CN118307662A

CN118307662A - Bifunctional protein and detection kit containing the bifunctional protein

Info

Publication number: CN118307662A
Application number: CN202211725232.4A
Authority: CN
Inventors: 饶星; 请求不公布姓名; 李临
Original assignee: Chemclin Diagnostics Corp
Current assignee: Chemclin Diagnostics Corp
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2024-07-09

Abstract

The invention provides a bifunctional protein, a detection kit containing the bifunctional protein and application of the bifunctional protein in photo-activated chemiluminescence detection. The bifunctional protein provided by the invention is provided with a first counterpart and a second marker, wherein the first counterpart is bridged to the protein through specific binding with the first marker, and the number of free amino groups of the protein is greater than or equal to 50. When the detection kit is used for detecting autoantibodies, interference factors in a reaction system can be removed, and a light excitation luminous signal is ensured.

Description

Bifunctional protein and detection kit containing same

Technical Field

The invention belongs to the field of in-vitro diagnosis, and particularly relates to a bifunctional protein, a detection kit containing the bifunctional protein, a use method of the detection kit and application of the detection kit in light-activated chemiluminescence detection.

Background

The photoexcitation chemiluminescence analysis is a homogeneous system chemiluminescence detection technology. At present, photo-activated chemiluminescence analysis has successfully established methods for detecting macromolecular antigens in a double-antibody sandwich mode, detecting specific antibodies in a double-antigen sandwich mode, detecting small molecular antigens in a double-antigen competition mode, detecting antibodies in a double-antibody competition mode and the like. However, photoexcitation chemiluminescence analysis has not been able to detect specific IgG antibodies, such as certain autoantibodies, perfectly in an indirect format.

Autoantibodies are important markers of autoimmune diseases, each of which is accompanied by characteristic autoantibodies. The existence of high-titer autoantibodies in the blood of patients is one of the characteristics of autoimmune diseases and is also an important basis for clinically diagnosing autoimmune diseases. The solid phase antigen-antibody to be detected-labeled anti-antibody mode, i.e. the indirect mode, is used as the first mode of autoantibody detection and is also the classical mode of autoantibody detection. The classical indirect method is carried out by adopting a two-step method, known antigens are combined with the antibody to be detected, the labeled anti-antibody (second antibody) is combined with the antibody to be detected, the non-specific (antigen-independent) antibody is separated and removed between the two steps, otherwise, the non-specific antibody is combined with the second antibody, a large amount of the second antibody is consumed, and the blank signal value is improved. However, the photo-activated chemiluminescence analysis is a homogeneous immunoassay mode, no separation washing link exists in the whole detection process, and the conventional photo-activated chemiluminescence analysis system cannot detect specific IgG antibodies, such as autoantibodies, in a classical mode.

Therefore, there is a need to develop a method that can be used for detection of autoantibodies by photo-activated chemiluminescent assay techniques.

Disclosure of Invention

Based on the defects existing in the prior art, the invention provides a novel semi-homogeneous immunoassay or wash light excitation chemiluminescence analysis, in particular to a method for detecting an autoantibody (IgG) by adopting an indirect mode based on a light excitation chemiluminescence platform, which can remove interference factors in a reaction system when used for detecting the autoantibody, and ensures a light excitation luminescence signal.

In order to achieve the purpose of the invention, the following technical scheme is adopted.

In a first aspect, the present invention provides a bifunctional protein having a first partner and a second label, the first partner being bridged to the protein by specific binding to the first label, the protein having a free amino group number of 50 or more.

The invention adopts protein containing a large amount of free amino acid, and the free amino acid is easier to be coupled in the photo-excitation chemiluminescence reaction. In some preferred embodiments, the protein has a free amino number of 50, 60, 70, 80, 90, 100, etc.

In some embodiments, each of the first counterparts is capable of specifically binding to a plurality of first labels; the first and second markers are different.

In some embodiments, the first and second markers have spacer arms 1. In some embodiments, the spacer 1 has a length of not less than 50 angstroms. In some embodiments, the spacer 1 is selected from PEG12, the spacer length of PEG12 beingIn other embodiments, the spacer 1 may employ an LC spacer consisting of an atom chain extended biotin valeric acid group. In other embodiments, the spacer 1 may employ PEG2, PEG4, PEG10, and the like.

In some embodiments, the protein is selected from bovine serum albumin. In some preferred embodiments, the protein is selected from Bovine Serum Albumin (BSA) and/or bovine serum gamma globulin (BGG). The bovine serum albumin BSA or bovine serum albumin (BGG) adopted by the application does not cross react with human, has low price and is easy to couple with free amino.

In some embodiments, the first label and/or the second label is selected from the group consisting of isothiocyanato Fluorescein (FITC), biotin (Biotin), 2, 4-Dinitrophenol (DNP), polyhistidine tag (His), and the like. In some embodiments, the first marker is selected from the group consisting of isothiocyanato fluorescein; the second marker is selected from activated biotin. In some embodiments, the isothiocyanato fluorescein is selected from FITC-NSH. In some embodiments, the activated biotin is selected from the group consisting of Bio-NSH.

In some embodiments, the first partner is a member of a specific binding pair member. In some embodiments, the specific binding pair member is an avidin-biotin system, preferably the avidin is selected from at least one of avidin, vitellin, streptavidin, neutravidin, and avidin-like molecules.

In a second aspect, the present invention provides a universal composition for a test kit, the composition comprising:

a bifunctional protein-coated separation vector of composition 1, the first aspect;

2, photosensitive microspheres coated with a second counterpart;

3, a luminous microsphere coated with a second antibody; the second partner is capable of specifically binding to the second label.

In some embodiments, the separation carrier is selected from magnetic microspheres or the interior surface of a reaction vessel. In some embodiments, the magnetic microspheres have a particle size of 200-1200nm, preferably 300-1000nm. In some embodiments, the magnetic microsphere is selected from the group consisting of amino superparamagnetic silica magnetic beads.

In a third aspect, the present invention provides a test kit comprising:

An agent comprising the composition of the second aspect, and

A reagent comprising a first antigen labelled with a first label, said first antigen being capable of specifically binding to an antibody to be tested.

In some embodiments, the first label that labels the first antigen has a spacer arm 2; preferably, the spacer arm 2 has a length greater than the spacer arm 1. In some embodiments, the spacer 2 is selected from PEG24, the spacer length of PEG24 beingIn other embodiments, the spacer 2 may employ an LC spacer consisting of an atom chain extended biotin valeric acid group. In other embodiments, the spacer 2 may employ PEG4, PEG10, or the like.

In some embodiments, the primary antibody has only one primary label binding site.

In some embodiments, the protein is selected from bovine serum albumin. In some preferred embodiments, the protein is selected from Bovine Serum Albumin (BSA) and/or bovine serum gamma globulin (BGG).

In some embodiments, the first label and/or the second label is selected from the group consisting of isothiocyanato Fluorescein (FITC), biotin (Biotin), 2, 4-Dinitrophenol (DNP), polyhistidine tag (His), and the like. In some embodiments, the first marker is selected from the group consisting of isothiocyanato fluorescein; the second marker is selected from activated biotin. In some embodiments, the isothiocyanato fluorescein is selected from FITC-NSH. In some embodiments, the activated biotin is selected from the group consisting of Bio-NSH. In the invention, FITC-NHS and Bio-NHS are combined with amino groups of the bifunctional proteins, and then the carrier is separated by carboxyl coating, so that the preparation is easy to separate.

In some embodiments, the first and second partners form a specific binding pair member, the first partner being one member of the specific binding pair member and the second partner being the other member of the specific binding pair member. In some embodiments, the specific binding pair member is an avidin-biotin system, preferably the avidin is selected from at least one of avidin, vitellin, streptavidin, neutravidin, and avidin-like molecules.

In some embodiments, the detection kit is a chemiluminescent detection kit.

In a fourth aspect, the present invention provides a detection method for detecting the presence or absence of an analyte in a sample to be detected using the detection kit according to the third aspect, the detection method comprising the steps of:

(1) Contacting a reagent containing the reagent of the composition 1 and a reagent containing a first antigen marked by a first marker with an antibody to be detected in a sample to be detected to form a first complex;

(2) Contacting a reagent comprising a composition 2 and a composition 3 with the first complex such that the composition 2 specifically binds to a second label in the first complex, and the composition 3 specifically binds to an antibody to be tested in a sample to be tested in the first complex, thereby forming a second complex;

(3) And (3) optically exciting the second compound, and detecting and analyzing an optical signal.

In some embodiments, the first antigen is selected from a natural antigen or a synthetic peptide fragment.

In some embodiments, the test sample comprises an autoantibody.

In some embodiments, in step (3), the wavelength of the photoexcitation is 660-700nm, preferably 680nm.

In a fifth aspect, the invention provides the use of a bifunctional protein of the first aspect, a composition of the second aspect, a kit of the third aspect or a detection method of the fourth aspect in a photoexcitation chemiluminescent assay.

In some embodiments, the excitation chemiluminescent detection is a photo-excitation chemiluminescent detection of the autoantibody. In some embodiments, the photoexcitation chemiluminescent detection is selected from qualitative or quantitative analysis of autoantibodies.

Compared with the prior art, the invention has the following beneficial effects:

(1) In the invention, the surface of the separation carrier for separation is provided with a bifunctional protein, the free amino groups of the bifunctional protein are easy to couple, and the bifunctional protein is provided with bifunctional characteristic markers, and the two marker binding reactions are combined in sequence as required without mutual interference.

(2) According to the invention, solid-liquid separation is realized by coating the separation carrier, so that interference factors in a reaction system are removed, and a light excitation luminous signal is ensured.

(3) In the present invention, it is suitable not only for the preparation of natural antigen-labeled biotin, but also for the detection of autoantibodies, such as anti-CCP antibodies, with synthetic peptide fragments (carrying only one biotin).

Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.

Drawings

FIG. 1 illustrates a schematic diagram of the mechanism of action of photoexcitation chemiluminescent detection according to some embodiments of the present application.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

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.

I. Terminology

The term photoexcitation chemiluminescence analysis is a homogeneous system chemiluminescence detection technology. For example, taking a double-antibody sandwich detection antigen as an example, an antibody participating in immune reaction is coated on the surface of a photosensitive microsphere (GG), and phthalocyanine substances are contained in the antibody; the other antibody is coated on the surface of a luminous microsphere (FG), and contains a dimethylthiophene derivative and an Eu chelate. The distance between the photosensitive microsphere and the luminous microsphere is shortened to be within 200nm by means of antigen-antibody combination, when the photosensitive microsphere is irradiated by 680nm excitation light, the photosensitive microsphere is activated and releases singlet oxygen with a high energy state to be transmitted to the luminous microsphere, the luminous microsphere emits light with the wavelength of 610nm after 4 mu s, and the concentration of an object to be detected is estimated through the light signal intensity.

The term "autoantibody" refers to an antibody against self tissues, organs, cells and cellular components, and may have a low level of autoantibodies in normal human blood, and if the level of autoantibodies is continuously increased, damage to the body may occur, inducing autoimmune diseases. Autoantibodies are important markers of autoimmune diseases, each of which is accompanied by characteristic autoantibodies. The existence of high-titer autoantibodies in the blood of patients is one of the characteristics of autoimmune diseases and is also an important basis for clinically diagnosing autoimmune diseases. For example, rheumatoid factors (rheumatoid factor, RF) are autoantibodies commonly found in serum of patients with rheumatoid arthritis (rheumatoid arthritis, RA), high-low RF aids in early diagnosis of RA, and RF is associated with clinical manifestations of the patient. The sensitivity of the anti-cyclic citrullinated peptide (CYCLIC CITR mu LLINATED PEPTIDE, CCP) antibody is equivalent to that of RF, but the specificity is higher, and the combined detection of the anti-cyclic citrullinated peptide and the CCP antibody is beneficial to improving the serological detection rate of RA patients. The combined detection of an anti-thyroperoxidase antibody (thyroid peroxidaseantibody, TPOAb) with an anti-thyroglobulin antibody (TG-Ab) is an important basis for diagnosing autoimmune thyroiditis.

The term "biotin" also known as vitamin H, coenzyme R, is a water-soluble vitamin, and also belongs to the vitamin B group, B7. It is an essential substance for the synthesis of vitamin C, an essential substance for the normal metabolism of fats and proteins. Is a necessary nutrient for maintaining natural growth and development of human body and normal human body function health. The term "activated biotin" refers to derivatives that can be chemically modified with the carboxyl groups of biotin to form various reactive groups. For example, bio-NSH (NHS-Biotin, C ₁₄H₁₈O₅N₃ S) is an N-hydroxysuccinimide Biotin that allows simple and efficient biotinylation of antibodies, proteins and any other primary amine-containing biomolecules in solution. Bio-NSH offers the possibility of labelling and detection, where steric hindrance of biotin binding is an important contributor. Since uncharged and containing simple alkyl chain spacer arms, bio-NSH is membrane permeable and can be used for intracellular labelling. Bio-NSH is the most commonly used biotinylation reagent. NHS-activated biotin can effectively react with primary amine groups (-NH ₂) in alkaline buffers to form stable amide linkages. Proteins or antibodies typically contain several primary amines, which can serve as targets for labeling, including the side chains of lysine residues and the N-terminus of each polypeptide.

The term 'isothiocyanato fluorescein (Fluorescein Isothiocyanate, FITC)' has the characteristics of high absorptivity, excellent fluorescence quantum yield, good water solubility and the like, and is a green fluorescein derivative which is most widely applied in biology, and an isothiocyanate group of the derivative can react with the amino terminal end or primary amine of protein so as to realize protein labeling including antibodies and lectin. In addition to use as protein markers, they can also be used as protein fluorescent tracers, labeled antibodies for rapid identification of pathogens, and for the microsequence of proteins and polypeptides (HPLC). FITC is a yellow-orange powder with a maximum excitation wavelength of 494nm. Upon excitation, a yellow-green fluorescence appears at a maximum emission wavelength of 520 nm. The term "FITC-NHS", fluorescein-N-hydroxysuccinimide, is a fluorescein-labeled active ester.

The term "N-hydroxysuccinimide ester (N-hydroxysuccinimide ester, succinimidyl ester, NHS)" is a chemical modification reagent that converts carboxyl groups to amine-reactive NHS esters for bioconjugation, crosslinking, labeling and immobilization methods.

The term "1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC)" is an organic compound having the formula C ₈H₁₇N₃. EDC is often used as a carboxyl activator in the preparation of amides from primary amines. EDC may also be used to activate phosphate groups. Such carbodiimides are commonly used in peptide synthesis; crosslinking of proteins and nucleic acids and the preparation of immunoconjugates. EDC is often used in combination with N-hydroxysuccinimide (NHS) or sulphonated-NHS to increase the efficiency of the coupling reaction.

The term luminescent microsphere refers to a microsphere, referred to as a label, that can undergo a chemical reaction to cause luminescence, such as by being converted to another microsphere that forms in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly or by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particles will be transitioned to an excited state to emit light.

The term photosensitive microsphere refers to polymer particles filled with photosensitive compounds formed on a substrate by coating functional groups, and the polymer particles can generate singlet oxygen under the excitation of light. In the application, the system is realized based on that the luminescent substances coated on the surfaces of the microspheres induce luminescent signals through light excitation and energy transfer, and the energy transfer is realized by leading the photosensitive microspheres and the luminescent microspheres to be close to each other depending on antigen-antibody combination.

II. Detailed description of the preferred embodiments

The photo-activated chemiluminescence analysis of autoantibodies can be performed in the following two ways by using a washing mode photo-activated chemiluminescence analysis. Firstly, a solid phase reaction plate is coated by means of the surface proteins of the luminous microspheres, a sample to be detected is added for incubation, and after washing, biotin antibody and streptavidin coated photosensitive microspheres are added according to a conventional mode. Secondly, marking bovine serum albumin by biotin, coating magnetic microspheres, and then combining streptavidin. During the test, biotin labeled antigen and a serum sample to be detected are added at the same time, and a known antigen-antibody complex to be detected is captured on the surface of the magnetic microsphere through biotin-avidin; unbound protein components, such as non-specific IgG components, are removed by magnetic field separation. Subsequently, luminescent microspheres coated with a second antibody, which binds to the antibody to be detected and photosensitive microspheres coated with streptavidin, which binds to the remaining biotin molecules on the surface of the antigen molecules, are added. Through the antigen-antibody combination and the avidin-biological combination, the photosensitive microsphere and the luminous microsphere are mutually close to each other to meet the condition of light excitation chemiluminescence, so as to perform excitation detection. However, these washing photoexcitation chemiluminescence assays have some drawbacks, such as labeling antigen molecules with biotin molecules, where biotin on the surface of the antigen molecules should preferentially bind streptavidin on the surface of the magnetic microsphere, and then bind avidin molecules on the surface of the photosensitive microsphere, and the two have a competitive relationship, and may affect each other; second, if synthetic peptide fragments, such as CCP, are used, only one biotin molecule is typically carried, and only streptavidin on the surface of the magnetic microsphere is bound, but not on the surface of the photosensitive microsphere.

In order to further solve the defects, the invention provides a novel semi-homogeneous immunoassay or wash light-activated chemiluminescence analysis, in particular to a method for detecting autoantibodies by adopting an indirect mode based on a light-activated chemiluminescence platform.

In some embodiments, the mechanism of action of the photoexcitation chemiluminescent assay is schematically shown in fig. 1.

In some embodiments, the bifunctional protein is bovine serum albumin BSA or bovine serum albumin-gamma BGG with a double tag, labeled isothiocyanato fluorescein (FITC-NHS, second tag) and activated biotin (Bio-NHS, first tag), respectively. Wherein, bovine serum albumin BSA or bovine serum albumin-gamma globulin BGG does not cross react with human, has low price and is easy to couple with free amino; besides two tags of isothiocyanato fluorescein (FITC-NHS) and activated biotin (Bio-NHS), other tags such as His, DNP and the like can be labeled.

Secondly, coating the magnetic microspheres or separating carriers such as the inner surface of a reaction container with the double-tag bovine serum albumin or bovine serum gamma globulin; thereafter, streptavidin (SA, first counterpart) is coupled again, thereby imparting the properties of Streptavidin (SA) and isothiocyanato Fluorescein (FITC) to the surface of the isolated carrier.

During detection, adding biotin-labeled antigen (Bio-Ag, antigen labeled by a first marker), wherein a serum sample to be detected (containing autoantibodies to be detected and Ab 1) is subjected to incubation, wherein the antigen is bound by the antibody to be detected, SA on the surface of the separation carrier is bound by biotin molecules, and a biotin-antigen-autoantibody to be detected (Bio-Ag-Ab 1) complex is fixed on the surface of the separation carrier; adsorbing the magnetic microspheres by a magnetic field, and then absorbing and removing the supernatant solution under negative pressure to remove non-specific IgG components; adding anti-FITC antibody (Ab 3) coated photosensitive microsphere (second counterpart coated photosensitive microsphere) and mouse anti-human IgG (Ab 2) coated luminescent microsphere solution (second antibody coated luminescent microsphere); the former is combined with FITC on the surface of the separation carrier, and the latter is combined with an antibody to be detected (Ab 1), so that the photosensitive microsphere and the luminous microsphere are promoted to be close to each other (for example, the distance is less than 200 nm); finally, the chemiluminescence process is started by light excitation (for example, at 680 nm), the luminous microsphere generates a light signal (for example, 610 nm), the signal intensity is in direct proportion to the content of the antibody to be detected (Ab 1), and the light signal can be used for qualitative or quantitative analysis.

The detection kit mainly comprises the following three processes:

(1) Specific antigen-antibody reaction process

And (3) carrying out standing incubation on the solution containing the bifunctional protein coated magnetic microsphere, the specific antigen and the sample to be tested, wherein the antibody to be tested in the sample to be tested and the specific antigen carry out immune binding reaction in the process.

(2) Solid-liquid separation interference elimination process

Placing the reaction liquid in a magnetic field, wherein the reaction liquid is subjected to solid-liquid separation of magnetic microspheres and liquid, and the liquid is sucked and discarded, so that substances, such as other immunoglobulins, which do not participate in affinity reaction on the surfaces of the magnetic microspheres in the reaction liquid can be removed; then removing the magnetic field, vibrating and uniformly mixing to redisperse the magnetic microspheres, wherein the dispersed magnetic microsphere solution only contains the magnetic microspheres coated with the antibody to be detected-bifunctional protein.

(3) Specific signal value generation process

The method comprises the steps of standing and incubating a luminous microsphere Anti-IgG-FG solution coated by a mouse Anti-human IgG antibody, a photosensitive microsphere Anti-FITC-GG solution coated by a mouse Anti-FITC antibody and a dispersed magnetic microsphere solution, wherein in the process, the mouse Anti-human IgG antibody and an antibody to be detected on the surface of the magnetic microsphere generate an antigen-antibody immune binding reaction, meanwhile, the mouse Anti-FITC antibody and FITC on the surface of the magnetic microsphere generate an antigen-antibody immune binding reaction, the distance between the photosensitive microsphere and the luminous microsphere is shortened due to the double immune reaction on the surface of the magnetic microsphere, when the distance is less than 200nm, light excitation is carried out at 680nm, a chemiluminescence process is started, and the luminous microsphere generates a light excitation luminous chemical signal.

The main reagents used in the following examples:

(1) The preparation method, composition structure and content of the photosensitive microspheres and luminescent microspheres used as the present invention can be found in example 1 of chinese patent CN100429197C (which is incorporated herein by reference in its entirety).

(2) Murine anti-FITC antibodies were supplied by Shanghai's biosciences, inc.

(3) Murine anti-human IgG monoclonal antibodies were supplied by fepeng biosystems, inc.

(4) FITC is supplied by SIGMA.

(5) Bio-NHS is supplied by thermofisher.

(6) Bio-PEG12-NHS is supplied by thermofisher.

(7) FITC-PEG-NHS is supplied by the Biotechnology Co.Ltd.

(8) BSA is provided by Shanghai ze bioengineering limited.

(9) The 300nm size amino superparamagnetic silica beads are provided by Shanghai Seiki Biotech Co.

Example 1

(1) 5Mg BSA protein was added to a centrifuge tube, 0.1M NaHCO ₃ labeling buffer at pH 8.0 was added, 10. Mu.l of Bio-PEG12-NHS biotin solution at a concentration of 10mg/ml was added, and 10. Mu.l of FITC-NHS fluorescein isothiocyanate solution at a concentration of 5mg/ml was added and mixed rapidly. The reaction was carried out overnight at 25-40rpm on a vertically rotating mixer at 2-8 ℃. Putting the marked solution into a dialysis bag, putting the dialysis bag into a beaker, adding 100 times of 0.1M pH 7.4PBS dialysis buffer solution into the beaker, putting the beaker on a magnetic stirrer, and dialyzing at 2-8 ℃. The dialysate is changed at least 1 time, and the dialysis is performed for at least 4 to 5 hours each time. The dialyzed protein is sucked out and transferred into a clean centrifuge tube, and the protein concentration is measured by sampling.

(2) Taking 1ml of the protein with the volume of 5mg/ml prepared in the step (1), adding 30 μl of the streptavidin SA solution with the volume of 2mg/ml, uniformly mixing, placing a centrifuge tube on a vertical rotary mixer at 37 ℃, and uniformly mixing for 30 minutes at 25-40 rpm. And centrifuging to remove unconjugated SA raw material, and obtaining the bifunctional protein.

Different bifunctional proteins were prepared using different tags and proteins according to the procedure described above:

Bovine serum albumin labeled with biotin and fluorescein isothiocyanate (Bio-BSA-FITC);

bovine serum albumin labeled with biotin and fluorescein isothiocyanate (Bio-PEG 12-BSA-PEG 12-FITC) containing spacer PEG12;

Bovine serum gamma globulin labeled with biotin and fluorescein isothiocyanate (Bio-BGG-FITC);

Bovine serum gamma globulin labeled with biotin and fluorescein isothiocyanate (Bio-PEG 12-BGG-PEG 12-FITC) containing a spacer arm PEG12.

Example 2

(1) Photosensitive microspheres coated with a second counterpart: mouse anti-FITC antibody coated aldehyde group photosensitive microsphere

5Mg of the photosensitive microspheres and 0.1mg of the mouse anti-FITC antibody are uniformly mixed by using 0.1M buffer solution with pH 9.6CB, a centrifuge tube is placed at 37 ℃ and uniformly mixed at 25-40rpm on a vertical rotating mixer for overnight, 75mg/ml of Gly solution is added for sealing, and the mixture is reacted at 25-40rpm on the vertical rotating mixer for 1 hour. The unconjugated antibody raw material is removed by centrifugation, the microspheres are washed, and a 0.1M pH 9.6CB buffer solution is added to obtain 60 mug/ml working solution of the mouse anti-FITC antibody coated photosensitive microspheres.

(2) Luminescent microspheres coated with a secondary antibody: mouse anti-human IgG monoclonal antibody coated aldehyde group luminous microsphere

5Mg of the luminescent microspheres and 0.1mg of the mouse anti-human IgG monoclonal antibody are uniformly mixed by using 0.1M buffer solution with pH 9.6CB, then placed at 37 ℃, uniformly mixed by a vertical rotating mixer at 25-40rpm for overnight, 75mg/ml of Gly solution is added for sealing, and the mixture is reacted for 1 hour at 25-40rpm on the vertical rotating mixer. And (3) centrifuging to remove unconjugated antibody raw materials, cleaning the microspheres, and adding 0.1M pH 9.6CB buffer to obtain 25 mug/ml mouse anti-human IgG monoclonal antibody coated luminescent microsphere working solution.

(3) The bifunctional protein-coated amino magnetic microsphere prepared in example 1

Adding 5mg of prepared bifunctional protein into a centrifuge tube, adding 0.1M pH 5.0MES activation buffer solution, adding 30 mug EDC/mg magnetic beads as an activating agent, uniformly mixing 50 mug NHS/mg magnetic beads, placing the centrifuge tube on a vertical rotary mixer for uniformly mixing at 25-40rpm for 30 minutes, adding 50mg of 300 nm-specification amino superparamagnetic silica magnetic beads, placing the centrifuge tube on the vertical rotary mixer for uniformly mixing at 25-40rpm for overnight at 37 ℃. And (3) collecting magnetism of the magnetic microspheres, then absorbing and discarding supernatant, and removing unconjugated bifunctional protein to obtain 25 mug/ml bifunctional protein coated magnetic microsphere working solution.

(4) A first antigen labeled with a first label: preparation of specific antigen (Bio-PEG 24-CCP)

The peptide fragment antigen CCP was synthesized from exocrine according to the amino acid sequence of cyclic citrullinated peptide and Bio-PEG24 was attached at the end of the peptide fragment. 1mg/mlBio-PEG24-CCP working solution was obtained.

Example 3

The detection method comprises the following detection steps:

(1) 200 mu L of the bifunctional protein-coated magnetic microsphere working solution prepared in the example 2,50 mu L of Bio-PEG24-CCP working solution and 10 mu L of serum sample to be detected are added into a reaction cup, uniformly mixed and incubated for 30 minutes;

(2) Separating by using a magnetic field, and sucking away supernatant solution under negative pressure; (the non-solid-liquid separation method does not perform this step);

(3) Adding 100 mu L of mouse anti-human IgG monoclonal antibody coated luminous microsphere working solution, mixing uniformly and incubating for 15 minutes;

(4) 150 mu L of mouse anti-FITC antibody coated photosensitive microsphere working solution is added, evenly mixed and incubated for 15 minutes;

(5) Exciting the detection light signal; and calculating a result according to the calibration function.

Example 4

10 Normal (negative) and 10 case (positive) samples were tested using the reagents prepared in example 2 and the solid-liquid separation method of example 3, and the results are shown in table 1 below.

TABLE 1

When the S/CO of the test result is more than or equal to 1, judging that the test result is positive; and when the test result S/CO is less than 1, judging as negative.

As can be seen from the results of Table 1, the results of the 4 groups of bifunctional proteins were identical, and the normal groups were all judged negative, and the case groups were all judged positive. The detection effect of the low-value sample is good.

Example 5

The reagent prepared by the bifunctional protein SA-Bio-PEG12-BSA-PEG12-FITC of the invention is used for detecting 5 groups of normal (negative) samples and 4 groups of case (positive) samples, the experimental group is subjected to solid-liquid separation, the control group is not subjected to solid-liquid separation, and the results are shown in the following table 2.

TABLE 2

As can be seen from the results in table 2, among the four positive samples, the low concentration positive sample 1 and the positive sample 2 of the experimental group were both judged to be positive; the control group was judged to be negative with respect to the low-concentration positive samples 1 and 2, and the detection sensitivity was insufficient.

The step of solid-liquid separation is added, and the interference substances in the reaction system can be removed, namely, the immunoaffinity reaction of the CCP antigen antibody is not interfered, and the signal value is kept at a higher level. Therefore, the invention realizes solid-liquid separation by coating the magnetic microspheres, removes interference factors in a reaction system and ensures a light excitation luminous signal.

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims

1. A bifunctional protein, characterized in that the protein carries a first counterpart and a second marker, the first counterpart is bridged to the protein by specifically binding to the first marker, and the number of free amino groups of the protein is greater than or equal to 50.

2 . The protein according to claim 1 , wherein each of the first pairing substances can specifically bind to a plurality of first markers; and the first markers and the second markers are different.

3. The protein according to claim 1, characterized in that the first marker and the second marker have a spacer arm 1; preferably, the length of the spacer arm 1 is not less than 50 angstroms.

4. The protein according to any one of claims 1 to 3, characterized in that the protein is selected from bovine serum globulin, preferably bovine serum albumin or bovine serum gamma globulin; and/or

The first marker and/or the second marker is/are selected from fluorescein isothiocyanate, biotin, 2,4-dinitrophenol, and a polyhistidine tag.

5. A universal composition for a detection kit, characterized in that the composition comprises:

Composition 1, a separation carrier coated with a bifunctional protein according to any one of claims 1 to 4;

Composition 2, photosensitive microspheres coated with a second counterpart;

Composition 3, luminescent microspheres coated with a secondary antibody;

The second partner can specifically bind to the second label.

6. The composition according to claim 5, characterized in that the separation carrier is selected from magnetic microspheres or the inner surface of a reaction container.

7. A detection kit, characterized in that the kit comprises:

A reagent containing the composition according to claim 5 or 6, and

A reagent containing a first antigen labeled with a first marker, wherein the first antigen can specifically bind to the antibody to be detected.

8. The detection kit according to claim 7, characterized in that the first marker for marking the first antigen has a spacer arm 2; preferably, the length of the spacer arm 2 is greater than that of the spacer arm 1;

Preferably, the first antigen has one and only one first marker binding site.

9. A method for detecting whether an analyte is present in a sample using the detection kit according to claim 7 or 8, characterized in that the detection method comprises the following steps:

(1) contacting a reagent containing composition 1 and a reagent containing a first antigen labeled with a first marker with a test antibody in a test sample to form a first complex;

(2) contacting a reagent containing component 2 and component 3 with the first complex, so that component 2 specifically binds to the second marker in the first complex, and component 3 specifically binds to the antibody to be tested in the sample to be tested in the first complex, thereby forming a second complex;

(3) Exciting the second complex with light, detecting and analyzing the light signal.

10. Use of the bifunctional protein according to any one of claims 1 to 4, the composition according to claim 5 or 6, the detection kit according to claim 7 or 8, or the detection method according to claim 9 in photochemiluminescence detection, especially in photochemiluminescence detection of autoantibodies.