to achieve the desired specificity and sensitivity due to the specificity of the detection mode. HBV PreS1 also has good correlation with HBV DNA (D.Buffello, 2000, 96%; Kuijpers, 1989, 88%; Theilman, 1986). In the diagnosis method for HBV PreS1 antigen, the currently developed or reported PreS1 immunological diagnosis reagent considers that PreS1 has low absolute concentration relative to HBsAg, and the conventional double PreS1 antibody sandwich method is difficult to achieve ideal sensitivity, and the detection mode is usually adopted to detect the whole LHBs (namely, the detection is mediated by the HBsAg on the LHBs, a specific antibody aiming at the HBsAg is used as a capture antibody or a report antibody, and then the specific antibody aiming at the PreS1 is matched with the capture antibody or the report antibody), so that the detection sensitivity of the PreS1 is effectively amplified, but the detection of the PreS1 completely depends on the HBsAg, an independent detection system is not formed, the false occurrence of the PreS1 caused by HBsAg mutation possibly occurs, and the dynamics of the PreS1 infected by mutant virus or different subtype viruses cannot be accurately reflected.

The development of a simple, accurate and high-sensitivity direct ELISA diagnostic reagent for HBV present infection has urgent practical significance in the aspects of HBV infection epidemic control, HB patient antiviral curative effect and disease course prognosis.

Therefore, there is still a lack in the art of effective methods for detecting hepatitis B virus.

Summary of The Invention

An object of the present invention is to establish a simple and effective HBcAg detection method without sample pretreatment process and a high-sensitivity method for detecting Pres1 by a double Pres1 antibody sandwich method, and to make up for the defect of false negative possibly caused by single HBcAg or Pres1 mutation or omission by jointly detecting HBcAg and Pres1 patterns, to construct a combined (HBcAg and Pres1) detection pattern highly related to HBV DNA, and to apply the pattern to qualitative or quantitative immunological diagnosis such as enzyme-linked immunosorbent assay, chemiluminescence, time resolution, colloidal gold assay, enzyme-linked immunosorbent assay, etc.

The invention relates to an immunological diagnostic method which is established by jointly detecting the pre-S1 antigen/core antigen of hepatitis B virus and is highly relevant to a nucleic acid detection method.

The invention relates to a kit for jointly detecting pre-S1 antigen/core antigen of hepatitis B virus and a preparation method thereof.

Specifically, the invention relates to an enzyme linked immunosorbent assay kit and a chemiluminescence assay kit for jointly detecting the pre-S1 antigen/core antigen of hepatitis B virus and a preparation method thereof.

The combined detection mode not only effectively embodies the complementary advantages of the combined detection of the two antigens, but also embodies the high correlation of the new index, namely the nucleic acid related antigen, formed by the combined detection result with the index of the nucleic acid of the hepatitis B virus.

In addition, the invention discloses a method for detecting the pre-S1 antigen by a high-sensitivity double-pre-S1 antibody sandwich for the first time, which is different from other published methods for detecting the pre-S1 antigen.

In a particularly preferred embodiment of the above method, the present invention uses for the first time a series of buffer solutions containing different amounts of different types of surfactants for splitting viruses, greatly improving the detection sensitivity of the combined index of the pre-S1 antigen and the core antigen and the single index of the pre-S1 antigen.

Drawings

FIG. 1 shows the construction of recombinant plasmid pT-2147 x 3 with triple tandem of preS1 region 21-47AA fragment.

FIG. 2 shows the SDS-PAGE results of the expressed and purified recombinant protein GST-2147 x 6. The results showed that the purity of the target protein after purification was over 85%. Lane 1: protein marker; lane 2: expressed pGEX-2147 x 6; lane 3: purified pGEX-2147 x 6.

FIG. 3 shows the correlation of chemiluminescence detection with fluorescent quantitative PCR. The virus content and the luminescence intensity of each specimen were logarithmized and subjected to linear correlation analysis as described in example 14.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The nomenclature used herein and the procedures in cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratories are those well known and widely used in the relevant arts.

The invention relates to an immunological diagnostic method which is established by jointly detecting the pre-S1 antigen/core antigen of hepatitis B virus and is highly relevant to a nucleic acid detection method. In one embodiment of the present invention, the combined pre-S1 antigen/core antigen has a distinct advantage over the single pre-S1 and core antigens by comparing pre-S1, core antigen alone with pre-S1 antigen/core antigen (enzyme linked immunosorbent assay) combined with pre-S1 antigen/core antigen (enzyme linked immunosorbent assay).

In yet another embodiment of the present invention, the pre-S1 antigen/core antigen (ELISA) for combined detection of hepatitis B virus is compared with conventional hepatitis B two-half assay, which can accurately characterize a sample containing a portion of the virus, but fails to accurately characterize such a sample.

In another embodiment of the present invention, the method for detecting pre-S1 antigen/core antigen of hepatitis B virus is applied to chemiluminescence diagnosis, and the quantitative result of the detection of pre-S1 antigen/core antigen of hepatitis B virus has better linear correlation with the virus content.

In another embodiment of the present invention, the detection method of the present invention is implemented by enzyme-linked immunosorbent assay. Correspondingly, the invention provides a preparation method of a related enzyme-linked immunosorbent assay kit and a kit prepared by the method.

In yet another embodiment of the present invention, the detection method of the present invention is carried out using a chemiluminescence method. Accordingly, the present invention provides a method for preparing a related chemiluminescent kit and the kit prepared thereby.

The above aspects and others are explained in more detail below.

Combined detection of pre-S1 antigen and core antigen of hepatitis B virus (combined detection for short)

In one aspect, the invention relates to a method for detecting hepatitis b virus in a sample or detecting hepatitis b virus infection comprising the combined detection of pre-S1 antigen and core antigen of hepatitis b virus.

Herein, for the purpose of the present invention, "combined detection" includes that the detection of the pre-S1 antigen and the detection of the core antigen are performed separately or in combination, and are performed simultaneously and sequentially in any order, as long as the signal processing for both the detection of the pre-S1 antigen and the detection of the core antigen are performed in combination.

The joint signal processing means that when the sample is judged to be positive or negative for hepatitis B virus or hepatitis B virus infection, the signal obtained by the former S1 antigen detection and the signal obtained by the core antigen detection are combined into a single index for judgment. For example, the signals detected by two antigens may be simply added and compared with a predetermined value. Alternatively, the signals from the two antigen detections may be combined into a new single indicator by appropriate processing or operation (e.g., weighting one or both signals), and compared to a predetermined value. This can improve sensitivity, detection rate, and the like. This may be due to avoiding missed tests, for example due to single antigen a negatives. The present invention is not limited by such a mechanism.

Thus, the detection of the pre-S1 antigen and the detection of the core antigen may be independent of each other at a stage prior to signal processing, i.e. they may occur at the same or different times, in the same or different assays. For example, the detection of the pre-S1 antigen and the detection of the core antigen may be performed by the same method or by different methods; for example, the detection of one antigen is performed using one of the following methods, while the detection of the other antigen is performed using a different (or the same) method: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemiluminescence, and time resolution. Preferably both antigens are detected using the same method.

For example, detection of the pre-S1 antigen may occur before, after, or simultaneously with detection of the core antigen.

When both antigens are detected by the same method, they may be carried out in the same or different test batches, in the same or different reaction vessels (e.g., reaction wells), on the same or different solid supports. Both may employ the same or different signal generators.

In a preferred embodiment, the two antigens are detected simultaneously, in combination. For example, the detection of two antigens is performed simultaneously in the same reaction vessel (e.g., reaction well). This can be implemented, for example, by the following method: the capture antibodies that capture both antigens are immobilized on the same support, and capture of both antigens is performed simultaneously in the same medium using the same viral lysate, optionally with simultaneous binding and detection of the second antibody in the same medium. Alternatively, the method may be implemented, for example, as follows: the capture antibodies that capture the two antigens are immobilized on different supports, the two supports are then mixed and the capture of the two antigens is performed simultaneously in the same medium, optionally with the binding and detection of the second antibody in the same medium, using the same viral lysate.

In a preferred embodiment of the method of the invention, the sample is an isolated biological sample from the individual to be tested, preferably the sample is serum.

In another embodiment, the method is used to determine the presence or absence of hepatitis B virus nucleic acid in serum.

In another embodiment, the combined detection of the hepatitis b virus pre-S1 antigen and the core antigen is achieved by one or more methods selected from the following: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemiluminescence, time-resolved assay; enzyme-linked immunosorbent assay or chemiluminescence assay is preferred.

In another embodiment, the pre-S1 antigen and the core antigen are detected simultaneously in the same reaction vessel (e.g., reaction well).

In another embodiment, the pre-S1 antigen is detected with the core antigen using a double antibody sandwich.

In another preferred embodiment, no pre-treatment of the sample, e.g. serum, is required. For example, the virus need not be denatured beforehand.

In one embodiment, the lysis of hepatitis b virus and the capture of the pre-S1 antigen and the core antigen are performed simultaneously in the same reaction vessel (e.g., reaction well) in the same medium. In this embodiment, preferably, the lysate for the combined detection according to the invention is used as the medium.

In a specific embodiment of the method of the invention, the combined detection method of the invention comprises the following steps:

a) providing a first anti-HBV PreS1 antibody and a second anti-HBV PreS1 antibody that can specifically bind to hepatitis b virus pre-S1 antigen (HBV PreS1), and a first anti-HBc antibody and a second anti-HBc antibody that can specifically bind to hepatitis b virus core antigen (HBc);

b) binding a first anti-HBV PreS1 antibody and a first anti-HBc antibody (both collectively referred to as a first antibody) as capture antibodies to the same solid support to form a first antibody-support conjugate; preferably, the carrier is an ELISA plate;

c) contacting a sample to be tested with a first antibody-carrier conjugate such that hepatitis b virus pre-S1 antigen and core antigen, if present, in the sample are captured to form an antigen-first antibody-carrier conjugate; the sample may be pretreated or not; preferably, the sample has not been previously treated for viral lysis, and the contacting of the sample to be tested with the first antibody-carrier conjugate is performed under conditions that allow viral lysis and antigen capture; more preferably, the contacting of the sample to be tested with the first antibody-carrier conjugate is performed in a lysis solution for combined assay according to the present invention;

d) contacting a second anti-HBV PreS1 antibody and a second anti-HBc antibody (both collectively referred to as the second antibody) with the product (carrier) of step c) under conditions that allow the second antibody to bind to the captured antigen;

e) detecting the amount of bound second antibody.

The antigen-first antibody-carrier conjugate of step c) includes hepatitis b virus pre-S1 antigen-first anti-HBV PreS1 antibody-carrier conjugate and core antigen-first anti-HBc antibody-carrier conjugate.

In step d), if hepatitis b virus pre-S1 antigen and core antigen are present, a second antibody-antigen-first antibody-carrier conjugate will be formed, including a second anti-HBV PreS1 antibody-hepatitis b virus pre-S1 antigen-first anti-HBV PreS1 antibody-carrier conjugate, and a second anti-HBc antibody-core antigen-first anti-HBc antibody-carrier conjugate

The first anti-HBV PreS1 antibody and the second anti-HBV PreS1 antibody may be the same or different and may be monoclonal or polyclonal, preferably different monoclonal antibodies. The first anti-HBc antibody and the second anti-HBc antibody may be the same or different and may be monoclonal antibodies or polyclonal antibodies, preferably different monoclonal antibodies.

Step e) detecting the amount of the bound second antibody can be performed by, for example, the following method: providing a first signal generator capable of binding effectively to said second anti-HBV PreS1 antibody and a second signal generator capable of binding effectively to said second anti-HBc antibody, the signal intensity produced by the first and second signal generators being related only to the amount of second anti-HBV PreS1 antibody and second anti-HBc antibody, respectively; contacting the first and second signal generators with a second antibody; the signal generated by the signal generator is detected. The first and second signal generators may be the same or different. In a preferred embodiment, the first and second signal generators are the same, or the signals generated by the first and second signal generators are the same, and can be detected in the same way; more preferably, the sum of the signals generated by the first and second signal generators is detected as an index of the determination result.

Step e) detecting the amount of bound second antibody can also be performed, for example, by the following method: the second antibody is directly labeled with a signal generator, and a signal generated by the signal generator is detected. The second anti-HBV PreS1 antibody and the second anti-HBc antibody may be labeled with the same signal generator, or different signal generators. In a preferred embodiment, the second anti-HBVPreS 1 antibody and the second anti-HBc antibody labeled signal generator are the same, or they generate the same signal and can be detected in the same way; more preferably, the sum of the signals generated by the second anti-HBV PreS1 antibody and the second anti-HBc antibody-labeled signal generator is detected as an index of the judgment result.

In the method of the present invention, the signal generator may be any substance that can be detected by an appropriate method, and examples thereof include an enzyme, a fluorescein, a chemiluminescent substance, an isotope, and a rare earth element.

In a preferred embodiment, the immunoassay is performed using monoclonal antibodies against the hepatitis B virus pre S1 antigen and against the core antigen.

In a preferred embodiment, the monoclonal antibody against hepatitis b virus pre-S1 antigen is selected from the group consisting of 3H5, 7H11, 2a7, 4D11, 6F1, 13G2, and 16F 5; preferably, HBV PreS1 is detected using 7H11 monoclonal antibody as the primary anti-HBV PreS1 antibody and 4D11-HRP as the secondary anti-HBVPreS 1 antibody pair.

In a preferred embodiment, the method further comprises the steps of: lysing the virus in the sample with a lysis solution prior to contacting the sample to be tested with the first antibody-carrier conjugate, wherein the lysis solution is preferably a virus lysis solution for the combined detection according to the invention.

In another embodiment, washing, drying, etc. steps may also be included between each step (e.g., between steps b) and c), and/or c) and d), and/or d) and e).

The invention also relates to a viral lysate useful for the combined detection of the hepatitis b virus pre-S1 antigen and the core antigen, comprising a surfactant selected from the group consisting of: chaps, sulfobetain8-18 (SB8, SB10, SB12, SB14, SB16, SB18), Brij 35, Tween series and/or HP-beta-CD, preferably selected from SB14, SB16, SB18, HP-beta-CD, TWEEN20, TWEEN40, TWEEN60, TWEEN 80.

In the above-mentioned virus lysate, preferably, the surfactant may be a combination of one or more (2, 3, 4, 5) of the above surfactants, such as one or a combination of two or three of HP, TWEEN series and SB series. For example, the HP + TWEEN series, the TWEEN series + SB series, the HP + SB series, or the HP + TWEEN series + SB series, preferably HP + TWEEN40, HP + TWEEN40+ SB14, HP + TWEEN40+ SB16, HP + TWEEN40+ SB18, HP + TWEEN40+ SB18, HP + TWEEN20+ SB14, HP + TWEEN60+ SB14, HP + TWEEN80+ SB 14. Preferably, the surfactant may be HP: 0.5% -4%; tween series: 0.1% -6%; SB 14%: 1% -10%; SB 16: 0.5% -1%; SB 18: 0.5% -1%; more preferably, HP: 1% -2%; tween series: 0.5% -2%; SB 14: 2% -8%; SB 16: 1 percent; SB 18: 1 percent; such as: 1% -2% of HP + 0.5% -1% of Tween + 1% -2% of SB 14.

Preferably, the surfactant is selected from the group consisting of the surfactants set forth in lyss 1-lyss 24 shown in table 4, more preferably lyss 1, lyss 10, lyss 11, lyss 12, lyss 13, lyss 14, lyss 15, lyss 16, lyss 17, lyss 18, lyss 19, lyss 20, lyss 21, lyss 22, lyss 23, and lyss 24, more preferably lyss 10, lyss 13, lyss 16, lyss 17, lyss 18, lyss 19, lyss 20, lyss 21, and lyss 24, and most preferably lyss 10, 13, 16, 18.

Preferably, the buffer is selected from citrate buffer, phosphate buffer, Tris buffer, amino acid buffer, acetate buffer, HEPES buffer, carbonate buffer. The buffer can be one or a combination of several (2, 3, 4 and 5) of the above buffers. The buffer is preferably citrate buffer solution or phosphate buffer solution. The pH range and concentration range of the buffer may be: phosphate buffer solution: 10-50mM pH 6.0-7.0, preferably 20mM pH 7.4; citrate buffer: 10-50mM pH 3.0-6.6 preferably 10mM pH 6.6; tris buffer: 10-50mM pH 7.0-9.0, preferably 20mM pH 7.0; acetate buffer solution: 10-50mM pH 3.8-6.0, preferably 10mM pH 6.0.

Optionally, the lysis solution may also contain one or more (e.g.1, 2, 3, 4, 5, 6-10) further ingredients, such as reducing agents (e.g.DTT, DTE, beta-mercaptoethanol), chelating agents (e.g.EDTA), osmo-regulators (e.g.salts such as NaCl; sucrose). For example, the lysis solution may further comprise one or a combination of the following components: beta-mercaptoethanol: 0.01% -1%, preferably 0.1% -0.5%, more preferably 0.4%; DTT: 1-30mM preferably 5-10mM, more preferably 5 mM; DTE: 1-30mM preferably 5-10mM, more preferably 5 mM; EDTA: 5mM-20mM preferably 5 mM; NaCl: more preferably, 50mM-300 mM: 50-150mM, more preferably 100 mM; sucrose: 2% to 20%, preferably 5% to 10%, more preferably 10%.

Preferably, the lysate is selected from lyss 1-lyss 24 shown in table 4, more preferably, the lysate is selected from lyss 1, lyss 10, lyss 11, lyss 12, lyss 13, lyss 14, lyss 15, lyss 16, lyss 17, lyss 18, lyss 19, lyss 20, lyss 21, lyss 22, lyss 23, and lyss 24, more preferably lyss 10, lyss 13, lyss 16, lyss 17, lyss 18, lyss 19, lyss 20, lyss 21, and lyss 24, most preferably, the lysate is selected from: lysis 10-citrate buffer pH6.6, 2% SB14, 5mM DTE, 100mM NaCl, 5mM EDTA; lysis 13-citrate buffer pH6.6, 2% SB16, 5mM DTE, 100mM NaCl, 5mM EDTA; lysis 16-citrate buffer pH6.6, 2% SB18, 5mM DTE, 100mM NaCl, 5mM EDTA; and lysis 18-citrate buffer pH6.6, 2% HP, 0.5% Tw40, 0.4% Me, 10% sucrose.

The virus lysate for jointly detecting the pre-S1 antigen and the core antigen of the hepatitis B virus can also be used for singly detecting the core antigen or singly detecting the pre-S1 antigen. Therefore, the invention also provides the virus lysate for detecting the core antigen. The invention also provides the virus lysate for detecting the pre-S1 antigen. The invention also provides application of the lysate in detecting core antigen and application of the lysate in detecting pre-S1 antigen.

Lysates have been reported for use only in detecting core antigens and typically use NP40 for disrupting the viral envelope, but these known lysates are not suitable for use in combined detection of the pre-S1 antigen with core antigens, such as CN 1352392A.

An important advantage of the virus lysate of the present invention is that it can be used as a medium for capturing viral antigens. The virus lysate of the present invention has the characteristic of sufficiently lysing the virus but being sufficiently mild so as not to substantially inactivate the antibody coated on the vector. The use of the virus lysate of the present invention allows for efficient antigen capture while lysing the virus. However, the present invention is not limited by this mechanism of action.

Thus, in a particularly preferred embodiment, the viral lysis and the capture of the antigens (the pre-S1 antigen and the core antigen) are performed simultaneously in the same reaction medium using the viral lysate for combined detection of the invention, without the need to pre-lyse the virus prior to antigen capture. This is also an important advantage of the present invention.

The invention also relates to a kit for combined detection, which comprises reagents for combined detection of the hepatitis B virus pre-S1 antigen and the core antigen according to the combined detection method.

In a specific embodiment, the combined detection kit of the present invention comprises an anti-HBVPreS 1 antibody (preferably comprising two different anti-HBV PreS1 antibodies, particularly monoclonal antibodies) and an anti-HBc antibody (preferably comprising two different anti-HBc antibodies, particularly monoclonal antibodies), and/or a virus lysate for combined detection according to the present invention.

Preferably, at least one of the anti-HBV PreS1 antibody and at least one anti-HBc antibody of the antibodies is labeled with or effectively binds to a signal generator. The anti-HBVPreS 1 antibody and the anti-HBc antibody-labeled signal generator or conjugated signal generator may be the same or different, and preferably may be detected by the same detection method. The antibody is preferably a monoclonal antibody. Preferably the anti-HBV PreS1 antibody is selected from: 3H5, 7H11, 2a7, 4D11, 6F1, 13G2 and 16F5, in particular 7H11 and 4D 11.

The kit preferably further comprises a reagent suitable for detecting the antigen-antibody reaction (e.g., a detection reagent for detecting a second antibody).

In a preferred embodiment, the combined detection kit according to the invention comprises:

(1) a solid phase carrier on which a first anti-HBV PreS1 antibody and a first anti-HBc antibody are immobilized; and/or (2) a viral lysate used in the combined detection of the present invention; and (3) optionally a second anti-HBVPreS 1 antibody and a second anti-HBc antibody, labeled with a signal generator or not; and (4) optionally, a detection reagent for detecting the second antibody; and (5) optionally a buffer, a washing solution, and instructions for use (preferably instructions describing the method of the combined assay of the invention).

In a preferred embodiment, the solid phase carrier in the kit of the present invention is a multi-well reaction plate (e.g., ELISA plate) in which a first anti-HBV PreS1 antibody and a first anti-HBc antibody are immobilized in the same or different wells of the multi-well reaction plate.

The present invention also relates to a solid phase carrier on which an anti-HBV PreS1 antibody and an anti-HBc antibody are immobilized. Preferably, the solid phase carrier is a multi-well reaction plate (e.g., microplate, chemiluminescent plate) having an anti-HBV PreS1 antibody and an anti-HBc antibody immobilized in the same or different wells of the multi-well reaction plate. The antibody is preferably a monoclonal antibody, particularly a specific antibody disclosed herein.

The invention also relates to the use of the reagent for combined detection of the pre-S1 antigen and the core antigen of hepatitis B virus in the preparation of a kit for detecting hepatitis B virus infection. Preferably, the reagent is a reagent for combined detection as described in the present invention. Examples of the reagent for combined detection described in the present invention are an anti-HBV PreS1 antibody (preferably comprising two different anti-HBV PreS1 antibodies, particularly monoclonal antibodies) and an anti-HBc antibody (preferably comprising two different anti-HBc antibodies, particularly monoclonal antibodies), and/or a virus lysate for combined detection of the present invention. Specifically, (1) a solid phase carrier on which a first anti-HBV PreS1 antibody and a first anti-HBc antibody are immobilized; and/or (2) a viral lysate used in the combined detection of the present invention; and (3) optionally a second anti-HBV PreS1 antibody labeled with a signal-producing substance or unlabeled and a second anti-HBc antibody; and (4) optionally, a detection reagent for detecting the second antibody; and (5) optionally a buffer, a washing solution, and instructions for use (preferably instructions describing the method of the combined assay of the invention).

Double antibody sandwich method for detecting hepatitis B virus pre-S1 antigen

In another aspect, the present invention relates to a method for detecting hepatitis b virus pre-S1 antigen using a double antibody sandwich method, characterized in that both antibodies (capture antibody and reporter antibody) used are anti-HBV PreS1 antibody specifically binding to hepatitis b virus pre-S1 antigen.

In a preferred embodiment of the method of the invention, no pretreatment of the sample is required.

In another preferred embodiment, the lysis of hepatitis B virus and the capture of the pre-S1 antigen are performed simultaneously in the same reaction vessel (e.g., reaction well) in the same medium.

In another preferred embodiment, a lysate used for the detection of the pre-S1 antigen described in the present invention is used as the medium.

In one embodiment, the method of the invention comprises:

a) providing a first anti-HBV PreS1 antibody and a second anti-HBV PreS1 antibody (the two antibodies may be the same or different, preferably different, especially monoclonal antibodies) that specifically bind to the pre-S1 antigen of hepatitis B virus;

b) binding the first anti-HBV PreS1 antibody as a capture antibody to a solid phase carrier to form a first anti-HBV PreS1 antibody-carrier conjugate; preferably, the carrier is an ELISA plate;

c) contacting a test sample with a first anti-HBV PreS1 antibody-carrier conjugate such that hepatitis b virus pre-S1 antigen, if present, in the sample is captured to form an antigen-first anti-HBV PreS1 antibody-carrier conjugate;

d) contacting a second anti-HBV PreS1 antibody with the product of step c) under conditions that allow the second anti-HBV PreS1 antibody to bind to the captured antigen to form a second anti-HBV PreS1 antibody-antigen-first anti-HBV PreS1 antibody-carrier conjugate;

e) the amount of the bound second anti-HBV PreS1 antibody was detected.

In a preferred embodiment, the step e) of detecting the amount of bound second antibody is performed by: providing a signal generator capable of binding effectively to said second anti-HBV PreS1 antibody, the intensity of the signal generated by the signal generator being related only to the amount of second anti-HBV PreS1 antibody; contacting the signal generator with a second antibody; the signal generated by the signal generator is detected.

In another preferred embodiment, the step e) of detecting the amount of bound second antibody is performed by: the second antibody is directly labeled with a signal generator, and a signal generated by the signal generator is detected.

Preferably, the signal generator is an enzyme, fluorescein, chemiluminescent substance, isotope, rare earth element.

In one embodiment, the detection of the hepatitis b virus pre-S1 antigen is achieved by one or more methods selected from the following: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemiluminescence, time-resolved assay; enzyme-linked immunosorbent assay or chemiluminescence assay is preferred.

In another embodiment, the present invention may further comprise washing, and/or drying steps between the steps (e.g., between steps b) and c), and/or c) and d), and/or d) and e)).

The invention also relates to a viral lysate useful for the detection of the pre-S1 antigen, comprising a surfactant selected from the group consisting of: chaps, sulfobetain8-18, Brij 35, TritonX100, Tween series and/or HP- β -CD, preferably selected from SB8, SB12, SB14, SB16, SB18, HP- β -CD, TWEEN20, TWEEN40, TWEEN60, TWEEN80, BRJ35, more preferably SB12, SB14, SB16, SB18, TWEEN 80.

Preferably, the surfactant may be a combination of one or more (2, 3, 4, 5) of the above surfactants, preferably SB16+ SB14, SB18+ SB 14. Preferably, the concentration of the surfactant may range from SB 14%: 1% -10%; SB 16: 0.5% -1%; SB 18: 0.5% -1%; more preferably: SB 14: 2% -8%; SB 16: 1 percent; SB 18: 1 percent; such as: 1% SB14+ 0.5% SB16 or 1% SB14+ 0.5% SB 18.

Preferably, the surfactant is selected from the group consisting of the A-P lysates, more preferably the C-M lysates and the P lysates shown in Table 2, more preferably the E, F, G, H, J lysates, most preferably the G lysates and the H lysates, or the surfactants described in the epsilon lysates, the zeta lysates, the eta lysates and the theta lysates shown in Table 6.

Preferably, the buffer is selected from citrate buffer, phosphate buffer, Tris buffer, amino acid buffer, acetate buffer, HEPES buffer, carbonate buffer. The buffer can be one or a combination of several (2, 3, 4 and 5) of the above buffers. The buffer is preferably citrate buffer solution or phosphate buffer solution. The pH range and concentration range of the buffer may be, phosphate buffer: 10-50mM pH 6.0-7.0, preferably 20mM pH 7.4; citrate buffer: 10-50mM pH 3.0-6.6 preferably 10mM pH 6.6; tris buffer: 10-50mM pH 7.0-9.0, preferably 20mM pH 7.0; acetate buffer solution: 10-50mM pH 3.8-6.0, preferably 10mM pH 6.0.

Optionally, the lysis solution may also contain one or more (e.g.1, 2, 3, 4, 5, 6-10) further ingredients, such as reducing agents (e.g.DTT, DTE, beta-mercaptoethanol), chelating agents (e.g.EDTA), osmo-regulators (e.g.salts such as NaCl; sucrose). For example, the lysis solution may further comprise one or more components selected from the group consisting of: beta-mercaptoethanol: 0.01% -1%, preferably 0.1% -0.5%, more preferably 0.2%; DTT: 1-30mM preferably 5-10mM, more preferably 5 mM; DTE: 1-30mM preferably 5-10mM, more preferably 5 mM; EDTA: 5mM-20mM, preferably 5 mM.

Preferably, the lysate is selected from the group consisting of the A-P lysates shown in Table 2, more preferably the C-M lysates and the P lysates, more preferably the E, F, G, H, J lysates, and most preferably the G lysates and the H lysates; or selected from epsilon lysates, zeta lysates, eta lysates, and theta lysates shown in Table 6.

The present invention also relates to a kit for detecting hepatitis b virus pre-S1 antigen, comprising reagents for detecting hepatitis b virus pre-S1 antigen according to the method of the present invention.

In a preferred embodiment, the kit of the invention comprises: a first anti-HBV PreS1 antibody and a second anti-HBV PreS1 antibody (the two antibodies may be the same or different and may be monoclonal antibodies or polyclonal antibodies) which can be specifically combined with the pre-S1 antigen of the hepatitis B virus and/or a lysate for detecting the pre-S1 antigen of the hepatitis B virus of the present invention. The kit preferably further comprises a reagent suitable for detecting the antigen-antibody reaction (e.g., a detection reagent for detecting a second antibody). Preferably, at least one of the anti-HBV PreS1 antibodies is labeled with or is capable of binding efficiently to a signal generator. The antibody is preferably a monoclonal antibody. Preferably the anti-HBV PreS1 antibody is selected from: 3H5, 7H11, 2a7, 4D11, 6F1, 13G2 and 16F5, in particular 7H11 and 4D 11.

In one embodiment, the kit comprises: (1) a solid phase carrier on which a first anti-HBV PreS1 antibody is immobilized; and/or (2) a lysate of the present invention for detecting hepatitis b virus pre-S1 antigen; and (3) optionally a second anti-HBV PreS1 antibody, labeled or unlabeled with a signal generator; and (4) optionally, a detection reagent for detecting the second antibody; and (5) optionally a buffer, a washing solution, and an instruction for use (preferably, the instruction for use describes the method for detecting hepatitis B virus pre-S1 antigen of the present invention).

Preferably, the solid phase carrier is a porous reaction plate (e.g., microplate, chemiluminescent plate) having an anti-HBV PreS1 antibody immobilized in the pores thereof.

Use of a reagent for detecting hepatitis b virus pre-S1 antigen in the preparation of a kit for detecting hepatitis b virus infection; preferably, the reagent is a reagent for detecting hepatitis B virus pre-S1 antigen as described in the present invention. For example, the reagents include: a first anti-HBV PreS1 antibody and a second anti-HBV PreS1 antibody (the two antibodies may be the same or different and may be a monoclonal antibody or a polyclonal antibody) which specifically bind to the hepatitis b virus pre-S1 antigen and/or a lysate for detecting the hepatitis b virus pre-S1 antigen of the present invention, optionally a reagent suitable for detecting an antibody reaction to the antigen (e.g., a detection reagent for detecting a second antibody); in particular: (1) a solid phase carrier on which a first anti-HBV PreS1 antibody is immobilized; and/or (2) a lysate of the present invention for detecting hepatitis b virus pre-S1 antigen; and (3) optionally a second anti-HBVPreS 1 antibody, labelled or not labelled with a signal generator; and (4) optionally, a detection reagent for detecting the second antibody; and (5) optionally a buffer, a washing solution, and an instruction for use (preferably, the instruction for use describes the method for detecting hepatitis B virus pre-S1 antigen of the present invention).

The invention also relates to methods of lysing viruses, comprising lysing the virus under mild conditions without substantially denaturing proteins (e.g., without substantially inactivating antibodies, such as pre-S1 antigen or core antigen antibodies), preferably by lysing the virus with a lysate of the invention for detecting hepatitis b virus pre-S1 antigen or a lysate of the invention for combined detection; the virus is preferably hepatitis b virus.

By "substantially" is meant that at least 70%, such as at least 80%, at least 90%, at least 95% of the things occur. For example, "not substantially denaturating a protein" or "not substantially inactivating an antibody" means that at least 70%, e.g., at least 80%, preferably at least 90%, or at least 95% of the protein or antibody is not denatured or inactivated.

The invention also relates to a mild virus lysate that is capable of substantially not denaturing proteins (e.g., substantially inactivating antibodies, such as the pre-S1 antigen or the core antigen antibody), and lysing viruses.

The present invention also relates to a method for detecting hepatitis b virus pre-S1 antigen and/or core antigen, comprising: lysing the virus under mild conditions without substantially denaturing the proteins (e.g., without substantially inactivating antibodies, such as the pre-S1 antigen or the core antigen antibody), preferably lysing the virus using a lysate of the invention that detects the hepatitis B virus pre-S1 antigen or a lysate of the invention that detects in combination; detecting hepatitis B virus pre-S1 antigen and/or core antigen. The detection of the pre-S1 antigen and the core antigen of hepatitis B virus can be carried out by known methods or by the method disclosed by the invention.

In particular, the present invention relates to a method for detecting hepatitis b virus pre-S1 antigen comprising: lysing the virus under mild conditions that do not substantially inactivate the anti-pre-S1 antigen antibody, preferably by lysing the virus with a lysate of the invention that detects the pre-S1 antigen of hepatitis B virus or a lysate of the invention that detects in combination; hepatitis B virus pre-S1 antigen was detected. The invention also relates to a method for detecting hepatitis B virus core antigen, which comprises the following steps: lysing the virus under mild conditions that do not substantially inactivate the anti-core antigen antibody, preferably by using a lysate that is used in the combined detection of the invention or the lysate used in the detection of the core antigen in the examples; detecting hepatitis B virus core antigen.

In a specific embodiment, the invention relates to a method of lysing a virus in a sample comprising lysing the virus with a virus lysate of the invention. Correspondingly, the invention also provides a method for detecting the core antigen, which comprises the step of cracking the virus in a sample by using the lysate. The core antigen can then be detected using known methods or methods disclosed herein. The invention also provides a method for detecting the pre-S1 antigen, which comprises the step of cracking the virus in a sample by using the lysate. The pre-S1 antigen can then be detected using known methods or methods disclosed herein. Preferably, the virus is hepatitis B virus.

In another embodiment, the invention relates to a method of lysing a virus comprising lysing the virus with a lysate of the invention that detects hepatitis b virus pre-S1 antigen; the virus is preferably hepatitis b virus. The invention also provides a method for detecting the pre-S1 antigen, which comprises the step of cracking the virus in a sample by using the lysate. The pre-S1 antigen can then be detected using known methods or methods disclosed herein.

The invention will be further described with reference to specific embodiments and the accompanying drawings. The examples are intended to illustrate the invention in detail by way of example. The choice of reagents, concentrations of reagents, temperatures and other variables and the choice of vectors and hosts are only illustrative of the application of the invention and do not constitute a limitation of the invention.

Examples

One, the first step.The invention relates to the preparation of HBV PreS1 monoclonal antibody

Example 1 preparation of GST-2147 x 6 antigen for use as antigen

Template preparation

With protease K-phenol: the chloroform method, HBV DNA was extracted from an internally preserved serum specimen (No. 263) of a patient with chronic hepatitis B. Multiple primers were designed, and the HBV gene was isolated by PCR segmentation and cloned into pMD18-T vector (Takara NO. CK2401) for sequencing. The GenBank accession number of the assembled full-length HBV sequence is AF233236, the full-length genome of the HBV strain is 3068bp, and the HBV strain is adw2 subtype. Wherein the cloning plasmid pT-PreS contains the full-length PreS1 and PreS2 genes. The sequence is as follows:

atgggaggttggtcttccaaacctcgacaaggcatggggacaaatctttctgttcccaatcctctgggattctttcccgatcaccagttggaccctgcgttcggagccaactcaaacaatccagattgggacttcaaccccaacaaggatcactggccagaggcaaatcaggtaggagcgggagcattcgggccagggttcaccccaccacacggcggtcttttggggtggagccctcaggctcagggcatattgacaacagtgccagcagcacctcctcctgcctccaccaatcggcagtcaggaagacagcctactcccatctctccacctctaagagacagtcatcctcaggcc(PreS1)；

atgcagtggaactccacaacattccaccaagctctgctagatcccagagtgaggggcctatattttcctgctggtggctccagttccggaacagtaaaccctgttccgactactgcctcacccatatcgtcaatct tctcgaggactggggaccctgcaccgaac(PreS2)。

plasmid construction

An upstream primer 2147F1(ATG AGA TCT CCT CTG GGA TTC TTT CC) and a downstream primer 2147R1(TTA GGA TCC ACC TCC ACC CGG ATT AAA GTC CCAATC TGG ATT GTT) are designed, a cloned plasmid pT-PreS is used as a template, a gene sequence of a 21-47AA segment in a preS1 region is called out by a PCR method, a plasmid pT-2147 x1 with pMD-18T reversely inserted is used as a starting plasmid, and a plasmid pT-2147 x 2 with 21-47 segments in double tandem is obtained firstly. In a similar manner, a recombinant plasmid pT-2147 x 3 with a 21-47 fragment triple tandem was constructed on the basis of pT-2147 x1 and pT-2147 x 2 (see FIG. 1 for a detailed plasmid construction process). Then, recombinant plasmid pT-2147-6 with PreS1(21-47) in six-fold tandem was obtained using pT-2147-3 as the starting plasmid.

The recombinant plasmid pT-2147 x 6 which is obtained by the above six tandem reaction was subjected to double digestion with Bgl II and BcoR I, and the objective fragment 2147 x 6 was recovered by a gel recovery kit (Shanghai Hua-Tu-Tokyo bioengineering Co., Ltd., NO.W5212). The expression plasmid pGEX-20T is subjected to double enzyme digestion treatment by BamH I and EcoR I, and the vector is recovered by an ethanol precipitation method. And (3) connecting the target fragment with pGEX-20T by using DNA ligase, and carrying out enzyme digestion identification to obtain GST-2147 x 6 recombinant expression plasmid.

Expression and purification of polypeptide GST-2147 x 6

The constructed recombinant plasmid GST-2147 × 6 was transformed into expression strain E.coli ER2566 for expression. SDS-PAGE was used to analyze the expression. GST-2147 × 6 exists in soluble form.

The soluble recombinant protein GST-2147 x 6 was purified by Glutathieone-Sepharose 4B affinity chromatography. The SDS-PAGE electrophoresis result shows that the purity of the target protein exceeds 85 percent. (see FIG. 2)

Example 2 production of hybridoma cell lines 3H5, 7H11, 2A7, 4D11, 6F1, 13G2, 16F5

Mouse immunization and fusion of splenocytes with hybridomas

6-8 weeks old female Balb/c mice were immunized for the first time and each mouse was given an intramuscular injection of 5. mu.g of GST-2147X 6 recombinant antigen emulsified in Freund's complete adjuvant (total volume 50. mu.l). After 15 days, the second basic immunization is carried out, and the method comprises the steps of taking the same amount of antigen, emulsifying the antigen by using Freund incomplete adjuvant, and carrying out intramuscular injection. After 30 days, tail vein was injected with a booster of 5 μ g of antigen without adjuvant, and 72 hours after booster immunization, mice were sacrificed, blood was collected, spleen was taken to prepare spleen cell suspension (suspended in RPMI1640 medium), and cells were counted in a cell counting plate. The cultured SP2/0 mouse myeloma cells were collected in 1/6 numbers of spleen cells, mixed, centrifuged, and the spleen cells were fused with mouse myeloma cells SP2/0 using polyethylene glycol (PEG 1500). The cell suspension was mixed with equal volumes of feeder cells (Balb/c mouse macrophages and thymocytes) and then cultured in 96-well cell culture plates (200. mu.l/well) in a 5% carbon dioxide incubator (ESPEC BNA-311) at 37 ℃. After 3 days, half of the stock solution was changed with HT medium (xanthine (hypoxanthon, H)1.361mg + thymidine (thymidine, T)0.388mg, dissolved in RPMI1640(GIBCO Co.) medium at 100mL, 45-50 ℃ and sterilized by filtration). After 7 days, the plates were coated with HBV PreS 121-47 synthetic peptide, and the culture supernatant of hybridoma cells obtained from the 96-well cell culture plates was assayed by enzyme-linked immunosorbent assay (ELISA) as described below. For cell clones positive for ELISA, cloning was performed by limiting dilution.

ELISA detection method

1. Coating antigen: HBV PreS 121-47 synthesizes peptide.

The sequence is as follows: PLGFFPDHQLDPAFGANSNNPDWDFNP

2. Coating buffer solution: 0.05mol/L carbonate buffer (20.02 gNa) at pH9.6₂CO₃，2.52g NaHCO₃Deionized water to 1 liter).

3. Preparing a coating solution: dissolving HBV PreS 121-47 polypeptide in coating buffer solution at a concentration of 1 μ g/ml,

4. coating:

adding 100 mul of coating solution into each hole of a 96-hole enzyme label plate, coating for 2 hours at 37 ℃, and then transferring to 2-8 ℃ for 16 hours.

5. And (3) sealing:

wash 1 time with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween 20). Then 200. mu.l of blocking solution (20mM Na pH7.4 containing 20% calf serum and 1% casein) was added to each well₂HPO₄/NaH₂PO₄Buffer solution), sealing at 37 deg.C for 2 hr; and throwing off the confining liquid. Drying and then putting into an aluminum foil bag for storage at 2-8 ℃.

6. And (3) detection:

adding into sample hole100 μ l of cell culture supernatant, each microplate equipped with 1-well positive control (mouse anti-GST-2147 × 6 polyclonal antiserum (obtained as in example 2), diluted 1: 100), and 1-well negative control (fresh cell culture), incubated at 37 ℃ for 30 min; washing with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20) for 5 times, patting dry, and adding 100 μ l of enzyme diluent (20mM Na pH7.4 containing 20% calf serum and 1% casein)₂HPO₄/NaH₂PO₄Buffer solution) of horseradish peroxidase-labeled goat anti-mouse immunoglobulin (HRP-GAM Ig, DAKO Co.) diluted 1: 1000, and incubated at 37 deg.C for 30 min; washing with PBST (20mM PB7.4, 150mM NaCl, 0.1% Tween20) washing solution for 5 times, beating to dry, adding A, B color development solution (provided by Beijing Wantai biological pharmaceutical Co., Ltd.) 50 μ l each, and developing at 37 deg.C for 10 min; add 50. mu.l stop solution (2M H)₂SO₄) The reaction was terminated, and A in each well was detected on a microplate reader (TECAN, Sunrise)₄₅₀Value, usually in A₄₅₀Values more than 2 times higher than the negative control were considered positive.

Obtaining and purifying monoclonal antibodies

1. Obtaining ascites of monoclonal antibody

Take 10 weeks old healthy Balb/c mice, inject the paraffin oil, each 0.5 ml. After 2-7 days, the cloned hybridoma cells are collected, centrifuged to remove the supernatant, added with serum-free medium (1640HT), and adjusted to a cell density of 2X 10⁵～2×10⁶One per ml, 0.5ml per mouse. The abdomen of the mouse is enlarged 7-10 days later, and ascites begins to be collected. Centrifuging at 3000rpm for 15 min, sucking the liquid of the middle clear part, filtering with 0.45 μm microporous membrane for sterilization, packaging, and storing at-20 deg.C.

2. Monoclonal antibody acquisition

The treated ascites was treated with 0.02mol/L PBS (81ml 0.2 mol/LNa) pH7.4₂HPO₄，19ml 0.2mol/L NaH₂PO₄20ml of 3mol/L NaCl, water to 100ml) was diluted twice, and saturated ammonium sulfate (concentration of 50% saturation) was slowly added dropwise with stirringAnd staying overnight at 2-8 ℃. After centrifugation at 12000rpm for 15 minutes at 4 ℃ and discarding of the supernatant, the pellet was dissolved in PBS 2 times the volume of the original ascites. And slowly adding saturated ammonium sulfate dropwise while stirring to enable the concentration of the ammonium sulfate to reach 33% saturation, and standing overnight at 2-8 ℃. The mixture was centrifuged at 12000rpm for 15 minutes at 4 ℃ to collect the supernatant. And (3) dropwise and slowly adding saturated ammonium sulfate while stirring, wherein the concentration reaches 50% saturation, and standing overnight at 2-8 ℃. Centrifuge at 12000rpm for 15 minutes at 4 ℃ and discard the supernatant. Dissolving the precipitate in a proper amount of PBS, placing into a dialysis belt, adding into 50-100 times of 0.02M PB buffer solution containing pH7.4, desalting for about 12 hours at 2-8 deg.C under stirring, and replacing the dialysate for more than 3 times. The purity of the monoclonal antibody can reach about 80 percent.

3. Monoclonal antibody purification

The dialyzed mAb was purified by DE52 column (Whatman No.4057050) under FPLC and the breakthrough peaks were collected. The purity of the monoclonal antibody can reach more than 90 percent. Storing the purified monoclonal antibody at-20 ℃.

7 hybridoma cell lines (3H5, 7H11, 2A7, 4D11, 6F1, 13G2 and 16F5) (not deposited) secreting anti-HBV PreS1 monoclonal antibody were selected by the above method

And II, performing secondary treatment.The HBV PreS1 monoclonal antibody preliminary identification

Example 3 screening of high affinity HBV PreS1 coated monoclonal antibody and enzyme-labeled monoclonal antibody

Preparing HBV PreS1 enzyme-labeled monoclonal antibody:

the modified sodium periodate method is adopted. Take the 20mg monoclonal antibody 3H5 as an example: accurately weighing HRP40mg, adding 0.2M acetate buffer solution (2 ml) with pH of 5.6, dissolving, and adding 0.06M NaIO₄2ml of the solution is reacted for 20 minutes at room temperature; adding 2ml of 0.16M glycol-10% NaCl solution, reacting at room temperature for 20 minutes, filling the enzyme solution into a dialysis bag, and dialyzing overnight against 0.001M acetate buffer solution with pH 4.0; adding 0.8ml of 2M carbonate buffer solution with the pH value of 9.6, adding 20mg of 3H5 monoclonal antibody, and stirring and reacting for 2 hours at the temperature of 4 ℃; adding newly prepared NaBH₄0.4ml of solution (5mg/ml) is stirred and reacted for 2 hours at 4 ℃; saturated dropwise (NH)₄)₂SO₄9.2ml of solution; stirring and reacting for 30 minutes at 4 ℃, and centrifuging for 20 minutes at 3500 rpm at 4 ℃; the supernatant was discarded, and the precipitate was dissolved in 2ml of 0.02M phosphate buffer pH7.4, dialyzed against 0.02M phosphate buffer pH7.4 for 24 hours, added with 2ml of glycerol, mixed well and stored at-20 ℃.

3H5, 7H11, 2A7, 4D11, 6F1, 13G2 and 16F5 monoclonal antibodies were HRP-labeled, respectively, in the manner described above.

Preparing an enzyme label plate:

1. preparing a coating solution:

the HBV PreS1 monoclonal antibody was applied to 20mM PB buffer (Na) pH7.4₂HPO₄/NaH₂PO₄Buffer, final concentration 20mM, pH 7.4) at a final concentration of 5. mu.g/ml.

2. Coating:

adding 100 mul of coating solution into each hole of a 96-hole enzyme label plate, and coating for 16-24 hours at the temperature of 2-8 ℃.

3. And (3) sealing:

3H5, 7H11, 2A7, 6F1, 13G2 and 16F5 monoclonal antibodies were coated separately in the manner described above.

The experimental method comprises the following steps:

1. preparing a specimen:

HBV virus positive (PCR detection) clinical serum specimen and HBV virus negative clinical serum specimen.

2. Sample adding:

taking the coated enzyme labelOne plate was plated and 50. mu.l of lysis solution (Na containing 1% TritonX 100) was added to each well₂HPO₄/NaH₂PO₄Buffer solution with final concentration of 20mM and pH value of 7.4), adding 50 μ l of corresponding specimen into each well, shaking and mixing, and placing in an incubator at 37 ℃ for reaction for 1 hour.

3. Adding an enzyme:

the reacted microplate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 100. mu.l of enzyme diluent (20mM Na having pH7.4 containing 20% calf serum and 1% casein) was added to each well₂HPO₄/NaH₂PO₄Buffer solution) 1: 1000 diluted enzyme-labeled monoclonal antibody (adopting NaIO)₄The corresponding monoclonal antibody is subjected to HRP labeling by an oxidation labeling method) and placed in an incubator at 37 ℃ for reaction for 30 minutes.

4. Color development:

the reacted enzyme-labeled plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 50. mu.l of each A, B color reagent was added to each well, and the plate was reacted at 37 ℃ for 15 minutes in an incubator.

5. Termination, reading:

adding stop solution (2M H) into each hole of the enzyme label plate after reaction₂SO₄)50 μ l and the A450/630 value of each well was measured on a microplate reader.

And (3) carrying out orthogonal detection on each coated monoclonal antibody and the enzyme-labeled monoclonal antibody by the method, and obtaining a P/N (ratio of the detection mean value of the positive sample to the detection mean value of the negative sample) value. See Table 1

TABLE 1 statistics of P/N ratio of each monoclonal antibody to enzyme-labeled monoclonal antibody

6. And (4) analyzing results:

as can be seen from the above table, the 7H11 monoclonal antibody coating and 4D11-HRP pairing detect HBV PreS1 as the best pairing.

And thirdly, performing the operation of the device.The invention relates to the establishment of a reaction system

Example 4 establishment of high sensitivity double front S1 antibody sandwich detection front S1 antigen system elisa plate preparation:

1. preparing a coating solution:

the HBV PreS1 monoclonal antibody 7H11 was diluted with 20mM PB buffer, pH7.4, to a final concentration of 5. mu.g/ml.

2. Coating:

3. And (3) sealing:

4. Preparing lysis solution containing different surfactants:

table 2: preparation of lysate of different components A-P

A, lysis solution: 2% Chaps +5mM EDTA +20mM PB7.4

B, lysis solution: 2% Chaps +5mM EDTA +20mM PB7.4+ 0.2% Me

C, cracking liquid: 2% HP + 0.2% Me +20mM PB7.4

D, cracking liquid: 2% SB8+ 0.2% Me +5mM EDTA +20mM PB7.4

E, lysis solution: 2% SB12+ 0.2% Me +5mM EDTA +20mM PB7.4

F, lysate: 2% SB14+ 0.2% Me +5mM EDTA +20mM PB7.4

G, lysate: 1% SB16+ 0.2% Me +5mM EDTA +20mM PB7.4+ 0.5% SB14

H, cracking liquid: 1% SB18+ 0.2% Me +5mM EDTA +20mM PB7.4+ 0.5% SB14

I, lysis solution: 2% SB14+5mM EDTA +20mM PB7.4

J lysis solution: 2% Tw80+ 0.2% Me +5mM EDTA +20mM PB7.4

K, lysis solution: 2% Tw60+ 0.2% Me +5mM EDTA +20mM PB7.4

L, lysate: 2% Tw40+ 0.2% Me +5mM EDTA +20mM PB7.4

M lysis solution: 2% Tw20+ 0.2% Me +5mM EDTA +20mM PB7.4

N, cracking liquid: 2% TX100+ 0.2% Me +5mM EDTA +20mM PB7.4

O lysis solution: 2% BRJ35+ 0.2% Me +5mM EDTA +20mM PB7.4

Lysis solution P: 1% BRJ35+ 0.2% Me +5mM EDTA +20mM PB7.4

Note:

HP: HP-beta-CD, Hydroxypropyl-beta-Cyclodextrin (Hydroxypropyl-beta-Cyclodextrin); SB 8: the amount of sulfobetain8 is such that,

3-(Octodecyldimethylammonic)propanesulfonate；SB10：sulfobetain10，

3-(Decyldimethylammonic)propanesulfonate；SB12：sulfobetain12，

3-(N，N-Dimethyldodecylammonio)-propanesulfonate；SB14：sulfobetain14，

3-(N，N-Dimethylmyristylammonio)-propanesulfonate；SB16：sulfobetain16，

3-(N，N-Dimethylpalmitylammonio)-propanesulfonate；SB18：sulfobetain18，

3-(N，N-Dimethylstearylammonio)-propanesulfonateChaps：

3- (3- (cholamidopropyl) dimethylammonio) -1-propanesulfonateMe: beta-mercaptoethanol; tw 80: tween-80; tw 40: tween-40; tw 20: tween-20; TX 100: TritonX 100; BRJ 35: brij 35 (polyoxyethylene lauric acid acyl ether 35); 20mM PB 7.4: na (Na)₂HPO₄/NaH₂PO₄Buffer, final concentration 20mM, pH7.4

The experimental method comprises the following steps:

1. preparing a specimen:

HBV virus positive (PCR detection) clinical serum specimen, normal human serum specimen.

2. Sample adding:

taking the coated ELISA plate, adding 50 mul of lysis solution with different formulas into each hole, adding 50 mul of corresponding sample into each hole (adding only 50 mul of corresponding sample without any lysis solution as a non-lysis control), shaking and mixing uniformly, and placing in a 37 ℃ incubator for reacting for 1 hour.

3. Adding an enzyme:

the reacted microplate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 100. mu.l of enzyme diluent (20mM Na having pH7.4 containing 20% calf serum and 1% casein) was added to each well₂HPO₄/NaH₂PO₄Buffer solution) diluted 1: 1000 4D11 enzyme-labeled monoclonal antibody, and placing the monoclonal antibody in an incubator at 37 ℃ for reaction for 30 minutes.

4. Color development:

5. Termination, reading:

adding stop solution (2M H) into each hole of the enzyme label plate after reaction₂SO₄)50 μ l, and detecting A in each well on a microplate reader_450/630The value is obtained.

6. Calculating the detection titer of the positive specimen:

the positive specimen detection titer refers to diluting the positive specimen by times until the positive specimen can not be detected as positive (the positive judgment refers to that the negative specimen A corresponding to the same cracking condition is used for positive judgment_450/630X 2.1 is Cutoff, the positive detection result is judged to be positive when the Cutoff is larger than or equal to the Cutoff, and the negative detection result is judged to be negative otherwise), and the maximum dilution multiple of the positive detection result of the sample is the detection titer of the positive sample.

The experimental results are as follows:

TABLE 3 statistics of detection data for different lysates of A-P

In the experimental results, it can be seen that the use of a series of buffer solutions containing different amounts of different kinds of surfactants can effectively split viruses, and improve the detection sensitivity of the pre-double pre-S1 antibody detection pre-S1 antigen, and the effective components include: chaps, sulfobetain8-18, Brij 35, Tween series, HP-beta-CD, TritonX100, and the like.

Example 5 establishment of Pre-S1 antigen and core antigen Joint detection lysis System

Preparation of lysates from different combination detection formulations

Lysates of 24 different components were prepared as shown in Table 4

Preparing an enzyme label plate:

1. preparing a coating solution:

the 7H11 monoclonal antibody and the anti-HBc monoclonal antibody G8 (Xiamen Bosheng Biotech Co., Ltd. No.050824) were diluted with 20mM PB buffer solution at pH7.4 to give final concentrations of 5. mu.g/ml and 4. mu.g/ml, respectively.

2. Coating:

3. And (3) sealing:

The experimental method comprises the following steps:

7. preparing a specimen:

8. Sample adding:

taking a coated ELISA plate, adding 50 mu l of different-formula lysis solutions into each hole, adding 50 mu l of corresponding samples into each hole (4 positive serums and 4 negative serums which are the same under each lysis condition), shaking, mixing uniformly, and placing in a 37 ℃ incubator for reaction for 1 hour.

9. Adding an enzyme:

the reacted microplate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 100. mu.l of enzyme diluent (20mM Na having pH7.4 containing 20% calf serum and 1% casein) was added to each well₂HPO₄/NaH₂PO₄Buffer solution) 4D11 enzyme (HRP) -labeled monoclonal antibody and enzyme (HRP) -labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotech Co., Ltd., NO.04042901) diluted at 1: 1000 were placed in an incubator at 37 ℃ and reacted for 30 minutes.

10. Color development:

11. Termination, reading:

The method is used for detecting 24 lysates with different formulas, and the P/N (ratio of the detection mean value of the positive sample to the detection mean value of the negative sample) value is obtained. See Table 5

12. And (4) analyzing results:

as can be seen from the above table, lysis10, lysis13, lysis16, lysis18 are the best lysate formulations.

Example 6 Effect analysis by adding lysis System to the conventional hepatitis B Virus Pre-S1 antigen assay

The traditional detection mode of the pre-S1 antigen of the hepatitis B virus is also generally to adopt a double-antibody sandwich ELISA method, but the detection sensitivity of the pre-S1 antigen is improved by means of the high abundance of the HBsAg on hepatitis B virus particles by using anti-HBsAg (signal-labeled anti-HBsAg) as a capture antibody (reporter antibody) and matching anti-PreS1(anti-PreS1) labeled with a signal as a corresponding reporter antibody (capture antibody).

Preparing an enzyme label plate:

1. preparing a coating solution:

the HBV PreS1 monoclonal antibody 4D11 was diluted with 20mM PB buffer solution with pH7.4 to a final concentration of 5. mu.g/ml; the purchased anti-HBsAg (Xiamen Bosheng Biotech) was diluted with 20mM PB buffer pH7.4 to a final concentration of 3. mu.g/ml.

2. Coating:

adding 100 mul of coating solution into each hole of a 96-hole enzyme label plate, and coating for 16-24 hours at the temperature of 2-8 ℃; the ELISA plate coated by the 4D11 monoclonal antibody is marked as an A plate, and the ELISA plate coated by the anti-HBsAg monoclonal antibody is marked as a B plate.

3. And (3) sealing:

the coated A and B plates were washed 1 time with PBST wash (PBS containing 0.1% Tween 20). Then 200. mu.l of blocking solution (20mM Na pH7.4 containing 20% calf serum and 1% casein) was added to each well₂HPO₄/NaH₂PO₄Buffer solution), sealing at 37 deg.C for 2 hr; and throwing off the confining liquid. Drying and then putting into an aluminum foil bag for storage at 2-8 ℃.

4. Preparing lysis solution containing different surfactants:

table 6: preparation of alpha-o different component cracking liquid

The alpha lysis solution is: 2% Chaps +5mM EDTA +20mM PB7.4

The beta lysis solution is: 2% HP +20mMPB7.4

Gamma lysis solution is: 2% SB8+5mM EDTA +20mM PB7.4

Delta lysate is: 2% SB12+5mM EDTA +20mM PB7.4

The epsilon lysis solution is: 2% SB14+5mM EDTA +20mM PB7.4

The zeta lysis solution was: 1% SB16+5mM EDTA +20mM PB7.4+ 0.5% SB14

Eta lysate is: 1% SB18+5mM EDTA +20mM PB7.4+ 0.5% SB14

The theta lysate is: 2% SB14+5mM EDTA +20mM PB7.4

The iota lysate is: 2% Tw80+5mM EDTA +20mM PB7.4

The kappa lysate is: 2% Tw60+5mM EDTA +20mM PB7.4

The lambda lysate is: 2% Tw40+5mM EDTA +20mM PB7.4

μ lysis was: 2% Tw20+5mM EDTA +20mM PB7.4

Nu lysis solution is: 2% TX100+5mM EDTA +20mM PB7.4

The xi lysate is: 2% BRJ35+5mM EDTA +20mM PB7.4

The lysate is: 1% BRJ35+5mM EDTA +20mM PB7.4

Note: the chemical names of the medicines are shown in example 4 and example 5

The experimental method comprises the following steps:

5. preparing a specimen:

6. Sample adding:

taking the coated A plate and B plate, adding 50 mul of lysate with different formulas into each well, adding 50 mul of corresponding sample into each well (the non-lysis control is only 50 mul of corresponding sample without any lysate), shaking and mixing uniformly, and placing in a 37 ℃ incubator for reacting for 1 hour.

7. Adding an enzyme:

the reacted A and B plates were washed 5 times with PBST wash solution, and 100. mu.l of enzyme diluent (20mM Na of pH7.4 containing 20% calf serum and 1% casein) was added to each well of the A plate₂HPO₄/NaH₂PO₄Buffer solution) HRP-labeled anti-HBsAg (1: 10000); mu.l of enzyme diluent (20mM Na pH7.4 containing 20% calf serum and 1% casein) was added to each well of the B plate₂HPO₄/NaH₂PO₄Buffer solution) HRP-labeled 4D11 (1: 1000) was placed in an incubator at 37 ℃ for 30 minutes.

8. Color development:

the reacted A plate and B plate were washed 5 times with PBST wash solution, 50. mu.l of A, B color reagent was added to each well, and the reaction mixture was incubated at 37 ℃ for 15 minutes.

9. Termination, reading:

adding 50 mul of stop solution into each hole of the reacted A plate and B plate, and detecting A of each hole on a microplate reader_450/630The value is obtained.

10. Calculating the detection titer of the positive specimen:

The experimental results are as follows:

table 7 statistics of lysate detection data for different fractions α ° o ″

And (4) analyzing results:

the results of the above experiments show that: the sensitivity of epsilon lysate, zeta lysate, eta lysate and theta lysate to the traditional detection system of pre-S1 antigen of hepatitis B virus is greatly improved

Diagnostic kit related to the invention

Example 7 preparation of diagnostic kit for detecting hepatitis b virus pre-S1 antigen (HBV PreS1) and detection method thereof (enzyme linked immunosorbent assay).

Preparation of the kit

The HBV PreS1 monoclonal antibody 7H11, 4D11 (other anti-pre-S1 antigen antibody can be used) hepatitis B virus pre-S1 antigen diagnostic kit contained in the invention comprises the following components and is prepared:

1. preparing an enzyme label plate:

a coating solution of appropriate concentration was prepared as in example 3 and applied to the microplate.

2. Sample lysis solution:

lysates of F or G or H as in example 4

3. Preparing an enzyme labeling reagent:

HRP-labeled 4D11 was prepared as an enzyme-labeled reagent at a suitable concentration (1: 1000) in 20mM Na pH7.4 containing 20% calf serum and 1% casein as diluents₂HPO₄/NaH₂PO₄And (4) a buffer solution.

4. Negative and positive control, color development reagent A, B solution, 20 × concentrated washing solution, and stop solution (2 MH)₂SO₄)：

Supplied by Beijing Wantai biological pharmaceutical Co.

Kit detection method

The detection method of the diagnostic kit for detecting hepatitis B virus pre-S1 antigen (HBV PreS1) is as follows:

1. preparing: the reagent is placed at room temperature (18-30 ℃) and is balanced for 15-30 minutes.

2. Preparing liquid: 50ml of concentrated washing solution (20X) are diluted to 1000ml with distilled or deionized water for further use.

3. Numbering: the corresponding micropores of the sample are numbered in sequence.

4. Cracking: add 50. mu.l of sample lysate (F or G or H lysis solution as in example 4) to the corresponding well using a pipette.

5. Sample adding: respectively adding 50 mul of sample to be detected into the corresponding holes, and shaking and mixing uniformly.

6. And (3) incubation: incubation was carried out with a sealing plate membrane for 60 minutes at 37. + -. 1 ℃ after sealing.

7. Washing: and removing the sealing plate film after incubation, sucking liquid in the holes, washing for 5 times by using a washing solution (1x), and soaking for 30-60 seconds each time.

8. Adding an enzyme: add 100 μ l enzyme labeling reagent into corresponding holes except blank hole.

9. And (3) incubation: incubation was carried out for 30 minutes at 37. + -. 1 ℃ with a membrane seal plate.

10. Washing: and removing the sealing plate film after incubation, sucking liquid in the holes, washing for 5 times by using a washing solution (1x), and soaking for 30-60 seconds each time.

11. Color development: 50 mul of each color developing agent A, B liquid is added into each hole, the mixture is mixed by gentle shaking, and the mixture is placed behind a sealing plate and a membrane sealing plate for color development at 37 +/-1 ℃ in a dark place for 15 minutes.

12. And (4) terminating: stop solution (2M H) was added to each well₂SO₄)50 μ l, mix well with gentle shaking.

13. And (3) determination: setting the wavelength of a microplate reader to 450/630nm for detection, and determining the OD value of each well.

14. And (4) judging a result:

critical value: cut Off (c.o.) - > 0.105

And (4) judging a result: (S-absorbance per well)

Negative result (S/C.O. < 1): the sample is negative when the absorbance value is less than Cut Off value, which indicates that HBV PreS1 antigen is not detected in the sample.

Positive result (S/C.O.. gtoreq.1): the positive result of the sample absorbance value being greater than or equal to Cut Off value represents the detection of HBV PreS1 antigen in the sample.

Example 8 preparation of a diagnostic kit for detecting hepatitis B virus core antigen (HBcAg) and a detection method thereof (enzyme linked immunosorbent assay).

Preparation of the kit

The hepatitis B virus core antigen diagnostic kit comprises the following components and is prepared:

1. preparing an enzyme label plate:

anti-HBc monoclonal antibody G8 (Xiamen Bosheng Biotech Co., Ltd. No.050824) was diluted with 20mM PB buffer solution at pH7.4 to a final concentration of 4. mu.g/ml.

2. Sample lysis solution:

lysis10 or lysis13 or lysis16 or lysis18 as in example 5

3. Preparing an enzyme labeling reagent:

the purchased HRP-labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotech Co., Ltd., NO.04042901) was prepared into an enzyme-labeled reagent with a suitable concentration (1: 1000 dilution) of 20mM Na containing 20% calf serum and 1% casein and having a pH of 7.4₂HPO₄/NaH₂PO₄And (4) a buffer solution.

Negative and positive control, color development reagent A, B solution, 20 × concentrated washing solution, and stop solution (2M H)₂SO₄)。

Kit detection method

The detection method of the diagnostic kit for detecting hepatitis B virus core antigen (HBcAg) is as follows:

4. Cracking: mu.l of the sample was pipetted into the corresponding well for lysis (lysis 10, lysis13, lysis16, lysis18 as in example 5).

7. Washing: and removing the sealing plate film after incubation, sucking liquid in the holes, washing for 5 times by using a washing solution, and soaking for 30-60 seconds each time.

10. Washing: and removing the sealing plate film after incubation, sucking liquid in the holes, washing for 5 times by using a washing solution, and soaking for 30-60 seconds each time.

14. And (4) judging a result:

critical value: cut Off (c.o.) - > 0.105

And (4) judging a result: (S-absorbance per well)

Negative result (S/C.O. < 1): the sample is negative when the absorbance value is less than the Cut Off value, which means that no HBcAg is detected in the sample.

Positive result (S/C.O.. gtoreq.1): the positive result is that the absorbance value of the sample is greater than or equal to the Cut Off value, which represents that the HBcAg is detected in the sample.

Example 9 preparation of diagnostic kit (enzyme linked immunosorbent assay) for combined detection of pre-S1 antigen and core antigen of hepatitis B virus and detection method thereof

Preparation of the kit

The invention comprises the following components and preparations of a pre-S1 and core antigen diagnostic kit (enzyme linked immunosorbent assay) for jointly detecting hepatitis B virus:

1. preparing an enzyme label plate:

7H11 (other anti-pre-S1 antigen antibody may also be used) was coated on the microplate in combination with the purchased anti-HBc monoclonal antibody G8 (Xiamen Bosheng Biotechnology Co., Ltd. No.050824)) (other anti-HBc antibody may also be used) diluted to an appropriate concentration (final concentrations of 5. mu.g/ml and 4. mu.g/ml, respectively) with 20mM PB buffer pH7.4 as in example 4.

2. Preparing a sample lysate:

lysis10 or lysis13 or lysis16 or lysis18 as in example 5

3. Preparing an enzyme labeling reagent:

4D11-HRP (other anti pre-S1 antigen antibody may also be used) and HRP-labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotech Co., Ltd., No.04042901)) (other anti-HBc antibody may also be used) were formulated as in example 4 into enzyme-labeled reagents at appropriate concentrations (1: 1000 dilution each) in 20mM Na containing 20% calf serum and 1% casein at pH7.4₂HPO₄/NaH₂PO₄And (4) a buffer solution.

4. Negative and positive control, color development reagent A, B solution, 20 × concentrated washing solution, and stop solution (2 MH)₂SO₄)。

Kit detection method

The detection method of the diagnostic kit for jointly detecting the pre-S1 antigen and the core antigen (HBcAg) of the hepatitis B virus is as follows:

4. Diluting: add 50. mu.l of sample dilution to the corresponding well using a pipette.

14. And (4) judging a result:

critical value: cut Off (c.o.) - > 0.105

And (4) judging a result: (S-absorbance per well)

Negative result (S/C.O. < 1): the sample is negative when the absorbance value is less than Cut Off value, which indicates that HBV PreS1 and HBcAg are not detected in the sample.

Positive result (S/C.O.. gtoreq.1): the positive result of the sample absorbance value being greater than or equal to Cut Off value represents that HBV PreS1 and/or HBcAg are detected in the sample.

And fifthly.Diagnostic kit applications and result analysis related to the present invention

Example 10 parallel comparison of the Combined detection of hepatitis B Virus Pre-S1 antigen and core antigen (nucleic acid-related antigen) with the detection of hepatitis B Virus nucleic acid, and the detection of the same Pre-S1 antigen alone

Kit preparation

Hepatitis b virus pre-S1 antigen diagnostic kit (enzyme linked immunosorbent assay) was prepared as in example 7; preparation of hepatitis b virus core antigen diagnostic kit (enzyme linked immunosorbent assay) as in example 8; the diagnostic kit (enzyme linked immunosorbent assay) for the combined detection of the pre-S1 antigen and the core antigen of the hepatitis B virus is prepared as in example 9. Commercial individual hepatitis B virus pre-S1 antigen diagnostic kits (enzyme linked immunosorbent assay) were purchased from Shanghai Fuxing Chang Zheng medicine science and technology Co., Ltd. (NO.3150480) and Shanghai alpha Biotech Co., Ltd., respectively.

The hepatitis B virus nucleic acid quantitative PCR detection reagent is purchased from Shenzhen P-based bioengineering company, Inc. (NO.06010121), Yingke innovation (Xiamen) science and technology company, Inc. and Daan Gen corporation (NO. # DA-B051).

The hepatitis B virus two-in-one and two-in-one enzyme-linked immunoassay reagent is purchased from Beijing Wantai biological pharmaceutical industry Co Ltd (WB2196), Yingke innovative creation (Xiamen) science and technology Co Ltd (IPT21001, IPT21201, IPT21301, IPT21401 and IPT21501) and Shanghai actual science and technology Co., Ltd (NO.267\265\266\ 263).

Detection specimen

116 clinical hepatitis B virus infection-related patient serum samples collected from the traditional Chinese medicine hospital in Xiamen city and the first people hospital in Quanzhou city in 2005-2006 for 3 months, and are preserved at-20 ℃ in a freezing way.

Detecting items

The hepatitis B virus nucleic acid quantitative PCR detection is carried out on each serum specimen, three reagents of different manufacturers (Shenzhen P ji bioengineering, Hill. Ke Xin (Xiamen) science and technology limited and Danan Gen corporation) are adopted for parallel detection, and the detection program and the result judgment are strictly carried out according to the specifications of the reagents. The qualitative judgment of the hepatitis B virus nucleic acid is based on the same detection result of the two reagents.

Two-in-one and two-half enzyme-linked immunoassay of hepatitis B virus is carried out on each serum specimen, three reagents of different manufacturers (Beijing Wantai biological medicine industry Co., Ltd., Yingke New creation (Xiamen) science Co., Ltd., Shanghai actual science Kowa biological technology Co., Ltd.) are adopted for parallel detection, and detection procedures and result judgment are strictly carried out according to the specifications of the reagents. The qualitative judgment of the hepatitis B virus HBsAg, HBsAb, HBeAg, HBeAb and HBcAb is based on the same detection result of the two reagents.

The same type of commercialized hepatitis B virus pre-S1 antigen enzyme-linked immunoassay is carried out on each serum specimen, two different manufacturer reagents (Shanghai Fuxing Chang Zheng medicine science and technology Co., Ltd. and Shanghai alpha Biotechnology Co., Ltd.) are adopted for parallel detection, and the detection program and result judgment are strictly carried out according to the specifications of the reagents.

For each serum sample, hepatitis B virus pre-S1 antigen detection, hepatitis B virus core antigen detection, and combined detection of hepatitis B virus pre-S1 antigen and core antigen (collectively referred to as nucleic acid-associated antigens) disclosed in examples 6, 7, and 8, respectively, were performed. The detection procedure and the judgment of the results were carried out exactly as described in the corresponding examples.

The result of the detection

Statistics are performed after all the items to be tested are completed, as shown in Table 8.

Table 8: statistical results of 116 clinical hepatitis B serum

Note: "reagent made at home 1": hepatitis B virus pre S1 antigen diagnostic kit (enzyme linked immunosorbent assay) produced by Shanghai alpha biotechnology company

"reagent 2 made in China": hepatitis B virus pre S1 antigen diagnostic kit (enzyme linked immunosorbent assay) produced by Shanghai Fuxing Changzhe medicine science and technology Limited company

Reagent for "joint test result": the present invention discloses a detection reagent (called HBV combo ELISA) combining hepatitis B virus pre-S1 antigen and core antigen, as described in example 8 "PreS 1" reagent: the invention discloses a hepatitis B virus pre-S1 antigen detection reagent, such as example 6

"Core" reagent: the invention discloses a hepatitis B virus core antigen detection reagent, as example 7

The calculation method of the sensitivity and the coincidence rate of the sensitivity and the nucleic acid comprises the following steps:

sensitivity-total number of detected positives/total number of true positives (nucleic acid positives)

Nucleic acid coincidence rate (total number of test samples-total number of two types of test results not coincident)/total number of test samples

And (4) analyzing results:

as can be seen from the statistical results in table 2, the hepatitis b virus pre-S1 antigen detection reagent according to example 7 has a great advantage in sensitivity compared to the commercial pre-S1 antigen detection reagent because the hepatitis b virus pre-S1 antigen detection reagent according to example 7, unlike the conventional commercial reagent, uses a double pre-S1 antibody sandwich method detection mode, instead of following the conventional detection mode by relying on the whole LHBs (i.e., by HBsAg-mediated detection on LHBs, using a specific antibody against HBsAg as a capture antibody or a reporter antibody, and pairing with a specific antibody against PreS1), forms an independent pre-S1 antigen detection reagent, does not cause the detection of the pre-S1 antigen on virus Dane particles due to mutation of HBsAg on LHBs, and has a high correlation with hepatitis b virus nucleic acid.

It can also be seen from table 2 that under the same detection conditions, the combined detection of PreS1 and HBcAg has greater advantages than the detection of PreS1 and HBcAg alone, regardless of the sensitivity or the nucleic acid coincidence rate, and the combined detection of HBcAg and PreS1 makes up for the possible false negative defect caused by single HBcAg or PreS1 mutation or omission. This is illustrated visually by the results of the specific tests on the partial serum samples listed in Table 9.

From the above analysis, it was found that the detection of the pre-S1 antigen alone is not sufficient to show the high correlation with the detection of hepatitis B virus nucleic acid, and that the new indicator formed by the combined detection of the pre-S1 antigen and the core antigen shows the high correlation with the detection of hepatitis B virus nucleic acid (97.41% in this example).

Example 11 Combined detection of Pre-S1 antigen and core antigen (nucleic acid-related antigen) of hepatitis B Virus and comparative analysis of hepatitis B Virus "two-and-a-half" (HBsAg, HBsAb, HBeAg, HBeAb, HBcAb)

Kit preparation

The kit for the combined detection of pre-S1 antigen and core antigen of hepatitis B virus (enzyme linked immunosorbent assay) was prepared as described in example 9, and a kit prepared by a similar method can be used.

The hepatitis B virus nucleic acid quantitative PCR detection reagent is purchased from Shenzhen P.P.Shiji bioengineering, Inc., Yingke innovation (Xiamen) science and technology, Inc. and Daan Gen corporation.

The hepatitis B virus two-in-one and two-in-one enzyme-linked immunoassay reagent is purchased from Beijing Wantai biological pharmaceutical industry Co., Ltd, Yingke creative (Xiamen) science Co., Ltd and Shanghai actual Korea biological technology Co., Ltd.

Detection specimen

116 clinical hepatitis B virus infection-related patient serum samples collected from traditional Chinese medicine hospitals in Xiamen city and first people hospitals in Quanzhou city in 2005-2006 for 3 months, and serum samples collected from hepatitis B virus infection-related blood donors at Xiamen blood station, and stored at-20 ℃ for freezing.

Detecting items

The hepatitis B virus nucleic acid positive, "two-and-a-half" HBsAg negative and semi-abnormal pattern specimens were confirmed by screening PCR, and the combined hepatitis B virus pre-S1 antigen and core antigen (collectively referred to as nucleic acid-related antigens) as disclosed in example 9 of the present invention were detected. The procedure for detection and the judgment of the result were carried out exactly as described in example 9.

Test results and analysis

Statistics are taken after all the items to be tested are completed, as shown in Table 10.

It can be seen from the above table that in the samples of the conventional hepatitis B virus "two-half and two-half" test for judging whether the non-virus replication carrier (HBeAg negative) or the non-B infectious virus infected carrier (HBsAg negative), a large amount of virus still exists in the samples, the samples can be detected well by the combined test of the former S1 and the core antigen, and a correct judgment and understanding can be provided for whether the clinical hepatitis patient carries the virus, so that the treatment scheme can be determined well. Particularly for the conventional blood station test item of surface antigen (HBsAg), the combined test of the former S1 and the core antigen can reduce the transfusion transmission of hepatitis B caused by surface antigen variation or missed test.

Example 12 comparative assay for the Combined detection of the Pre-S1 antigen and core antigen (nucleic acid-associated antigen) of hepatitis B Virus with conventional Single primer quantitative PCR of hepatitis B Virus nucleic acid

Kit preparation

The diagnostic kit (enzyme linked immunosorbent assay) for the combined detection of the pre-S1 antigen and the core antigen of the hepatitis B virus is prepared as in example 9.

Detection specimen

314 HBsAg-positive hepatitis B virus infection serum samples collected from China biological products institute in 2005 at 12 months were stored at-20 ℃.

Detecting items

The hepatitis B virus nucleic acid quantitative PCR detection is carried out on each serum specimen, three reagents of different manufacturers (Shenzhen P ji bioengineering, Hill. Ke Xin (Xiamen) science and technology limited and Danan Gen corporation) are adopted for parallel detection, and the detection program and the result judgment are strictly carried out according to the specifications of the reagents. The qualitative judgment of the negative of the hepatitis B virus nucleic acid is that the detection results of the three reagents are negative, and the quantitative PCR negative is obtained.

Screening and quantitative PCR were performed to confirm that the HBV nucleic acid was negative and the HBsAg was positive in two and a half pairs, and the combined HCV pre-S1 antigen and core antigen (collectively referred to as nucleic acid-related antigen) disclosed in example 5 of the present invention were detected. The procedure for detection and the judgment of the results were carried out exactly as described in example 5. And performing nested PCR detection on the samples (method reference: Marie-ane Pet et al, JMV 2001).

Test results and analysis

And (4) counting results after all the items to be detected are completed, wherein 39 HBsAg positive serum samples are counted, and the hepatitis B virus nucleic acid negative serum samples are confirmed by quantitative PCR. Of the 39 specimens, 31 were positive in the combined test,

after the 31 specimens were subjected to the split-type PCR, 9 of the specimens were confirmed to be positive for hepatitis B virus nucleic acid.

From the above results, it is difficult to express the presence of virus in a non-replicating state because only replicated virus but not provirus can be detected by conventional serum PCR, and there is a possibility that some omission of detection may occur in conventional quantitative PCR detection reagents due to various limitations because single primers are used and nested PCR with high sensitivity is not used. Clinically, hepatitis symptoms of hepatitis B patients are usually obvious, but the conventional PCR detection is nucleic acid negative.

The mode of jointly detecting the pre-S1 antigen and the core antigen of the hepatitis B virus can make up part of defects of conventional serum PCR to a certain extent.

Example 13 specificity assay for the Combined detection of hepatitis B Virus Pre-S1 antigen and core antigen (nucleic acid-associated antigen) reagents

Kit preparation

A diagnostic kit (enzyme linked immunosorbent assay) for the combined detection of the pre-S1 antigen and the core antigen of hepatitis B virus was prepared as described in example 9.

Detection specimen

5976 serum samples of healthy blood donors collected from Xiamen CDC, Beijing blood station and Shaoxing blood station in 2005-2006-3 months are frozen and preserved at-20 ℃.

Detecting items

Each serum was tested for the combined hepatitis B virus pre-S1 antigen and core antigen (collectively referred to as HBV combo ELI SA) as disclosed in example 9. The procedure for detection and the judgment of the result were carried out exactly as described in example 9.

Results and analysis

And (5) counting results after all the items to be detected are finished, wherein the counting results are shown in a table 11.

Origin of specimen	Number of negative specimen	Number of negative samples in the Joint test	Specificity of
Origin of specimen	Number of negative specimen	Number of negative samples in the Joint test	Specificity of	Building door CDC	4554	4547	99.90％
Shaoxing blood station	428	427	99.80％	Building door CDC	4554	4547	99.90％
Shaoxing blood station	428	427	99.80％	Beijing blood station	994	991	99.70％
Total up to	5976	5965	99.82％	Beijing blood station	994	991	99.70％

Table 11: specificity analysis of Combined detection

From the above results, the detection reagent for detecting the combined hepatitis b virus pre-S1 antigen and the core antigen (collectively referred to as nucleic acid-related antigen) disclosed by the present invention has good specificity.

Example 14 Combined detection of hepatitis B Pre-S1 antigen and core antigen for use in chemiluminescence

Preparation of chemiluminescent kit

The invention comprises the following components and preparations of a pre-S1 and core antigen diagnostic kit (chemiluminescence method) for jointly detecting hepatitis B virus:

1. preparing a chemiluminescent plate:

7H11 (others may be used) was combined with a commercial anti-HBc monoclonal antibody to coat daughter chemiluminescent plate (Xiamen New America, NO. C0004) diluted G8 (Xiamen Biotech, Inc. No.050824) (other antibodies against pre-S1 antigen and anti-core antigen may be used) to appropriate concentrations (final concentrations of 5. mu.g/ml and 4. mu.g/ml, respectively) as in example 3.

2. Sample lysis solution:

as in lysis10 or lysis13 or lysis16 or lysis18 in example 5.

3. Preparing an enzyme labeling reagent:

4D11-HRP was mixed with a commercial HRP-labeled anti-HBs monoclonal antibody, HRP-labeled anti-HBc monoclonal antibody CZ (Xiamen Bosheng Biotech Co., Ltd., No.04042901) (other antibodies against pre-S1 antigen and anti-core antigen may be used) as in example 2 to prepare a suitable concentration (1: 10000 enzyme labeling reagent, 20mM Na pH7.4 containing 20% calf serum and 1% casein in a diluent₂HPO₄/NaH₂PO₄And (4) a buffer solution.

4. Luminescent agent (PIERCE NO.1856155), washing solution (PBS solution containing 1% Tween20)

Kit detection method

The detection method of the diagnostic kit (chemiluminescence method) for jointly detecting the pre-S1 antigen and the core antigen (HBcAg) of the hepatitis B virus is as follows:

4. Cracking: add 50. mu.l of sample lysate to the corresponding well using a pipette.

11. Luminescence: add 100 μ l of luminescent agent into each well, and place in chemiluminescence apparatus to measure the luminescence intensity of each well.

Analysis of results

20 positive hepatitis B specimens containing different amounts of virus were sampled and subjected to fluorescent quantitative PCR measurement, and the results of the measurement by the chemiluminescence method are shown in Table 12. Since both the fluorescence quantitative PCR quantification and the luminescence intensity are close to logarithmic distribution, we performed linear correlation analysis after respectively logarithmizing the virus content and the luminescence intensity of each specimen as shown in FIG. 3.

TABLE 12 correlation of chemiluminescence detection with fluorescence quantitative PCR

As can be seen from Table 12 and FIG. 3, the combined detection method of pre-S1 and core antigen of hepatitis B virus has a high correlation with virus content. The quantitative detection of the combined hepatitis B virus pre-S1 and core antigen can be used for estimating the nucleic acid load of the sample.

Claims

1. A method for detecting hepatitis b virus comprising detecting in a sample a combination of hepatitis b virus pre-S1 antigen and core antigen.

2. The method of claim 1, wherein said combined detection of hepatitis b virus pre-S1 antigen and core antigen is achieved by one or more methods selected from the group consisting of: enzyme-linked immunosorbent assay, immunodiafiltration, immunochromatography, chemiluminescence, and time resolution.

3. The method of claim 1, wherein the pre-S1 antigen and the core antigen are detected simultaneously in the same reaction vessel.

4. The method of claim 1, wherein the sample is not pretreated.

5. The method of any one of claims 1-4, wherein the lysis of the hepatitis B virus and the capture of the pre-S1 antigen and the core antigen are performed simultaneously in the same reaction vessel in the same medium.

6. The method of any one of claims 1-4, comprising the steps of:

a) providing a first anti-HBV PreS1 antibody and a second anti-HBV PreS1 antibody that can specifically bind to a hepatitis b virus pre-S1 antigen, and a first anti-HBc antibody and a second anti-HBc antibody that can specifically bind to a hepatitis b virus core antigen;

b) binding a first anti-HBV PreS1 antibody and a first anti-HBc antibody as capture antibodies to the same solid phase carrier to form a first antibody-carrier conjugate;

c) contacting a sample to be tested with a first antibody-carrier conjugate such that hepatitis b virus pre-S1 antigen and core antigen, if present, in the sample are captured to form an antigen-first antibody-carrier conjugate;

d) contacting a second anti-HBV PreS1 antibody and a second anti-HBc antibody with the product of step c) under conditions that allow the second antibody to bind to the captured antigen to form a second anti-HBV PreS1 antibody/second anti-HBc antibody-hepatitis b virus pre-S1 antigen/core antigen-first anti-HBVPreS 1 antibody/first anti-HBc antibody-carrier conjugate;

e) detecting the amount of bound second antibodies, i.e., second anti-HBVPreS 1 antibody and second anti-HBc antibody.

7. The method of claim 6, wherein in step c) the sample has not been subjected to prior viral lysis treatment and the contacting of the sample to be tested with the first antibody-carrier conjugate is performed under conditions that allow viral lysis and antigen capture.

8. The method of claim 6, wherein the step e) of detecting the amount of the bound second antibody is performed by: providing a first signal generator capable of binding effectively to said second anti-HBV PreS1 antibody and a second signal generator capable of binding effectively to said second anti-HBc antibody, the signal intensity produced by the first and second signal generators being related only to the amount of second anti-HBVPreS 1 antibody and second anti-HBc antibody, respectively; contacting the first and second signal generators with a second antibody; the signal generated by the signal generator is detected.

9. The method of claim 6, wherein the step e) of detecting the amount of the bound second antibody is performed by: the second antibody is directly labeled with a signal generator, and a signal generated by the signal generator is detected.

10. The method of claim 8 or 9, wherein the signal generator is an enzyme, a chemiluminescent substance, an isotope, or a rare earth element.

11. The method of any one of claims 1-4, wherein the immunoassay is performed using monoclonal antibodies against the hepatitis B virus pre-S1 antigen and against the core antigen.

12. The method of claim 11, wherein the monoclonal antibody against hepatitis b virus pre-S1 antigen is selected from the group consisting of 3H5, 7H11, 2a7, 4D11, 6F1, 13G2, and 16F 5.

13. The method of claim 12, wherein HBVPreS1 is detected using 7H11 mab as the primary anti-HBVPreS 1 antibody and 4D11-HRP as the secondary anti-HBV PreS1 antibody pairing.

14. The method of claim 6, further comprising the step of: the lysis solution is used to lyse the virus in the sample prior to contacting the sample to be tested with the first antibody-carrier conjugate.

15. A viral lysate useful in the method of any one of claims 1-14, comprising a surfactant selected from the group consisting of: chaps, sulfobetain8-18 selected from SB8, SB10, SB12, SB14, SB16 and SB18, Tween series and/or HP-beta-CD, or a combination of one or more thereof.

16. The virus lysate of claim 15, wherein the surfactant is selected from the group consisting of SB14, SB16, SB18, HP- β -CD, TWEEN 80.

17. The virus lysate of claim 15, wherein the surfactant is one or a combination of two or three of HP, TWEEN and SB series.

18. The virus lysate of claim 15, wherein the surfactant is HP + TWEEN40, HP + TWEEN40+ SB14, HP + TWEEN40+ SB16, HP + TWEEN40+ SB18, HP + TWEEN40+ SB18, HP + TWEEN20+ SB14, HP + TWEEN60+ SB14, or HP + TWEEN80+ SB 14.

19. The virus lysate of claim 15, wherein the surfactant is HP: 0.5% -4%; tween series: 0.1% -6%; SB 14%: 1% -10%; SB 16: 0.5% -1%; SB 18: 0.5% -1% or a combination thereof.

20. The virus lysate of claim 15, wherein the surfactant is HP: 1% -2%; tween series: 0.5% -2%; SB 14: 2% -8%; SB 16: 1 percent; SB 18: 1% or a combination thereof.

21. The virus lysate of claim 15, wherein the surfactant is 1% -2% HP + 0.5% -1% Tween + 1% -2% SB 14.

22. Virus lysate according to claim 15, wherein the lysate additionally comprises one or more additional components selected from the group consisting of reducing agents, osmo-regulators.

23. The virus lysate of claim 15, wherein the lysate is selected from the group consisting of lyss 1, lyss 10, lyss 11, lyss 12, lyss 13, lyss 14, lyss 15, lyss 16, lyss 17, lyss 18, lyss 19, lyss 20, lyss 21, lyss 22, lyss 23, and lyss 24, as shown in table 4.

24. The viral lysate of claim 15, wherein the lysate is selected from the group consisting of: lysis 10-citrate buffer pH6.6, 2% SB14, 5mM DTE, 100mM NaCl, 5mM EDTA; lysis 13-citrate buffer pH6.6, 2% SB16, 5mM DTE, 100mM NaCl, 5mM EDTA; lysis 16-citrate buffer pH6.6, 2% SB18, 5mM DTE, 100mM NaCl, 5mM EDTA; and lysis 18-citrate buffer pH6.6, 2% HP, 0.5% Tw40, 0.4% Me, 10% sucrose.

25. The method of claim 5, wherein a lysis solution according to any one of claims 15-24 is used as the medium.

26. The method of claim 6, wherein in step c) the contacting of the sample to be tested with the first antibody-carrier conjugate is performed in a lysis solution according to any one of claims 15 to 24;

27. the method of claim 7, wherein the lysate is a viral lysate of any one of claims 15-24.

28. A kit comprising reagents for the combined detection of hepatitis b virus pre-S1 antigen and core antigen according to the method of any one of claims 1-14.

29. The kit according to claim 28, wherein the anti-HBV PreS1 antibody and anti-HBc antibody, and/or the virus lysate according to any one of claims 15 to 24 are contained.

30. The kit of claim 28 comprising two different anti-HBV PreS1 antibodies and two different anti-HBc antibodies.

31. The kit of claim 28, wherein said kit further comprises reagents suitable for detecting said antigen-antibody reaction.

32. The kit of claim 28 wherein at least one of the antibodies anti-HBVPreS 1 antibody and at least one anti-HBc antibody are labeled with or are capable of binding efficiently to a signal generator, and the anti-HBV PreS1 antibody and anti-HBc antibody labeled signal generator or bound signal generator may be the same or different.

33. The kit according to claim 28, wherein the anti-HBV PreS1 antibody and the anti-HBc antibody-labeled signal generator or the combined signal generator are detected by the same detection method.

34. The kit of claim 28, wherein the antibody is a monoclonal antibody;

35. the kit as claimed in claim 28, wherein the anti-HBV PreS1 antibody is selected from the group consisting of: 3H5, 7H11, 2a7, 4D11, 6F1, 13G2, and 16F 5.

36. The kit of claim 29, comprising: (1) a solid phase carrier on which a first anti-HBV PreS1 antibody and a first anti-HBc antibody are immobilized; and/or (2) a viral lysate according to any one of claims 15 to 24; (3) optionally a second anti-HBV PreS1 antibody labeled or unlabeled with a signal generator and a second anti-HBc antibody; (4) optionally a detection reagent for detecting the second antibody; and (5) optionally buffers, washing solutions, instructions for use.

37. The kit of claim 36, wherein the solid phase carrier is a multi-well reaction plate, and the first anti-HBV PreS1 antibody and the first anti-HBc antibody are immobilized in the well of the multi-well reaction plate.

38. Use of a reagent for combined detection of hepatitis b virus pre-S1 antigen and core antigen in the preparation of a kit for the detection of hepatitis b virus infection.

39. The use of claim 38, wherein the agent is an agent as described in any one of claims 28-37.