WO2008053900A1 - Highly sensitive immunological analysis method and immunological analysis reagent for hepatitis b virus - Google Patents

Abstract

The object is to provide: a highly sensitive immunological analysis method for a hepatitis B virus; a highly sensitive immunological analysis reagent for a hepatitis B virus; and an immunological analysis kit for a hepatitis B virus, each of which utilizes an antigen-antibody reaction. The object is achieved by conducting an antigen/antibody reaction in which an antigen is contacted with an antibody in the presence of a saponin, a bile salt or an anionic surfactant. Specifically disclosed is an immunological analysis method for a hepatitis B virus by a sandwich method involving the step of contacting an antigen with a soluble antibody, wherein a saponin, a bile salt or an anionic surfactant is preferably allowed to exist in the reaction system. Further disclosed are: a highly sensitive immunological analysis reagent for a hepatitis B virus; and an immunological analysis kit for a hepatitis B virus, each of which utilizes an antigen-antibody reaction.

Description

 Specification

 Highly sensitive immunological analysis method and reagent for immunological analysis of hepatitis B virus

 [0001] The present invention relates to a highly sensitive immunological analysis method for hepatitis B virus and a reagent for immunological analysis. According to the present invention, by performing an antigen-antibody reaction in the presence of saponin, hepatitis B virus (hereinafter referred to as “HBV”) antigen in a test sample (hereinafter also referred to as a specimen) Can be detected or quantified with high sensitivity.

 Background art

 [0002] Blood used for blood transfusion often causes post-transfusion infection. HBV is a causative virus for post-transfusion hepatitis and is transmitted by blood transfusion during surgery. Therefore, it is extremely important to diagnose the presence or absence of blood virus infection by screening blood for transfusion.

 Such infection diagnosis methods include immunological analysis methods such as antibody test methods for detecting antibodies against viruses present in test samples, and antigen test methods for detecting antigens such as viruses, or viruses. There are gene detection methods for detecting these genes.

 [0003] Of these diagnostic methods, immunological analysis methods are highly sensitive and specific, but they are still insufficient for screening and monitoring of infectious diseases that are less sensitive than gene detection methods. I can't say that. For example, in blood donation blood screening, in order to diagnose HBV infection, in addition to HBs antigen test, HBs antibody test, and HBc antibody test, 20 blood donated blood is pooled into one sample for nucleic acid amplification test ( Nucleic acid Amplification Test (hereinafter referred to as NAT) has been screened, and blood that was determined to be negative by the HBs antigen test but was determined to be HBV positive by NAT has been found.

[0004] Gene detection methods include the NAT and DNA probe methods, which are currently widely used in clinical practice. NAT test methods such as PCR and TMA are highly sensitive detection methods for detecting gene fragments. However, for example, when extracting HBV genomic DNA from a sample, the processing time required for the method is 2 hours, and multiple operation steps are required. This is very complicated. Furthermore, the complexity of the operation increases the chances of contamination with other positive specimens, increasing the possibility of generating false positive specimens. In addition, there is a problem that skill of the operator is required to obtain a stable quantitative value. In recent years, the development of automated equipment has been used to prevent contamination and shorten the DNA extraction processing time.However, since it still requires expensive equipment, it is generally popular except for facilities that process samples in large quantities. Not done. In addition, since the DNA primer must match the target gene, it is necessary to use several types of primers, and the cost per test is higher than the immunological analysis method.

[0005] Compared to genetic testing methods that have these problems, immunological analysis methods can provide stable results with fewer problems of contamination, and automation is progressing so much. There are advantages such as not requiring skill and low cost. However, there is a problem that the detection sensitivity is low compared with the gene detection method, and various methods for increasing the sensitivity of the reaction have been studied. As a method for increasing the sensitivity of an immunological analysis method, for example, an antibody test method improves an antigen for detecting an antibody, and an antigen test method improves an antibody that binds to an antigen. I have been. In the detection step, attempts have been made to increase sensitivity by improving the chromogenic substrate and the luminescent substrate.

 [0006] Further, in the step of reacting an antigen and an antibody, a method of increasing sensitivity by adding various compounds has been considered. For example, it has been reported that the addition of hydrophilic polymers such as polybulurpyrrolidone (Japanese Patent No. 3468763) and pullulan (Japanese Patent No. 3396231) increases the sensitivity of measurement of immune reaction. By adding these hydrophilic polymers to the step in which the antigen and the antibody are brought into contact with each other, it is predicted that the concentration of the antigen 'antibody in the reaction solution will be concentrated. As a result, the contact between the antigen and the antibody in the reaction solution is expected. Efficiency can be increased and sensitivity can be improved. However, these methods are considered to increase the non-specific signal at the same time, and it is not possible to obtain a sufficient increase in sensitivity.

[0007] Patent Document 1: Japanese Patent No. 3468763 Patent Document 2: Japanese Patent No. 3396231

 Disclosure of the invention

 Problems to be solved by the invention

[0008] The conventional hepatitis B virus immunological analysis method uses an immunological analysis method that is less sensitive than the gene detection method. There was a case of virus contamination. Such false negative blood can be used for blood transfusions, which can cause post-transfusion infection. Surprisingly, the present inventors have conducted extensive research on increasing the sensitivity of the hepatitis B virus immunoassay in order to reduce false negatives of such an immunological assay. It was found that the sensitivity of the immunological analysis method is increased by performing an antigen-antibody reaction in which an antigen and an antibody are contacted in the presence of saponin, bile acid, or an anionic surfactant.

 The present invention is based on these findings.

 Means for solving the problem

[0009] Therefore, the present invention includes a sandwich method comprising a step of contacting a hepatitis B virus antigen and an anti-hepatitis B virus antibody in the presence of a saponin, a bile salt, or an anionic surfactant. Relates to a method for immunological analysis of hepatitis B virus.

 In a preferred embodiment of the immunological analysis method according to the present invention, the anti-hepatitis B virusless antibody is a soluble antibody.

 In another preferred embodiment and embodiment of the immunological analysis method according to the present invention, the saponin is a triterpenoid saponin, in particular, a killer saponin or a tea seed-derived saponin.

 Further, the present invention provides a reagent for hepatitis B virus immunological analysis by a sandwich method, wherein a reagent for contacting a hepatitis B virus antigen with an anti-hepatitis B virus antibody is used as a saponin, bile salt, or The present invention also relates to a reagent for hepatitis B winores immunological analysis characterized by containing an anionic surfactant.

 The preferred embodiment of the reagent according to the present invention! /, For the embodiment! /, Is that the anti-hepatitis B virus antibody is a soluble antibody.

In another preferred embodiment and embodiment of the reagent according to the present invention, the saponin is a triterpenoid. It is a saponin, in particular, a saponin derived from quilla or a saponin derived from tea seed. The present invention further relates to a hepatitis B virus immunological analysis kit containing a reagent for hepatitis B virus immunological analysis.

 Note that “analysis” in the present specification includes both “detection” for determining the presence or absence of an analysis target compound and “quantification” for determining the presence of the analysis target compound. In addition, “test” in the present specification includes both “detection” for determining the presence or absence of an antigen or antibody and “quantification” for determining the amount of an antigen or antibody.

 Further, the “hepatitis B virus immunological analysis method” according to the present invention can be used as a “diagnosis method of hepatitis B virus”.

 The invention's effect

 [0010] According to the present invention, it is possible to detect and quantify HBV antigens or antibodies with high sensitivity in comparison with the hepatitis B virus immunological analysis method by the sandwich method. Moreover, according to the present invention, it is possible to increase the sensitivity of the hepatitis B virus immunological analysis method without increasing nonspecific binding between the antigen and the antibody.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0011] Saponins are a group of glycosides widely contained in plants, and have a structure in which an oligosaccharide is bound to a triterpene skeleton or a steroid skeleton, such as digitalis and soybean.

, Croaker, redwood, savonso, grape, olive, ginseng, yellowwood, senega, yellow squirrel, Japanese apricot, Japanese hornbill, licorice, yellow sage, tochibanin ginseng, daisaw tree, Japanese apricot, green, red, black, red, black Etc. Saponins are roughly classified into triterpenoid saponins and steroid saponins according to the type of sapogenins. Sapogenin is an aglycone (non-sugar part) of saponin, and there are triterpenes and steroids.

[0012] A triterpenoid saponin is a glycoside having aglycone of a triterpene (sometimes referred to as a triterpenoid) such as a terpene hydrocarbon or alcohol having 30 carbon atoms. Triterpenodosaponin may also be referred to as triterpene saponin. Examples of the sugar bonded to triterpene include glucose, rhamnose, arabinose, galactose, xylose, pentose, and hexose. Used in the method of the present invention The triterpenoid saponins that can be used are not particularly limited as long as they are glycosides of triterpenes. Saponin, licorice-derived saponin, Hirosanega-derived saponin, saponin-derived saponin, Tochiba carrot-derived saponin, Japanese scorpion-derived saponin, arabi-derived saponin, honey-butterfly-derived saponin, egonoki-derived saponin, modama-derived saponin, jujube saponin, jujube Examples include tea seed-derived saponins, quilla-derived saponins, and the like. In particular, killer saponins and tea seed saponins are preferred.

 [0013] Steroidal saponins are glycosides having steroids as aglycones, and are particularly distributed in plants such as the liliaceae, the genus Hydrangeaceae, and the genus Camellia. Examples of the sugar bonded to the steroid include glucose, rhamnose, arabinose, galactose, xylose, pentose, and hexose. The steroid saponin that can be used in the method of the present invention is not particularly limited as long as it is a steroidal glycoside. Specifically, amoronin, digitonin, di-succin, gitonin, cammonin, noronin, sanoressa Mention may be made of saponins, sumilonins, tigonins, trilarins, trilins, euconins. Steroid saponins also include glycosides having aglycone as a steroid alkaloid, which is a steroid containing a nitrogen atom.

 [0014] The concentration of the saponin that can be used in the hepatitis B virus immunoassay method of the present invention in the reaction solution in which the antigen and the antibody are brought into contact with each other is not particularly limited, but preferably the lower limit is 0.001. % (Mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the reaction solution. Moreover, it is preferably from 0 · 01 to 10% (mass / volume percent), more preferably from 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. % Mass / volume percent). Strength S enhances the reaction between antigen and antibody over a wide concentration range.

[0015] In the method for immunoassay of hepatitis B virus of the present invention, the step of contacting an antigen and an antibody in the presence of bile acid can also be performed using the method for immunoassay of hepatitis B virus. Sensitivity can be increased. Bile acids are produced in the liver of mammals, amphibians, reptiles, birds or fish, and in the form of sodium salts, the main component of bile is the gallbladder. It helps to absorb fat from the intestinal tract by making it into micelles by its surface-active action. Bile acid has a structure in which a hydroxyl group is bonded to a steroid skeleton, and has a hydrophobic steroid skeleton and a hydrophilic group such as a hydroxyl group in the same molecule, and thus exhibits a surface-active action. Specific examples of bile acids that can be used in the method for immunological analysis of hepatitis B virus of the present invention include cholic acid, deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, litho-monorelic acid, Examples include taurocholic acid, taurodeoxycholic acid, apocholic acid, glycocholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, taurodehydrocholic acid, taurolysocholic acid, tauronoresoxycolic acid, etc. Examples of the salt include sodium salt. Particularly preferred are cholic acid, deoxycholic acid, and salts thereof.

 [0016] The concentration of the bile acid antigen that can be used in the hepatitis B virus immunoassay method of the present invention in the reaction solution in which the antibody is brought into contact with the antibody is not particularly limited, but preferably the lower limit is 0. 001% (mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the solution. Moreover, it is preferably 0.01 to 10% (mass / volume percent), more preferably 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. 2% mass / volume percent). It is possible to enhance the antigen-antibody reaction over a wide concentration range.

[0017] Further, in the method for immunological analysis of hepatitis B virus of the present invention, the step of contacting an antigen and an antibody in the presence of an anionic surfactant can also be performed. It is possible to increase the sensitivity of the virus immunological analysis method. Examples of the anionic surfactant include alkylbenzene-based alkylaryl sulfonates, higher alcohol-based alkyl sulfates, alkyl ether sulfates, monoalkyl phosphates, and alpha-olefin alpha. Examples thereof include olefin sulfonate and normal paraffin-based alkane sulfonate. Preferably, sodium lauryl sulfate (NLS), sodium dodecyl sulfate (SDS), 1-octane sodium sulfonate, 1 sodium decane sulfonate, 1-sodium sodium decane sulfonate, sodium 1-dodecane sulfonate, etc. Can do. [0018] The concentration of the anionic surfactant that can be used in the hepatitis B virus immunological analysis method of the present invention in the reaction solution in which the antigen and the antibody are contacted is not particularly limited, but the lower limit is 0. 001% (mass / volume percent) or more, the upper limit being the concentration that can be dissolved in the solution, preferably 0.01 to 10% (mass / volume percent), more preferably 0 02-5% (mass / volume percent), most preferably 0.05-0.8% (mass / volume percent).

 [0019] In the method for immunological analysis of hepatitis B virus of the present invention, the step of bringing a hepatitis B virus antigen into contact with an anti-hepatitis B virus antibody comprises saponin, cholic acid, and an anionic surfactant. By conducting the reaction in the presence of a mixture of two or more of these, it becomes possible to measure the antigen of hepatitis B violets more sensitively. In particular, a mixture of saponin and an anionic surfactant is preferred! The effects obtained by adding saponin and the effects obtained by adding an anionic surfactant can be obtained, and in some cases, a synergistic effect can be obtained.

 [0020] As the saponin and anionic surfactant used in the mixture, all of the saponin and anionic surfactant which can be used alone can be used. In addition, the concentration of saponin and anionic surfactant is determined by the force used at the same concentration as that used alone.

 [0021] The method for immunological analysis of hepatitis B virus by the sandwich method of the present invention comprises a method for treating hepatitis B virus antigen and anti-hepatitis B virus antibody in the presence of saponin, bile acid, or anionic surfactant. A step of contacting. The presence of saponin, bile acid, or anionic surfactant in the liquid phase where the antibody and the antigen bind to each other enhances the binding between the antigen and the antibody. In the hepatitis B virus immunological analysis method of the present invention, the antigen may be an antigen immobilized on a carrier or a soluble antigen. The antibody may be either an antibody immobilized on a carrier or a soluble antibody, but is preferably a soluble antibody. In the present specification, “soluble antibody” means an antibody not immobilized on a carrier.

Yes

[0022] The immunoassay method for hepatitis B virus immunoassay by the sandwich method of the present invention includes an antigen test for measuring an antigen using an antibody specific to the antigen from the viewpoint of the measurement target in the sample. It is possible to classify into an acupuncture method and an antibody test method for measuring a specific antibody against an antigen. Furthermore, the two-step method can be classified into a forward sandwich method and a reverse sandwich method.

[0023] The procedure of the antigen measurement method is not particularly limited as long as it is a method for detecting or measuring an antigen in a test sample. For example, the procedure can be performed as follows.

 First, a capture antibody that binds to the antigen to be measured is immobilized on a carrier such as a microplate or beads. After that, blocking with bovine serum albumin is performed to prevent non-specific adsorption to the capture antibody or carrier. A test sample containing the antigen to be measured is added to the plate or beads on which the capture antibody is immobilized, together with the primary reaction solution, and the capture antibody and the target antigen are brought into contact with each other to bind (antigen test primary reaction step). . Thereafter, antigens and contaminants not bound to the capture antibody are washed with a washing solution. Next, an antibody that recognizes the captured antigen and a labeled antibody bound with an enzyme such as horseradish peroxidase (HRP) are added, and the labeled antibody is bound to the captured antigen (antigen test secondary reaction step). By this reaction, an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate. It is possible to detect the signal by washing the unbound labeled antibody with a washing solution, adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, and reacting the enzyme with the substrate.

[0024] Performing a step (antigen test primary reaction step) in which the immobilized capture antibody and the antigen in the test sample are contacted in the presence of the saponin, bile acid, or anionic surfactant. Thus, the reactivity of the antigen-antibody reaction can be enhanced. Further, in the presence of the saponin, bile acid, or anionic surfactant, an antigen captured by the capture antibody and a labeled antibody are contacted with each other (antigen test secondary reaction step). The reactivity of the antibody reaction can be enhanced. However, since the potentiating effect of saponin, etc., when the antibody is soluble is strong, the saponin, bile acid, or anionic surfactant is added in the antigen test secondary reaction step rather than the antigen test primary reaction step. It is preferable to make it exist. The antigen test primary reaction step and the antigen test secondary reaction step can be performed in a reaction solution usually used for antigen-antibody reaction. The buffer used for the reaction solution is Although it is not particularly limited as long as it can stably maintain the components necessary for the original antibody reaction and does not inhibit the antigen-antibody reaction, specifically, a phosphate buffer, Tris buffer, glycine buffer or It can be appropriately selected from conventionally known buffers such as a hepes buffer. The reaction solution also contains other components such as carrier proteins such as ushi serum albumin, antiseptics such as sodium azide to stabilize antigens and antibodies, and surface activity to suppress nonspecific reactions. It is also possible to contain an agent or the like.

[0025] The procedure of the antibody test method is not particularly limited as long as it is a method for detecting or measuring an antibody in a test sample. For example, it can be performed as follows.

 First, a capture agent that binds to the antibody to be measured is immobilized on a carrier such as a microplate or beads. After that, blocking with bovine serum albumin is performed to prevent non-specific adsorption to the capture antigen or carrier. A test sample containing the antibody to be measured is added to the plate or bead on which the capture antigen is immobilized, together with the primary reaction solution, and the capture antigen and the target antibody are brought into contact with each other to bind (antibody test primary reaction step). . Thereafter, antibodies and contaminants that have not bound to the capture antigen are washed with a washing solution. Next, an antibody that recognizes the captured antibody and a labeled antibody conjugated with an enzyme such as horseradish peroxidase (HRP) are added, and the labeled antibody is bound to the captured antibody (antibody test secondary reaction step). By this reaction, an immune complex of a capture antigen, an antibody, and a labeled antibody is formed on a carrier such as a microplate. It is possible to detect the signal by washing the unbound labeled antibody with a washing solution, adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, and reacting the enzyme with the substrate.

[0026] Performing a step (antibody test primary reaction step) in which the immobilized capture antigen and the antibody in the test sample are contacted in the presence of the saponin, bile acid, or anionic surfactant. Thus, the reactivity of the antigen-antibody reaction can be enhanced. In the antibody measurement method, the anti-hepatitis B virus antibody in the sample is soluble in the primary reaction step of the antibody test, and the saponin, bile acid, or anionic surfactant is present in the reaction solution. Therefore, it is possible to enhance the reactivity of antigen-antibody reaction. In the antibody testing method, the buffer solution, carrier protein, surfactant, etc. used in the antigen testing method can also be used. [0027] The antigen test method and the antibody test method can be performed by a two-step forward sandwich method, a reverse sandwich method, and a one-step method. The antigen test method and antibody test method exemplified above are test methods based on the forward sandwich method. The forward sandwich method, reverse sandwich method, and one-step method will be described below using the antigen test method as an example, but the antibody test method can also be performed by the forward sandwich method, reverse sandwich method, and one-step method. is there.

 The forward sandwich method has the same force as the procedure described as the antigen test method. Briefly, the capture antibody is immobilized on a carrier such as a microplate or a bead. Then, block with bovine serum albumin. The sample to be measured is added to the plate or beads on which the capture antibody is immobilized together with the primary reaction solution, and the capture antibody and the target antigen are brought into contact with each other and bound (forward primary reaction step). After this, the plate and beads are washed with a washing solution. Next, a labeled antibody is added, and the labeled antibody is bound to the captured antigen (forward secondary reaction step). By this reaction, an immunocomplex of a capture antibody, an antigen-labeled antibody is formed on a carrier such as a microplate. After washing with a washing solution, a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody is added, and the signal is detected by reacting the enzyme with the substrate.

[0029] In the reverse sandwich method, first, a capture antibody that binds to an antigen to be tested is immobilized on a carrier such as a microplate or a bead. After that, in order to prevent non-specific adsorption to the capture antibody and the carrier, blocking with bovine serum albumin is performed to prepare a solid-phase carrier. Next, the test sample containing the antigen to be tested is brought into contact with and bound to the labeled antibody in the reaction solution (reverse primary reaction step). The reaction solution containing the antigen-labeled antibody immune complex in the test sample is brought into contact with a carrier on which the capture antibody is immobilized (reverse secondary reaction step). By this reaction, an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate. Wash unbound antibody, antigen, labeled antibody, etc. with washing solution. Next, by adding a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody, the signal can be detected by reacting the enzyme with the antibody. In this reverse sandwich method, since the forward secondary reaction step is performed before the forward primary reaction step in the forward sandwich method, only one washing step is required. [0030] In the reverse sandwich method, in the presence of saponin, bile acid, or an anionic surfactant, a test sample containing an antigen to be measured is contacted with a labeled antibody in a reaction solution and allowed to bind (reverse) By performing the primary reaction step), it is possible to enhance the reactivity of the antigen-antibody reaction. In addition, in the presence of the saponin, bile acid, or anionic surfactant, a reaction solution containing an antigen-labeled antibody immune complex in a test sample is mixed with a carrier on which a capture antibody is immobilized. By performing the step of contacting (reverse secondary reaction step), the reactivity of the antigen antibody reaction can be enhanced. However, it is preferable that the saponin, bile acid, or anionic surfactant is present in the reverse primary reaction step in which the antibody is soluble.

 [0031] In the one-step method, first, a capture antibody that binds to an antigen to be measured is immobilized on a carrier such as a microplate or a bead. Thereafter, in order to prevent non-specific adsorption to the capture antibody or the carrier, blocking with bovine serum albumin or the like is performed to prepare a solid phase carrier. Next, the reaction solution is added to the immobilized carrier, and the test sample containing the antigen and the labeled antibody are added simultaneously, or the test sample containing the antigen is added, and then the labeled antibody is added immediately. The immobilized capture antibody, antigen and labeled antibody are simultaneously contacted and reacted (one-step reaction process). By this reaction, an immune complex of a capture antibody, an antigen, and a labeled antibody is formed on a carrier such as a microplate. Next, the unbound antibody, antigen, labeled antibody, etc. are washed with a washing solution. Finally, a chromogenic substrate or a luminescent substrate for the enzyme of the labeled antibody is added, and the signal can be detected by reacting the enzyme with the substrate. Even in the one-step method, the cleaning process is performed only once.

[0032] In the one-step method, in the presence of saponin, bile acid, or an anionic surfactant, a carrier on which a capture antibody is immobilized, a test sample containing an antigen, and a labeled antibody react with each other. By performing the (one-step reaction process), the reactivity of the antigen-antibody reaction can be enhanced. In this case, the saponin, bile, or both of the reactivity of the immobilized capture antibody and the antigen-antibody reaction of the antigen in the test sample, as well as the antigen-antibody reaction of the antigen and labeled antibody in the test sample are detected. The ability to add an acid or an anionic surfactant has a potentiating effect. It has a stronger enhancing effect on the reactivity of the antigen-antibody reaction between the antigen in the test sample and the labeled antibody in which the antibody is soluble. Conceivable. [0033] The hepatitis B virus antigen that can be used in the method for immunological analysis of hepatitis B virus by the sandwich method of the present invention is an epitope or antigenic determinant to which an antigen binding site of an anti-hepatitis B virus antibody binds. The antigen is not particularly limited as long as it has an antigen. For example, the antigen used to measure the anti-hepatitis B virus antibody in the test sample by the antibody detection method can be a native antigen of hepatitis B virus, or a combination produced using Escherichia coli or the like. It is also possible to use a replacement HBV antigen. Examples of hepatitis B virus antigens detected by the antigen testing method include HBs antigen, HBc antigen, HBe antigen and HBc-related antigen, and HBs antigen is particularly preferable. The HBs antigen includes a LargeS protein consisting of 389 to 400 amino acid residues, a MiddleS protein consisting of 281 amino acid residues, and a SmallS protein consisting of 226 amino acid residues. Antigenic determinants or epitopes of these HBs antigens include pre-S1 region, pre-S2 region, common antigenic determinant a and the like. However, the antigenic determinant or epitope utilized in the present invention is not limited to these, and the force S that utilizes the antigenic determinant or epitope of all HBs antigens.

 [0034] The anti-hepatitis B virus antibody used in the method for immunological analysis of hepatitis B virus by the sandwich method of the present invention is not particularly limited. Examples thereof include antibodies that recognize HBs antigen, HBc antigen, HBe antigen, and HBc-related antigen. Specific examples of antibodies that bind to the HBs antigen include antibodies that bind to peptides consisting of amino acids 51 to 60 of the SmallS protein consisting of 226 amino acid residues, such as the 6G6 antibody, 15; An antibody that binds to a peptide consisting of amino acid residues of ~ 170, such as an HBsl21 antibody, an antibody that binds to a peptide consisting of amino acid residues of 31 to 50, such as an HBsl23 antibody, 1 1;! An antibody that binds to a peptide consisting of residues, for example, an HBsl36 antibody, 11;! To an antibody that binds to a peptide consisting of amino acid residues Nos. 130 to 130, such as the HBs 128 antibody, amino acids residues Nos. 1-226 It recognizes structural epitopes such as the common antigenic determinant a that bind to the full-length peptide of HBs and do not bind to peptides consisting of 20 amino acid residues that overlap by 10 amino acid residues from the amino acid sequence of the HBs antigen. Body, for example, a HBs605C3 antibodies and SF124CS antibody.

The type of antibody is not particularly limited. For example, blood of mammals of humans and mice A monoclonal antibody secreted from a hybridoma cell obtained by cell fusion with a myeloma cell may also be used. Antibody is obtained by using proteases such as pepsin and papain to obtain antibody fragments such as F (ab ') and Fab.

 2

 I can do it. In general, heavy chains (H chains) of antibodies have heavy chains bonded to each other by S—S bonds, and the bonds are cleaved by a reducing agent. For the antibody in the present invention, these antibody fragments such as F (ab ′), Fab \ Fab, F (abc ′), Fabc ′ and the like can be used.

 2

 is there.

 [0035] The state of the antibody that binds to the antigen in the hepatitis B virus immunoassay method of the present invention may be either solid-phased on an insoluble carrier or the like, or may be in a soluble state. It is preferably soluble in the liquid. As used herein, “soluble antibody” means an antibody that is not immobilized on an insoluble carrier or the like during an antigen-antibody reaction. In this specification, the term “insoluble carrier” means a material for immobilizing an antibody or antigen such as a plate or bead, and a polymer compound such as dextran when preparing a labeled antibody. In some cases, an antibody, a labeling enzyme, and the like are bound, but such a polymer compound is not contained in the insoluble carrier.

[0036] Enzymes for labeling antibodies used in the hepatitis B virus immunoassay method of the present invention include alkaline phosphatase, β-galatatosidase, luciferase, and the like in addition to horseradish peroxidase (HRP). it can. In addition to enzymes, luminescent substances such as ataridinium derivatives, fluorescent substances such as europium, and radioactive substances such as I ¹²⁵ can be used as labeling substances. In addition, the substrate and the luminescence inducing substance can be appropriately selected in accordance with the labeling substance.

Furthermore, the labeled antibody in the present invention includes those bound with substances that can be used for detection of antigen-antibody reaction signals such as haptens, low molecular weight peptides, and lectins as detection markers. Haptens include biotin, dinitrophenyl (DNP) and FITC. For example, when biotin is bound to an antibody and a probe complex is prepared, an avidin having affinity for biotin is labeled with an enzyme such as HRP, a fluorescent substance such as fluorescein, or a luminescent substance such as an ataridinium derivative. Signals can be detected by color reaction, fluorescence, luminescence, etc. after reaction. [0037] The method for labeling the antibody is not particularly limited, and the antibody may be directly labeled with a label such as an enzyme, but the antibody and the label such as an enzyme are bound to a polymer compound such as dextran. Alternatively, the labeled antibody may be bound to a polymer compound such as dextran. Even when an antibody is bound to such a high molecular compound, it is included in the “soluble antibody” in the present specification as long as the labeled antibody is soluble in the reaction solution.

 [0038] The test sample analyzed by the hepatitis B virus immunological analysis method of the present invention may contain a hepatitis B virus antigen or anti-hepatitis B virus antibody measured by the immunological analysis method. As long as it is a test sample with a certain amount, it is not particularly limited, and examples thereof include biological fluids generally used for clinical diagnosis, such as blood, serum, plasma, or urine. Furthermore, in addition to the biological fluid, the culture supernatant of cells containing hepatitis B virus antigen or anti-hepatitis B virus antibody is also included in the test sample.

 [0039] The reagent for immunological analysis of hepatitis B virus by the sandwich method of the present invention may contain the saponin, bile acid, or anionic surfactant. In addition, the immunological analysis reagent can contain a mixture of two or more of saponin, cholic acid, and an anionic surfactant. In particular, it is preferable to include a mixture of saponin and an anionic surfactant.

 [0040] The hepatitis B virus immunological analysis reagent is not particularly limited as long as it is a reagent used in an antigen-antibody reaction in which an antigen is brought into contact with an antibody. For example, it may be a reagent used for the antigen test method or a reagent used for the antibody test method. Further, it may be a reagent used in the forward sandwich method, the reverse sandwich method, and the one-step method. Hepatitis B virus The buffer used for the immunological analysis reagent can be appropriately selected from conventionally known buffers such as phosphate buffer, Tris buffer, glycine buffer or hepes buffer. The reagent for immunological analysis of hepatitis B virus includes other components, for example, carrier proteins such as urine serum albumin for stabilizing antigens and antibodies, preservatives such as sodium azide, and non-preservatives. A surfactant or the like for suppressing a specific reaction can also be contained. Furthermore, either a hepatitis B virus antigen or an anti-hepatitis B virus antibody to be contacted during the antigen-antibody reaction can be contained in the reagent.

[0041] During the reaction of contacting the hepatitis B virus antigen and the anti-hepatitis B virus antibody, The concentration of saponin or bile acid in the hepatitis B virus immunological analysis reagent is not particularly limited, but preferably the lower limit is 0.001% (mass / volume percent) or more, and the upper limit is dissolved in the solution. This is the concentration that can be done. Also, preferably 0.01 to 10% (mass / volume percent), more preferably 0.02 to 5% (mass / volume percent), and most preferably 0.03-3. % Mass / volume percent). In addition, the concentration of the anionic surfactant of the reagent for immunological analysis of hepatitis B virus in the reaction of contacting the hepatitis B virus antigen and the anti-hepatitis B virus antibody is not particularly limited, but preferably The lower limit is 0.001% (mass / volume percent) or more, and the upper limit is the concentration that can be dissolved in the solution. Also, preferably from 0.01 to 10% (mass / volume percent), more preferably from 0.02 to 5% (mass / volume percent), and most preferably from 0.05 to 0.8. % (Mass / volume percent). In addition, in the case of a mixture of two or more selected from saponins, bile acids and anionic active agents, the concentration of each substance can be used at a concentration where each substance is added alone. It is. The concentration of saponin, bile acid, or anionic surfactant in the hepatitis B virus immunological analysis reagent should be the same as that in the reaction by selecting an appropriate mixing ratio with the test sample. It is possible to adjust to. In addition, it is possible to use at the above concentration by attaching a user manual or the like indicating the mixing ratio with the test sample.

[0042] The immunological analysis kit of the present invention may contain a reagent for hepatitis VIII virus immunological analysis containing saponin, bile salt, or an anionic surfactant. The reagent for hepatitis B virus immunological analysis may be provided in solution, but may also be provided in a lyophilized powder form. The kit for immunological analysis of the present invention can contain a hepatitis virus antigen, an anti-hepatitis virus antibody, a carrier such as an antigen or antibody-immobilized plate or beads, instructions for use, and the like.

 Example

 [0043] Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but these are not intended to limit the scope of the present invention.

The following hybridoma cell lines producing monoclonal antibodies used in the examples HBsl 21 [International Accession Number FERM BP-10697], HBsl 23 [International Accession Number FER M BP-10698], HBsl36 [International Accession Number FERM BP-10699], HBs605C3 [International Accession Number FERM BP-10701], SF124CS [International Accession Number FERM BP—l 0703] are effective as of October 12, 2006. In addition, Hypridoma cell line 6G6 [International Deposit No. FERM BP-10117] was established on September 9, 2004 by the National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology) (T 305-8566 Japan). The deposit was made internationally at Tsukuba, Ibaraki Pref.

[0044] <Example 1>

 In Example 1, in the presence of saponin, the antigen test secondary reaction step of the antigen test method was performed, and HBV antigen was measured.

 Add 100 L of anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBsl36 antibody equal volume mixture) to each well of 96-well microplate (FluoroNunk Module, Maxisope surface) at a concentration of S ^ g / mL. In addition, it was incubated at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well and blocked at 37 ° C. for 4 hours. After removing the blocking solution, add 25 L of reaction buffer (0.3 M BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 L of healthy human serum or HBV positive specimen to each well, and stir at room temperature. For 1 hour. After the reaction, the cells were washed 8 times with 0.05 mM Tween 20, 0.1% sodium dodecyl sulfate (SDS) in 10 mM phosphate buffer pH 7.3 (washing solution). Next, the alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF12 4CS antibody) was diluted with 10 mM phosphate buffer ρΗ7 · 3 containing 0.5% BSA and 2% mouse serum. SIGMA) was added at a reaction concentration of 0-3.2%. 100 L of this antibody dilution was added to each well and allowed to react for 1 hour at room temperature with stirring. After washing 8 times with the washing solution, 100 L of the substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Luminous CT—9000D). The results are shown in Table 1. In the table, “RLU” represents the Relative Light Unit, and “S / N ratio” was calculated by dividing the RLU of the HBV positive specimen by the RLU of the healthy person.

[0045] Healthy human serum was unaffected by the addition of saponin. On the other hand, HBV positive test When the saponin-containing reaction solution was used, the body was improved by 2.2 to 3.0 times the reactivity of the reaction solution not containing saponin.

[table 1]

 Saponin concentration 0 0.03% 0.05% 0.10% 0.20¾ 0.40% 0.80% 1.60% 3.20%

RLU Healthy human serum 75 68 81 75 74 70 76 64 42

S / N ratio HBV¾ sex specimen 1 70.0 107.6 110.7 155.5 181.2 200.2 178.2 203.7 190.3

HBV positive specimen 2 36.8 60.4 60.2 80.8 91.7 108.0 94.8 108.8 100.8

HBV positive specimen 3 9.2 13.9 15.6 19.8 18.8 24.2 21.2 23.7 22.2

HBV positive specimen 4 51.7 81.5 87.5 125.2 134.9 153.3 139.5 149.5 152.6

HBV positive specimen 5 52.3 78.2 85.7 118.8 135.1 143.9 137.2 152.1 152.1

HBV positive specimen 6 60.8 91.9 97.2 135.9 150.3 165.3 145.8 156.5 159.5

Example 2

 In Example 2, an antigen test secondary reaction step of the antigen test method was performed in the presence of bile acid, and HBV antigen was measured.

Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ^ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours. After removing the blocking solution, add 25 μL of reaction buffer (0.3% BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 μL of healthy human serum or HBV positive sample to each well, stirring The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, the plate was washed 8 times with lOmM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS). Next, alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF 124CS antibody) was diluted with lOmM phosphate buffer ρ 血清 7 · 3 containing 0.5% BSA and 2% mouse serum, and sodium cholate (WAKO) was added to the reaction solution. Alternatively, sodium deoxycholate (WAKO) was added so that the concentration during the reaction was 0-0.40%. 100 L of this antibody dilution was added to each well and allowed to react for 1 hour at room temperature with stirring. After washing 8 times with the washing solution, 10 O ^ L of substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Lum inous CT—9000D). The results are shown in Table 2. "RLU" and "S The “/ N ratio” was calculated in the same manner as in Example 1.

 [0048] Healthy human serum was not affected by the addition of sodium cholate or sodium deoxycholate. On the other hand, when sodium cholate or sodium deoxycholate-containing reaction solution was used, the reactivity of HBV positive specimens was improved.

 [0049] [Table 2]

 Sodium colate. Sodium deoxycolate Additive-free 0.025% 0.050% 0.100% 0.200% 0.400% 0.025% 0.050% 0.100% 0.200% 0.400% RLU Healthy human serum 87 70 60 76 66 53 77 80 59 53 57

S / N ratio HBV positive specimen 1 66.7 87.2 108. 9 96.6 29 6 99.0 108.1 145.5 186.2 161.2

HBV positive specimen 2 36.4 48.4 59. 7 52.8 68 0 53.7 55.7 74.8 98.4 92.7

HBVI1 sex specimen 3 9.0 12.7 14. 9 13.3 16 0 12.7 13.7 16.4 21.2 22.5

HBVI sex specimen 4 50.5 69.5 81.2 2 73.8 95 6 71.1 80.5 107.1 134.7 117.7

HBV fertile specimen 5 48.4 66.6 83. 5 70.4 94 2 68.4 70.7 116.4 126.9 120.8

HBV positive specimen 6 56.7 81.7 96. 2 81.2 08 4 80.5 76.1 122.8 139.2 123.1

Example 3

 In this example 3, the shadow 592222

 In the presence of an ionic surfactant 6 o 2542, the antigen test secondary reaction step of the antigen test method was performed, and HBV antigen was measured.

Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ^ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours. After removing the blocking solution, add 25 μL of reaction buffer (0.3% BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 μL of healthy human serum or HBV positive sample to each well, stirring The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, it was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS). Next, dilute alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF124CS antibody) with lOmM phosphate buffer ρΗ7 · 3 containing 0.5% BSA and 2% mouse serum, and react with NLS (WAKO) in the reaction solution. The concentration at the time was 0-0.8%, or SDS (WAKO) was added so that the concentration at the reaction was 0-0.4%. 100 L of this antibody dilution was added to each well and allowed to react for 1 hour at room temperature with stirring. Wash with washing solution 8 times, and add substrate solution (TROPIX, CDP-star with Emerald II) to each well. 100 L was added to the flask and incubated at room temperature for 15 minutes. Luminometer (DIA—IATRO

N, Luminous CT—9000D). The results are shown in Table 3. "R

“LU” and “S / N ratio” were calculated in the same manner as in Example 1.

[0051] Healthy human serum was unaffected by the addition of either the anionic surfactant NLS or SDS. On the other hand, when NLS or SDS-containing reaction solution was used, the reactivity of HBV positive samples was improved.

[0052] [Table 3]

 NLS SDS

 Additive-free 0.05% 0.10% 0.20% 0.40% 0.80% 0.05% 0.10% 0.20% 0.40% RLU Healthy human serum 40 19 15 26 26 40 22 14 15 25 S / N ratio HBV positive specimen 1 102.2 435.2 563.1 330.7 300.6 194.7 320.0 600.8 580.6 386.0 HBV positive specimen 2 52.2 215.8 307.0 167.2 163.7 103.1 158.9 293.7 327.5 199.9 HBV positive specimen 3 13.3 48.4 79.4 46.8 42.6 27.8 37.9 69.7 79.5 53.0 HBV positive specimen 4 69.6 296.8 394.7 218.0 208.9 125.8 228.6 372.9 361.3 227.8 HBV positive specimen 5 86.1 314.0 410.5 230.7 213.6 135.2 230.2 410.7 369.9 257.6 HBV positive specimen 6 84.1 318.6 416.9 233.0 231.2 141.9 234.1 437.1 412.6 261.8

[Example 4]

 In Example 4, an antigen test secondary reaction step of the antigen test method was performed in the presence of an anionic surfactant, and HBV antigen was measured.

Each anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and HBs136 antibody equal volume mixture) in a 96-well microplate ^ 1110 0 Nunk Module, Maxisope surface) at a concentration of 68/111 Add 100 L to the well and incubate at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C for 4 hours. After removing the blocking solution, add 25 L of reaction buffer (0 · 3M BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 L of healthy human serum or HBV positive specimen to each well, and stir The reaction was allowed to proceed for 1 hour at room temperature. After the reaction, the plate was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS). Next, alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (S F124CS antibody) was added to 0.5% BSA, 2% mouse serum, and 0.2% SDS, 1-octane sodium sulfonate, 1-decane sodium sulfonate, 1-un Sodium decane sulfonate or lOmM phosphate buffer pH 7.3 containing sodium 1-dodecane sulfonate After dilution, 100 L was added to each well, and the mixture was allowed to react at room temperature for 1 hour with stirring. The plate was washed 8 times with a washing solution, and 100 μL of a substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Luminous CT—9000D). The results are shown in Table 4. “RLU” and “S / N ratio” were calculated in the same manner as in Example 1.

 [0054] Healthy human serum was unaffected by the addition of SDS, an anionic surfactant, and various alkyl sulfonates. On the other hand, when SDS or various alkyl sulfonate-containing reaction solutions were used, the reactivity of HBV positive specimens was improved.

 [0055] [Table 4]

 1

 Addition SDS Mono-octane 1-decane 1-undecane 1-dodecane Additives Not added Sulfonic acid N a Sulfonic acid N a Sulfonic acid N a Sulfonic acid N a

RLU Healthy human serum 29 27 21 28 25 23

S / NJt HBV positive specimen 1 215.8 519.0 480.4 419.7 616.3 631.0

HBV positive specimen 2 112.0 278.7 251.4 211.9 358.6 322.6

HBV positive specimen 3 36.0 79.0 69.6 56.5 102.9 92.0

HBV positive specimen 4 142.8 326.9 321.9 270.1 374.8 357.3

HBV positive specimen 5 147.5 313.6 313.5 272.4 389.9 379.0

HBV positive specimen 6 159.7 346.1 325.1 286.6 408.6 431.1

Example 5

 In Example 5, an antigen test secondary reaction step of the antigen test method was performed in the presence of saponin or cholic acid, and HBV antigen was measured.

Anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and H Bsl36 antibody equivalent mixture)

100 L was added to each well of nk Module, Maxisope surface) and incubated at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, and blocking was performed at 37 ° C. for 4 hours. After removing the blocking solution, add 25 L of reaction buffer (0.3M BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 L of healthy human serum or HBV positive specimen to each well, and stir at room temperature. For 1 hour. After the reaction, it was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS). Next, alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF12 4CS antibody) was added to 0.5% BSA, 2% mouse serum and 0.4% saponin (from Kira; SIGM A and tea seeds; WAKO), 0.4% Cholic acid, 0.4% Tween20, or 0.4% CH APS containing 10 mM phosphate buffer pH 7.3 diluted, adding 100 L to each well, stirring The reaction was allowed to proceed for 1 hour at room temperature. After washing 8 times with the washing solution, 100 L of substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Luminous CT—9000D). The results are shown in Table 5. “RLU” and “S / N ratio” increased as in Example 1.

 [0057] Healthy human serum was not affected by the addition of various saponins or cholic acid.

 On the other hand, when various saponins or cholic acid-containing reaction solutions were used, the reactivity of HBV-positive samples was improved, but the addition of Tween 20 and CHAPS did not affect the reactivity.

 [0058] [Table 5]

 0.4% Kira 0.4% Tea

 No additive 0.4 <½ Cholic acid 0.4% Teen20 0.4% CHAPS Saponin Saponin

 RLU Healthy human serum 42 36 48 45 41 46

S / N ratio HBV positive specimen 1 109.7 343.4 186.7 138.8 95.2 104.6

HBV positive specimen 2 60.2 183.8 99.9 71.8 54.5 55.4

HBV positive specimen 3 16.9 45.1 27.5 21.3 16.4 16.5

HBV positive specimen 4 78.7 252.7 128.5 105.6 68.5 78.1

HBV positive specimen 5 80.0 257.5 136.4 110.4 69.2 83.3

HBV positive specimen 6 81.6 249.1 148.4 100.8 73.4 81.7

Example 6

 In Example 6, the antigen test secondary reaction step of the antigen test method was performed in the presence of saponin and an anionic surfactant, and HBV antigen was measured.

Anti-HBsAg monoclonal antibody (6G6 antibody, HBsl21 antibody, HBsl23 antibody, and H Bsl36 antibody equivalent mixture)

100 L was added to each well of nk Module, Maxisope surface) and incubated at 4 ° C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 10 mM phosphate buffer containing 0.5% sodium caseinate was added to each well, and blocking was performed at 37 ° C. for 4 hours. After removing the blocking solution, add 25 L of reaction buffer (0.3M BES containing 1% BSA / 2% Mouse serum, pH 7.2) and 75 L of healthy human serum or HBV positive specimen to each well, and stir at room temperature. For 1 hour. After the reaction, 0.05% Tween 20, 0.1% Washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing sodium decyl sulfate (SDS). Next, alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF12 4CS antibody) was added to 0.5% BSA 2% mouse serum and 0.4% saponin (from Kira; SIG MA), 0.1% NLS 0.2% deoxycholate, 0.4% The solution was diluted with 10 mM phosphate buffer ρΗ7 · 3 containing saponin and 0.1% NLS or 0.2% deoxycholate and 0.1% NLS, added to each well, and allowed to react at room temperature for 1 hour with stirring. After washing 8 times with the washing solution, 100 L of substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminescence intensity was measured with a luminometer (DIA—IATRON, Luminous CT—9000D). The results are shown in Table 6. “RLU” and “S / N ratio” were calculated in the same manner as in Example 1.

 [0060] The HBV antigen-positive specimen can be detected with higher sensitivity than the control solution when an antibody diluted with a solution containing saponin, NLS and deoxycholate is used, and NLS is detected in saponin or deoxycholate. When combined and used for dilution, detection can be performed with higher sensitivity than when each is used alone.In particular, the combined use of saponin and an anionic surfactant has the effect of enhancing the antigen-antibody reaction. Admitted.

 [0061] [Table 6]

jsiinai η _n ιοί, ΐ-0.4% Saponin 0.2% Deoxy 0.2% Deoxy No additive 0.4% τ Ho U% NLS ₊₀ .i¾N _L s Cholic acid Cholic acid + 0.1% NLS

RLU Blood loss of healthy people 50 35 26 24 30 29

S / N ratio HBV fertile sample 1 78.7 367.2 356.5 600.7 255.6 332.0

HBV positive specimen 2 48.6 220.1 216.9 348.1 161.2 205.4

HBV positive specimen 3 14.9 60.3 60.3 103.5 46.4 59.4

HBV positive specimen 4 58.4 259.9 247.2 398.6 185.3 228.1

HBV positive specimen 5 59.6 255.5 237.7 393.2 189.4 224.3

HBV positive specimen 6 68.8 266.6 281.1 417.2 197.8 247.3

Example 7

 In Example 7, in the presence of saponin, the antigen test primary reaction step of the antigen test method was performed, and the HBV antigen was measured.

To a 96-well microplate (FluoroNunk Module, Maxisope surface), add 150 L of anti-HBs Ag monoclonal antibody (equal mixture of HBs605C3 antibody, HBsl23 antibody, and HBsl28 antibody) to each well at a concentration of 3 g / mL, and 4 ° Incubated with C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 1 OmM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours. The After removal of the locking solution, 1.0% or 2.0% (the final concentration of the reaction solution is 0.25% or 0.5%) 3- (N, N- Dimethylmyristyl-ammonio) propanesulfonate (C 14APS; SIGMA), or 1.0% , Or 2.0% (the final concentration of the reaction solution is 0.25% or 0.5%) Reaction buffer containing 1% BSA / 2% Mouse serum (3% BES, ρΗ7 · 2) ) 25 L and normal human serum or HBV positive sample 75 were added to each well and allowed to react at room temperature for 1 hour with stirring. After the reaction, it was washed 8 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecyl sulfate (SDS). Next, alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF124CS antibody) is diluted with 10 mM phosphate buffer pH 7.3 containing 0.5% BSA and 2% mouse serum, and 100 μL is added to each well and stirred. For 1 hour at room temperature. After washing 8 times with the washing solution, 100 L of the substrate solution TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes.

 The luminescence intensity was measured with a luminometer (DIA—IATRON, Luminous CT—9000D), and the results are shown in Table 7. “RLU” and “S / N ratio” were calculated in the same manner as in Example 1.

Yes

 [0063] The HBV antigen-positive specimen is compared with the case where the reaction with the immobilized antibody is performed in the presence of the reaction buffer containing saponin, compared with the case where the reaction is performed with the reaction buffer without saponin. Improved reactivity. On the other hand, the reactivity was not improved by the addition of C14APS.

 [0064] [Table 7] No additive 0.25% C14APS 0.50% C14APS

 RLU Healthy human serum 137 180 146

 S / N ratio HBV positive sample 1 4.9 3.6 3.4

 HBV positive specimen 2 46.0 37.9 44.2

HBV positive specimen 3 22.7 21.9 24.4

HBV positive specimen 4 20.5 15.7 16.4 No additive 0.25% saponin 0.50% saponin

 RLU Healthy human serum 137 111 95

 S 舰 HBV positive specimen 1 3.8 5.9 5.7

 HBV positive specimen 2 39.6 56.6 71.2

HBV positive specimen 3 23.1 24.5 32.3

HBV positive specimen 4 17.4 18.3 18.9 [Example 8]

 In Example 8, in the presence of saponin, the antigen test primary reaction step of the antigen test method was performed, and the HBV antigen was measured.

 To a 96-well microplate (FluoroNunk Module, Maxisope surface), add 150 L of anti-HBs Ag monoclonal antibody (equal mixture of HBs605C3 antibody, HBsl23 antibody, and HBsl28 antibody) to each well at a concentration of 3 g / mL, and 4 ° Incubated with C. After washing twice with 10 mM phosphate buffer pH 7.3, 350 μL of 1 OmM phosphate buffer containing 0.5% sodium caseinate was added to each well, followed by blocking at 37 ° C. for 4 hours. After removal of the blocking solution, reaction buffer containing 0-10% (final concentration 0-2.5% in the reaction solution) of saponin (from Kiraja; SIGMA) (including 1% BSA / 2% Mouse serum, 0 · 3Μ BES PH 7.2) 25 μL of healthy human serum or 75 μL of HBV positive specimen was added to each well and allowed to react at room temperature for 1 hour with stirring. After the reaction, the cells were washed 8 times with a 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween20 and 0.1% sodium dodecinole sulfate (SDS). Next, dilute the alkaline phosphatase-labeled anti-HBsAg monoclonal antibody (SF124C S antibody) with 10 mM phosphate buffer ρΗ7.3 containing 0.5% BSA and 2% mouse serum, add 100 to each well, and stir. The reaction was allowed to proceed for 1 hour at room temperature. After washing 8 times with the washing solution, 100 μL of the substrate solution (TROPIX, CDP-star with Emerald II) was added to each well and incubated at room temperature for 15 minutes. Luminometer DIA—IATRON, Luminous CT—9000D) was used to measure the luminescence intensity and the results are shown in Table 8. “RLU” and “S / N ratio” were calculated in the same manner as in Example 1.

 [0066] The HBV antigen-positive specimen is compared with the case where the reaction with the immobilized antibody is performed in the presence of the reaction buffer containing saponin, compared with the case where the reaction is performed with the reaction buffer without saponin. Improved reactivity.

 [0067] [Table 8]

 Add saponin 0¾ 0.13% 0.25% 0.50% 0.75% 1.00% 1.25% 1.50% 1.75% 2.00% 2.25% 2.50%

R L U Healthy human serum 182 121 91 133 103 82 88 113 113 113 111 96

SZN ratio HBV positive specimen 1 32.5 52.1 62.6 44.0 48.0 61.5 55.5 44.2 47.1 43.0 41.5 64.4

HBV positive specimen 2 16.0 25.2 35.4 26.9 31.9 42.3 39.5 30.7 29.6 38.5 32.2 44.0

HBV positive specimen 3 51.5 74.5 94.2 75.5 95.2 166.4 114.8 103.0 92.8 96.1 94.8 119.0

HBV positive specimen 4 62.5 91.7 121.8 83.4 116.7 138.6 131.0 105.0 97.1 90.6 129.7 117.7 Industrial applicability The present invention provides a method for immunological analysis of hepatitis B virus by the Sandwich method for highly sensitive detection or quantification of hepatitis B virus antigens in blood. Diagnose the presence of infection and screen blood for transfusion quickly and accurately. In addition, the present invention can provide a highly sensitive reagent for hepatitis B virus immunological analysis and a hepatitis B virus immunological analysis kit.

 As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

Claims

The scope of the claims  [1] Sandwich method type B hepatitis virus comprising the step of contacting hepatitis B virus antigen with anti-hepatitis B virus antibody in the presence of saponin, bile salt, or anionic surfactant Immunological analysis method. [2] The hepatitis B virus immunological analysis method according to claim 1, wherein the anti-hepatitis B virus antibody is a soluble antibody. [3] The hepatitis B virus immunological analysis method according to claim 1 or 2, wherein the saponin is a triterpenoid saponin. 4. The method for immunological analysis of hepatitis B virus according to claim 3, wherein the triterpenoid saponin is killer saponin or tea seed saponin. [5] Reagent for immunological analysis of hepatitis B virus by sandwich method, which is a reagent for contacting hepatitis B virus antigen with anti-hepatitis B virus antibody, saponin, bile salt, or anionic interface A reagent for immunological analysis of hepatitis B virus, comprising an active agent.  6. The reagent for hepatitis B virus immunological analysis according to claim 5, wherein the anti-hepatitis B virus antibody is a soluble antibody. 7. The reagent for hepatitis B virus immunological analysis according to claim 5 or 6, wherein the saponin is a triterpenoid saponin. [8] The reagent for immunological analysis of hepatitis B virus according to [7], wherein the triterpenoid saponin is killer saponin or tea seed saponin. [9] A hepatitis B virus immunological analysis kit comprising the reagent for hepatitis B virus immunological analysis according to any one of! / In claim 5-8.