JP2003083976A - HBs antigen measurement reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a conventional HB having an anti-HBs antibody supported on a solid phase.
To provide an HBs antigen measurement reagent which has higher sensitivity and higher sample processing capacity than an s antigen measurement reagent, and which can cope with an escape mutant of hepatitis B virus. SOLUTION: In qualifying or quantifying an HBs antigen, a first biotinylated monoclonal antibody against a common antigenic determinant a of the HBs antigen and a first biotinylated monoclonal antibody against a pre-S2 antigen of hepatitis B are used.
A second enzyme-labeled monoclonal antibody against the common antigenic determinant a of the aqueous solution and the HBs antigen and a second enzyme-labeled monoclonal antibody against the pre-S2 antigen of hepatitis B
Use reagents that combine aqueous solutions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a qualitative assay for hepatitis B virus surface antigen (hereinafter sometimes abbreviated as “HBs antigen”) or a reagent for quantifying HBs antigen.

[0002]

2. Description of the Related Art Hepatitis B virus (hereinafter sometimes abbreviated as "HBV") is known as one of typical hepatitis viruses, and when infected, a serious case such as fulminant hepatitis occurs. There is also. Further, when persistent infection of HBV is established (chronic), if the symptom worsens, a serious disease such as cirrhosis or hepatocellular carcinoma may be accompanied. HBV infection is H
It is widely known that this is established when body fluids such as blood containing BV and semen contact the mucous membranes, or are directly exposed to the body by administration of blood containing HBV or blood products, needle stick accidents, and the like. Therefore, qualitative or quantitative determination of HBV is very important not only for diagnosing hepatitis B but also for preventing secondary infection such as screening of blood for transfusion and screening of blood product materials. The assay of HBV contained in blood or the like is generally performed by qualitatively or quantitatively determining the HBs antigen that reflects the amount of HBV. The qualitative or quantitative determination of the HBs antigen is generally performed by an immunological method, and the immunological method mainly adopted is the agglutination method (hereinafter, referred to as “PA
Method), a radioimmunoassay using a radioisotope labeled substance (hereinafter also abbreviated as “RIA method”), or a colorimetric or chemical method using an enzyme labeled substance. An enzyme immunoassay method utilizing luminescence (hereinafter sometimes abbreviated as "EIA method") can be mentioned. The PA method has been often used because it is easy to operate and easy to measure a large number of samples, but it has a problem that the detection sensitivity is limited and the window period becomes long. RIA
Although the method is superior to the PA method in terms of detection sensitivity, there is a problem in that handling is complicated due to the use of radioisotopes. On the other hand, the EIA method is relatively easy to operate and has excellent detection sensitivity and detection range. Therefore, it is expected that the EIA method will become the mainstream in future HBs antigen measurement. However, in the EIA method, it takes a long time to measure a sample, so that there remains a problem in terms of sample throughput.

The HBs antigen is measured by the EIA method by first contacting a sample with a solid phase on which an antibody specific to the HBs antigen is immobilized in a primary reaction to specifically capture the HBs antigen, and then in a secondary reaction with an enzyme. A sandwich method is generally used in which an antibody specific to the HBs antigen labeled with is contacted with the HBs antigen to bind to it, and then the HBs antigen is detected using a colorimetric substrate or a chemiluminescent substrate. As the solid phase in the above primary reaction, a polystyrene porous plate, magnetic particles, or a porous matrix such as cellulose is generally used.

In the EIA method disclosed in Japanese Patent Publication No. 3134231, a porous matrix using a glass fiber filter as a solid phase is used. Furthermore, in the EIA method,
A measuring instrument (hereinafter, also referred to as "reaction cup") in which an absorption layer for absorbing a solution that has passed through the solid phase is combined with the solid phase is used. In the EIA method, it is considered that the EIA method has a weak point because it is possible to continuously advance the reaction on the solid phase by separating the reagents and the like by sequentially injecting the sample and the reagents from the upper part of the reaction cup. The existing sample processing capacity has improved, and rapid measurement has become possible even with multiple samples. When the HBs antigen is measured by the EIA method, an antibody specific to the HBs antigen is directly immobilized on the glass fiber filter. However, when the antibody specific to the HBs antigen is directly immobilized on the glass fiber filter as described above, there is a problem that the sensitivity is insufficient especially for a sample containing a low concentration of HBs antigen.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly sensitive HBs antigen measuring reagent which has a high sample processing capacity and can measure a sample containing a low concentration of HBs antigen. .

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that in qualitatively or quantitatively determining HBs antigen, (1) a common antigen determinant of HBs antigen at an appropriate concentration. biotinylated monoclonal antibody against a and pre-S of hepatitis B virus at an appropriate concentration
A first aqueous solution containing a second biotinylated monoclonal antibody against two antigens, (2) a first enzyme-labeled monoclonal antibody against a common antigenic determinant a of HBs antigen at an appropriate concentration, and a hepatitis B virus pre-S2 at an appropriate concentration Second containing a second enzyme-labeled monoclonal antibody against the antigen
It was found that by the sandwich method combining aqueous solutions, it is possible to achieve high sensitivity while maintaining high sample processing capacity,
The present invention has been completed.

That is, the above objects and advantages of the present invention are as follows: (1) The first to the common antigenic determinant a of HBs antigen
Biotinylated monoclonal antibody and hepatitis B virus p
A first aqueous solution containing a second biotinylated monoclonal antibody against the re-S2 antigen in a weight ratio of 1 to 0.05 to 2 in this order, and a total concentration of 1 to 100 μg / mL. And (2) the first enzyme-labeled monoclonal antibody against the common antigenic determinant a of the HBs antigen and the second enzyme-labeled monoclonal antibody against the hepatitis B virus pre-S2 antigen in a weight ratio of 1 to 0.05. ~ 2, and the total of both is 0.5 to 50
This is achieved by a combination reagent for qualitatively or quantifying the presence of HBs antigen by the sandwich method, which comprises a combination of second aqueous solutions containing at a concentration of μg / mL.

Further, the above objects and advantages of the present invention are preferably the common antigenic determinant a recognized by the first biotinylated monoclonal antibody, and the common antigenic determinant a recognized by the first enzyme-labeled monoclonal antibody. This is achieved by the above combination reagent of the present invention, which differs from the site in the region.
Further, the above objects and advantages of the present invention are preferably
Pre- recognized by the second biotinylated monoclonal antibody
This is achieved by the above-mentioned combination reagent of the present invention in which the site within the S2 antigen region and the site within the pre-S2 antigen region recognized by the second enzyme-labeled monoclonal antibody are different.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below, but the substances, manufacturing methods and operating methods used in the present invention are not limited to the following. The HBs antigen applied to the present invention is not limited as long as it is a protein or peptide derived from the S gene region on the HBV genome. HBV has many subspecies (hereinafter, also referred to as “subtype”) and mutants, and the HBs antigen applied to the present invention depends on the origin of these subtypes and mutants. There is no limitation. Further, the HBs antigen in the present invention may be a complete molecular protein, a fragment protein, or a peptide. The HBs antigen is HBV with a chain length of about 3.2 Kb.
Hepatitis B virus (H
It is a structural protein that forms the outer shell of BV), and as described above, it is widely known that the HBs antigen concentration reflects the HBV amount.

In the present invention, the common antigenic determinant a is the antigenic determinant a of the HBs antigen consisting of the region of amino acids 111 to 156 from the N terminus in the HBs antigen protein.
Refers to. As described above, there are various subtypes of HBV, and these subtypes are roughly classified into four types according to the difference in three major antigenic determinants in the HBs antigen. The major antigenic determinants in the HBs antigen include an antigenic determinant a commonly present between subtypes, and an antigenic determinant d / y or an antigenic determinant w / r as an antigenic determinant different between subtypes. HBV subtypes are represented by a combination of major antigenic determinants present in each subtype. More specifically, the HBV subtype includes four types of subtypes represented by adw, adr, ayw, and ayr. The common antigenic determinant a is commonly present in all of the four HBV subtypes. The present invention is not limited at all even if the amino acid sequence of the region is changed or changed due to the influence of HBV mutation.

The first biotinylated monoclonal antibody in the present invention is the common antigenic determinant a of the HBs antigen.
Is specifically recognized and biotinylated (labeled with biotin)
The monoclonal antibody described above can be used without particular limitation. The first biotinylated monoclonal antibody may recognize any site as long as it is a region of the common antigenic determinant a of the HBs antigen. In the present invention, by using a monoclonal antibody that specifically recognizes the common antigenic determinant a of HBs antigen, it becomes possible to detect all of the above-mentioned four types of HBV subtypes. As the monoclonal antibody used in the present invention, a monoclonal antibody produced by a known method can be used without any limitation. Further, the antibody subclass of the monoclonal antibody is not particularly limited, but IgG or IgM is more preferably used in view of the detection sensitivity of HBs antigen. There is no limitation on the origin (animal species) of the monoclonal antibody, and known animal species such as mouse, rat and guinea pig can be used. As a method for biotinylating a monoclonal antibody, a known method can be used without any limitation. Generally, a method in which a biotinylated reagent and a monoclonal antibody aqueous solution are first contacted and reacted to bind biotin to the first monoclonal antibody, and then unreacted biotinylated reagent is removed and purified by gel filtration, dialysis, or the like. It has been known. This method is particularly preferably used because the reaction of biotinylation is mild and thus the attack on the monoclonal antibody is low and the procedure is simple, and the biotinylation reagent is commercially available and easily available. Hereinafter, biotinylation of a monoclonal antibody will be specifically described. Examples of the biotinylation reagent used in the reaction include biotin-N-hydroxysuccinimide ester and biotin-in which N-hydroxysuccinimide group or maleimide group is introduced into biotin.
Examples thereof include piperazinyl maleimide. When biotin-piperazinyl maleimide is used as the biotinylation reagent, it is necessary to expose the thiol group as F (ab) 2 antibody, Fab antibody, or the like using the monoclonal antibody with pepsin, papain, or the like. Considering this, biotin-N-hydroxysuccinimide ester is more preferably used as the biotinylation reagent. Further, the biotinylation reaction is achieved by contacting a biotinylation reagent with a monoclonal antibody aqueous solution. As the aqueous solution component used in the aqueous solution of the monoclonal antibody, a known buffer solution can be used without any limitation as long as it does not show reactivity with the biotinylation reagent. The pH of the aqueous solution is preferably weakly basic with a pH of 8 to 10 when biotin-N-hydroxysuccinimide ester is used as the biotinylation reagent in consideration of reaction efficiency and influence on the monoclonal antibody. Is. The reaction temperature is preferably around room temperature in consideration of reaction efficiency and influence on the monoclonal antibody.
Further, the reaction time depends on the reaction temperature, but when the biotinylation reaction is performed at room temperature, it is usually about 3 to 4 hours. Following the biotinylation reaction, the unreacted biotinylation reagent can be removed and purified by gel filtration, dialysis, or the like.
The unreacted biotinylated reagent may compete with the biotinylated monoclonal antibody, and therefore needs to be removed and purified. Examples of methods for removing and purifying the unreacted biotinylated reagent include gel filtration and dialysis. The gel filtration method is more preferable in consideration of simplicity and yield of the biotinylated monoclonal antibody. Se is used as a carrier for gel filtration used in the method.
Exclusion limit molecular weight of 10 such as phadex G-25
Although it is preferable to use a desalting carrier having a KD or less,
Considering simplicity, for example, Sephadex G-25
A pre-packed column for commercial desalting with a small capacity, such as PD-10 (manufactured by Amersham Pharmacia Co., Ltd.) in which is previously packed, is more preferably used.

The pre-S2 antigen applied to the present invention is
There is no limitation as long as it is a protein or peptide derived from the pre-S2 gene region on the HBV genome. The pr
The e-S2 antigen may be a complete molecular protein, a fragment protein, or a peptide. Furthermore, the pre-S2 antigen applied to the present invention is not limited by the difference in origin such as HBV subtype and mutant strain,
Further, the present invention is not limited at all even if the amino acid sequence of the region is changed or changed due to the influence of HBV mutation. The pre-S2 gene is a gene located before the S gene on the HBV genome, and the pre-S2 gene
The antigen is expressed with the HBs antigen on the surface of the hepatitis B virus coat. Therefore, it is known that the amount of pre-S2 antigen substantially correlates with the amount of HBs antigen.

The second biotinylated monoclonal antibody in the present invention can be used without particular limitation as long as it is a monoclonal antibody which specifically recognizes the pre-S2 antigen and is biotinylated. The second biotinylated monoclonal antibody is the above-mentioned pre-S.
Any part of the two antigens may be recognized. As the monoclonal antibody used in the present invention, a monoclonal antibody produced by a known method can be used without any limitation. Further, the antibody subclass of the monoclonal antibody is not particularly limited, but IgG or IgM is more preferably used in consideration of the detection sensitivity of HBs antigen. There is no limitation on the origin (animal species) of the monoclonal antibody, and known animal species such as mouse, rat and guinea pig can be used. As a method for biotinylating the second monoclonal antibody, a known method can be used without any limitation. More specifically, biotinylation may be carried out in the same manner as the above-mentioned first biotinylated monoclonal antibody.

In the present invention, the first biotinylated monoclonal antibody against the common antigenic determinant a of the HBs antigen and HB
A biotinylated monoclonal antibody having different specificity such as a second biotinylated monoclonal antibody against V-pre-S2 antigen is used in combination. Although HBV is a DNA virus, it once replicates DNA via RNA during the growth process, and is therefore known as a virus that is highly susceptible to mutation. When the mutation occurs on the S gene of HBV, an antigen different from the normal HBs antigen is expressed, and as a result, an antibody against the normal HBs antigen (hereinafter referred to as “HBs antibody”).
(Although it may be abbreviated), a mutant strain having an HBs antigen that cannot be recognized may also appear. Such a mutant strain is called an "escape mutant", and it is known that an HBs antibody carrier such as a past infected person or a vaccinated person can be infected. In the case of an escape mutant in which such a mutation occurs in a region related to the common antigenic determinant a of the S gene, it is possible that the first biotinylated monoclonal antibody against the common antigenic determinant a of the HBs antigen of the present invention cannot recognize the HBs antigen. There is a nature. Therefore, it is necessary to take measures to surely supplement even such an escape mutant. In the present invention, the second biotinylated monoclonal antibody against the pre-S2 antigen is used in combination with the first biotinylated monoclonal antibody against the common antigenic determinant a to take measures against the escape mutant. That is, as described above, p similar to the HBs antigen is expressed in the outer shell of HBV and shows a correlation with the amount of HBs antigen.
By using the second biotinylated monoclonal antibody against the re-S2 antigen in combination, the second biotinylated monoclonal antibody against the common antigenic determinant a of the HBs antigen cannot be detected by the second biotinylated monoclonal antibody in the subject containing the escape mutant. The monoclonal antibody can complement the reaction and detect HBV.

In the present invention, the first biotinylated monoclonal antibody against the common antigenic determinant a of the HBs antigen and HB
An aqueous solution containing a second biotinylated monoclonal antibody against V-pre-S2 antigen at a weight ratio of 1 to 0.05 to 2 in this order, and a total concentration of 1 to 100 μg / mL. 1 aqueous solution. The mixing ratio of the first biotinylated monoclonal antibody against the common antigenic determinant a of the HBs antigen and the second biotinylated monoclonal antibody against the HBV pre-S2 antigen is determined by the reaction between the first biotinylated monoclonal antibody and the second biotinylated monoclonal antibody. From the viewpoint of balance, the first biotinylated monoclonal antibody is preferably used in a weight ratio of 1 to the second biotinylated monoclonal antibody of 0.05 to 2. When the weight ratio of the first biotinylated monoclonal antibody is 1 and the second biotinylated monoclonal antibody is less than 0.05, the reactivity of HBV to the pre-S2 antigen becomes weak and the escape mutant cannot be detected. On the other hand, when the weight ratio of the first biotinylated monoclonal antibody to the second biotinylated monoclonal antibody is larger than 2, the reactivity of the HBs antigen to the common antigenic determinant a becomes weaker, and thus the reactivity with other HBs antigen reagents is decreased. Correlation deteriorates. The concentrations of the first biotinylated monoclonal antibody and the second biotinylated monoclonal antibody also depend on the amount of the analyte, but considering the detection sensitivity of the HBs antigen and the prevention of nonspecific reaction, the first biotinylated monoclonal antibody is used. And the total of the second biotinylated monoclonal antibody is 1 to
It is preferably 100 μg / mL. If the total concentration of the biotinylated monoclonal antibody is less than 1 μg / mL, the sensitivity will be insufficient, and if it exceeds 100 μg / mL, nonspecific reaction will increase. The pH of the first aqueous solution of the present invention is preferably adjusted by using an appropriate buffer. As the buffering agent, known buffering agents such as phosphate buffer and Good's buffer can be used without any limitation. The concentration of the buffering agent is not particularly limited, but it is preferable to use it at about 10 to 200 mM in consideration of the buffering capacity. Also, the pH is not particularly limited. Considering the efficiency of the antigen-antibody reaction and the storability of the aqueous solution, it is preferable to adjust the pH to 5-9. Furthermore, for the purpose of suppressing non-specific reaction, or improving reproducibility and preservability, the first aqueous solution contains salts such as sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium azide, and Microsides I to III.
Preservatives such as, mouse serum, goat serum, rabbit serum, human serum, animal serum such as fetal bovine serum, proteins such as bovine serum albumin and casein can be appropriately selected and added. The concentration of the additive is not particularly limited.
0.01 for salts such as sodium chloride and potassium chloride
~ 1M, sodium azide and Microside I ~ II
In the case of preservatives such as I, 0.01 to 0.1% by weight, in the case of animal serum such as mouse serum or goat serum, 0.1 to 50% by weight, protein such as bovine serum albumin or casein is 0% by weight. It is preferably set to 0.01 to 20% by weight.

As the first enzyme-labeled monoclonal antibody in the present invention, a monoclonal antibody that specifically recognizes the common antigenic determinant a of HBs and is enzyme-labeled is the same as the first biotinylated monoclonal antibody without any limitation. Can be used. In the present invention, by using a monoclonal antibody that specifically recognizes the common antigenic determinant a of HBs antigen, it becomes possible to detect all of the above-mentioned four types of HBV subtypes. The first enzyme-labeled monoclonal antibody may recognize any site as long as it is in the region of the common antigenic determinant a of the HBs antigen, but the first enzyme-labeled monoclonal antibody should not interfere with the binding of the first enzyme-labeled monoclonal antibody.
It is necessary to recognize a site different from the site in the common antigenic determinant a region recognized by the biotinylated monoclonal antibody.
As the monoclonal antibody used in the present invention, a monoclonal antibody produced by a known method can be used without any limitation. Also, the antibody subclass of the monoclonal antibody is not particularly limited.
Considering the detection sensitivity of HBs antigen, IgG or I
gM is more preferably used. There is no limitation on the origin (animal species) of the monoclonal antibody, and known animal species such as mouse, rat and guinea pig can be used. As the enzyme for labeling the monoclonal antibody, any known enzyme can be used without any limitation. Specific examples of the enzyme for labeling the monoclonal antibody include peroxidase, alkaline phosphatase, β-galactosidase and the like. Among them, peroxidase is preferably used because it has excellent storage stability and is easy to handle. As a method for enzyme-labeling the monoclonal antibody, any known method can be used without any limitation.
Exemplifying the method of enzyme-labeling a monoclonal antibody,
When using peroxidase as the enzyme, Ant
ibodies (Ed Harlow et al., 1988,
The glutaraldehyde method and the periodic acid method as described in p. The periodate method is used as a suitable method because the bond between the enzyme and the antibody is strong, the yield of the peroxidase-labeled antibody is good, and the operation is relatively simple.

As the second enzyme-labeled monoclonal antibody in the present invention, a monoclonal antibody which specifically recognizes the HBV pre-S2 antigen and is enzyme-labeled can be used without any limitation. The second biotinylated monoclonal antibody may recognize any part of the pre-S2 antigen described above, but the second biotinylated monoclonal antibody recognizes it so as not to interfere with the binding of the second enzyme-labeled monoclonal antibody. It is necessary to recognize a site different from the site in the pre-S2 antigen region. As the monoclonal antibody used in the present invention, a monoclonal antibody produced by a known method can be used without any limitation. Further, the antibody subclass of the monoclonal antibody is not particularly limited, but IgG or IgM is more preferably used in view of the detection sensitivity of HBs antigen. There is no limitation on the origin (animal species) of the monoclonal antibody, and known animal species such as mouse, rat and guinea pig can be used. Further, the kind of enzyme labeling the monoclonal antibody and the enzyme labeling of the monoclonal antibody may be performed in the same manner as the above-mentioned first monoclonal antibody. In the present invention, a second enzyme-labeled monoclonal antibody specific for pre-S2 antigen is used as HBs.
By using together with the first enzyme-labeled monoclonal antibody specific to the common antigenic determinant a of the antigen, even if the escape mutant cannot be recognized by the first enzyme-labeled monoclonal antibody, the reaction by the second enzyme-labeled monoclonal antibody can be satisfied. Can be dealt with. Also,
Even in the case of normal wild type HBV, the combined use of two enzyme-labeled monoclonal antibodies also has the effect of increasing the detection sensitivity of HBV.

In the present invention, the common antigenic determinant a of the HBs antigen is
Enzyme-labeled monoclonal antibody against HBV and p
An aqueous solution containing the second enzyme-labeled monoclonal antibody against the re-S2 antigen at a weight ratio of 1 to 0.05 to 2 in this order, and a total concentration of 0.5 to 50 μg / mL. The second aqueous solution is used. The mixing ratio of the first enzyme-labeled monoclonal antibody against the common antigenic determinant a of HBs antigen and the second enzyme-labeled monoclonal antibody against HBV pre-S2 antigen is determined by the reaction between the first enzyme-labeled monoclonal antibody and the second enzyme-labeled monoclonal antibody. From the standpoint of balance, the first enzyme-labeled monoclonal antibody is preferably used in a weight ratio of 1 to the second enzyme-labeled monoclonal antibody of 0.05 to 2. When the weight ratio of the first enzyme-labeled monoclonal antibody to 1 is less than 0.05 for the second enzyme-labeled monoclonal antibody, HBV pre-S
The reactivity to the two antigens becomes weak and it becomes difficult to detect the escape mutant. On the other hand, when the weight ratio of the first enzyme-labeled monoclonal antibody to the second enzyme-labeled monoclonal antibody is larger than 2, the reactivity of the HBs antigen to the common antigenic determinant a becomes weaker, so that the HBs antigen may not react with other HBs antigen reagents. Correlation deteriorates. In addition, the concentrations of the first enzyme-labeled monoclonal antibody and the second enzyme-labeled monoclonal antibody are the sum of the first enzyme-labeled monoclonal antibody and the second enzyme-labeled monoclonal antibody in consideration of the detection sensitivity of the HBs antigen and the prevention of nonspecific reaction. 0.5 ~ 50μg /
It is preferably in mL. If the total concentration of the enzyme-labeled monoclonal antibody is less than 0.5 μg / mL, the sensitivity will be insufficient, and if it exceeds 50 μg / mL, nonspecific reaction will increase.
In addition, the second aqueous solution of the present invention may be adjusted to pH by using an appropriate buffer.
Is preferably adjusted. As the buffering agent, known buffering agents such as phosphate buffer and Good's buffer can be used without any limitation. The concentration of the buffer is not particularly limited,
Considering the buffering capacity, it is preferable to use it at about 10 to 200 mM. Although the pH is not particularly limited, it is preferable to adjust the pH to 5 to 9 in consideration of the efficiency of the antigen-antibody reaction and the storability of the aqueous solution. further,
The second aqueous solution contains salts such as sodium chloride, potassium chloride, magnesium chloride and calcium chloride, sodium azide and Microsides I to III for the purpose of suppressing non-specific reactions or improving reproducibility and storage stability. Preservatives, mouse serum, goat serum, rabbit serum, human serum, animal serum such as fetal bovine serum, proteins such as bovine serum albumin and casein can be appropriately selected and added. The concentration of the additive is not particularly limited. In the case of salts such as sodium chloride and potassium chloride, about 0.01 to 1M, and in the case of preservatives such as sodium azide and Microcide I to III, 0.01 to 0.1% by weight of mouse serum and goat serum. In the case of such animal serum, 0.1 to 50% by weight, and in the case of proteins such as bovine serum albumin and casein, 0.01 to 2%.
It is preferably 0% by weight.

In the present invention, in order to qualitatively or quantitatively determine the presence of HBs antigen, a sandwich method using a combination reagent in which the first aqueous solution and the second aqueous solution are combined is applied.
As for the sandwich method applied here, known methods can be applied without any limitation as long as the combination reagent is used. Examples of the method for qualitatively or quantifying the presence of HBs antigen by the sandwich method are as follows: (1) contacting a test solution that may contain HBs antigen with a first aqueous solution of the present invention, and incubating under appropriate conditions. To form a HBs antigen-biotinylated monoclonal antibody complex, and then (2) the complex is contacted with a solid phase supporting a substance having an affinity for biotin to bind to the solid phase, (3) By washing away the biotinylated monoclonal antibody that has not bound to the solid phase and unreacted HBs antigen, and (4) contacting the second aqueous solution with the solid phase,
The HBs antigen bound to the solid phase was bound to the enzyme-labeled monoclonal antibody, and then (5) the solid phase was washed to remove the excess enzyme-labeled monoclonal antibody, and then (6) the reaction was performed. A method of qualifying or quantifying the presence of HBs antigen by measuring the enzyme activity of the complex can be mentioned. Here, the analyte solution that may contain HBs antigen (1) may be any solution that may contain HBs antigen, such as serum or plasma, or a buffer solution or protein-containing solution in which HBs antigen is mixed. Is mentioned. Further, as the conditions for contacting and incubating the test solution that may contain the HBs antigen with the first aqueous solution, a reaction temperature of 15 to 50 ° C. and a reaction time of about 1 to 15 minutes are preferable. If the reaction temperature is lower than 15 ° C, the reaction takes too long, and if the reaction temperature is higher than 50 ° C, protein denaturation may occur. If the reaction time is less than 1 minute, the sensitivity may be insufficient, and if it exceeds 15 minutes, the measurement time may be insufficient. Is not so preferable because it extends. The ratio when the test solution and the first aqueous solution are brought into contact with each other is not particularly limited, but considering the detection sensitivity of the HBs antigen or the reproducibility of the measurement, the first aqueous solution and the test solution are mixed in this order of volume. The ratio is preferably about 1 to 1-10. When the amount of the analyte solution is smaller than that of the first aqueous solution, the detection sensitivity of the HBs antigen is decreased, and conversely, when the amount of the analyte solution is large, the concentrations of the first and second biotinylated monoclonal antibodies in the first aqueous solution are set high. Therefore, the reproducibility is deteriorated because the influence of the liquid amount error of the first aqueous solution amount to be sampled at the time of measurement becomes large. Examples of the substance having an affinity for biotin in (2) include avidin and anti-biotin antibody. By immobilizing the HBs antigen-biotinylated monoclonal antibody complex thus formed by reacting in advance on the solid phase through the substance having an affinity for biotin, it is immobilized per unit area of the solid phase. Since it becomes possible to increase the amount of HBs antigen, the detection sensitivity of HBs antigen can be increased.
When an anti-biotin antibody is selected as a substance having an affinity for biotin, the reaction specificity can be further increased by allowing the first aqueous solution to coexist with the same species of serum as the animal species for which the anti-biotin antibody was obtained. it can. Further, the solid phase on which the substance having an affinity for biotin is carried is not particularly limited. Examples of materials that are preferably used include inorganic substances such as glass fiber and silica, and organic substances such as polystyrene and cellulose. Also,
As for the shape, a filter shape, a tube shape, a spherical shape, etc. are exemplified as a preferable shape. Furthermore, among these, if a solid phase material in which a filter for absorbing a reaction solution is connected to a solid phase of a filter material and a porous material such as glass fiber is used, a continuous reaction can be achieved without performing B / F separation. Even when the number of sample solutions is large, the measurement can be performed quickly and efficiently, so that it is more preferable in consideration of the sample processing capacity. The reaction temperature and reaction time in (2) above are 15 to 50 for the same reason as in (1).
C., and preferably 1 to 15 minutes. Also,
The washing solution used in (3) and (5) is one that can remove the biotinylated monoclonal antibody that has not bound to the solid phase and unreacted HBs antigen, or that can remove excess enzyme-labeled monoclonal antibody. It is not particularly limited as long as it is, but preferably Tween 20 or Tr
It is preferable to use a buffer solution containing a surfactant such as iton X-100. The concentration of the surfactant used here depends on the type of the surfactant, but is 0.0 if the cleaning effect is taken into consideration.
It is preferable to use it at about 5 to 1%. In addition, proteins such as casein and albumin, or animal serum may be added to the washing solution in order to enhance the washing effect. The reaction time and reaction temperature in (4) are 15 for the same reasons as in (1) and (2).
It is suitable to set the temperature to -50 ° C and for 1 to 15 minutes. When measuring the enzyme activity in (6), it is necessary to select a colorimetric, fluorescent, or luminescent substrate according to the characteristics of the labeling enzyme of the enzyme-labeled monoclonal antibody. For example, as a substrate used when peroxidase is selected as the labeling enzyme, 3,3 ′, 5,5′-tetramethylbenzidine (T
Examples include colorimetric substrates such as MBZ) and 1,2-phenylenediamine, fluorescent substrates such as 4-hydroxyphenylacetic acid and 3- (4-hydroxyphenyl) propionic acid, and luminescent substrates such as luminol. These appropriately selected enzyme substrates are used by dissolving them in an appropriate reaction medium. In addition, depending on the property of the selected enzyme, for example, when peroxidase is selected, an additive necessary for the reaction of the enzyme substrate such as hydrogen peroxide is added to the enzyme substrate solution. The enzyme substrate solution is brought into contact with the solid phase subjected to the operations up to (5) for reaction, and then, depending on the property of the enzyme substrate, for example, 3,
3 ', 5,5'-tetramethylbenzidine (TMBZ)
When is selected, colorimetric, fluorescent, or emission intensity is measured, such as measuring absorbance at a wavelength of 595 nm. Then, the obtained colorimetric, fluorescence, or emission intensity is
Contains HBs antigen of known concentration measured in advance, or H
Qualitative or quantitative determination of the presence of HBsAg contained in the sample is achieved by referring to the colorimetric, fluorescence, or luminescence intensity obtained from the sample solution containing no BsAg.

[0020]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 (1) First biotinylated monoclonal antibody against common antigenic determinant a of HBs antigen, and HBV pre-S
Preparation and Purification of Second Biotinylated Monoclonal Antibody Against Two Antigens Both the first biotinylated monoclonal antibody and the second biotinylated monoclonal antibody were prepared by the following procedure (hereinafter, biotin of the first biotinylated monoclonal antibody). The monoclonal antibody used for the biotinylation is referred to as “monoclonal antibody A”, and the monoclonal antibody used for the biotinylation of the second biotinylated monoclonal antibody is referred to as “monoclonal antibody B”). Here, a method for preparing a first biotinylated monoclonal antibody using the monoclonal antibody A is shown. As the monoclonal antibody A and the monoclonal antibody B, those manufactured by Special Immune Research Institute were used. First, a 10 mM HEPES buffer solution (pH 8.
5) was used to prepare a monoclonal antibody A solution having a total volume of 250 μL. Next, 30 μL of a biotinylation reagent (50 mM Biotin-AC5-Osu, manufactured by Dojindo Co., Ltd.) was added to the solution, mixed, and then incubated at 25 ° C. for 4 hours. Then, using a PD-10 column (manufactured by Amersham Pharmacia) equilibrated with a 10 mM phosphate buffer (pH 7.4, hereinafter sometimes referred to as “PBS”) in advance, unreacted in the reaction solution. Purification was carried out to remove the biotinylated reagent of. For the purification, first, 250 μL of the reaction solution was applied to a PD-10 column, then 2.25 mL of PBS was added to the PD-10 column, and then 3.5 mL of PBS was used to apply the first biotinylated monoclonal antibody (hereinafter, referred to as “1. Sometimes referred to as "-B antibody"). The second biotinylated monoclonal antibody (hereinafter sometimes referred to as "2-B antibody") was also produced by the same method as above.

(2) Preparation and Purification of First Peroxidase-Labeled Monoclonal Antibody Against Common Antigenic Determinant a of HBs Antigen and Second Peroxidase-Labeled Monoclonal Antibody Against HBV pre-S2 Antigen First Peroxidase-Labeled Monoclonal Antibody and Second Both preparations of peroxidase-labeled monoclonal antibody were carried out by the following procedure (hereinafter, the monoclonal antibody used to prepare the first peroxidase-labeled monoclonal antibody was used as “monoclonal antibody C” and the second peroxidase-labeled monoclonal antibody). The monoclonal antibody is referred to as "monoclonal antibody D"). Here, a method for producing a first peroxidase-labeled monoclonal antibody using the monoclonal antibody C is shown. As the monoclonal antibody C and the monoclonal antibody D, those manufactured by Special Immune Research Laboratories were used. First, 5 mg of peroxidase (RZ> 3.0, manufactured by Toyobo Co., Ltd.) was dissolved in 1.2 mL of water, and then the peroxidase solution contained 0.1 M sodium periodate.
0.3 mL of mM phosphate buffer (pH 7.0) was added, and the mixture was allowed to stand at 25 ° C. for 20 minutes. Then, the reaction solution is 1 m
The solution was dialyzed against M sodium acetate / acetate buffer (pH 4.0) at 4 ° C. overnight to obtain the dialysate, and a periodate-activated peroxidase solution was obtained. Then 10 mg / mL
20 mM carbonate buffer solution (pH 9.5) containing monoclonal antibody C is prepared, 0.5 mL of the solution is mixed with the periodate-activated peroxidase solution, and the mixture is mixed at 25 ° C. for 2 hours.
Stir for hours. Next, 0.1 mL of a 4 mg / mL sodium borohydride aqueous solution was added to the reaction solution, and the mixture was allowed to stand at 4 ° C. for 2 hours. Then, the reaction solution was dialyzed against PBS overnight to obtain a first peroxidase-labeled monoclonal antibody (hereinafter sometimes referred to as "1-P antibody"). A second peroxidase-labeled monoclonal antibody (hereinafter sometimes referred to as "2-P antibody") was also prepared by the same method as above.

(3) Preparation of first aqueous solution The 1-B antibody concentration and the 2-B antibody concentration prepared in (1) were respectively 20 μg / mL, that is, 1-B antibody and 2-B antibody.
The antibody ratio was 1: 1 in this order, the total concentration of 1-B antibody and 2-B antibody was 40 μg / mL, and the goat serum concentration and mouse serum concentration were 20% and 1%, respectively.
The first aqueous solution was prepared by adding 0% to PBS.

(4) Preparation of the Second Aqueous Solution The 1-P antibody concentration and the 2-P antibody concentration prepared in (2) are adjusted to 5 μg / mL, that is, the 1-P antibody and the 2-P antibody are The ratio is 1 to 1 in this order so that the total concentration of 1-P antibody and 2-P antibody is 10 μg / mL, the mouse serum concentration, and fetal calf serum (FCS).
A second aqueous solution was prepared by adding PBS to the respective concentrations of 10% and 3%.

(5) Measurement of test solution Human serum containing HBs antigen or not and human serum containing HBV escape mutant were used as test solutions in the following measurements. In addition, the following measurements were performed using a commercially available analyzer ID-1000 (manufactured by Toyobo Co., Ltd.). First, 50 μL of the sample solution and the first aqueous solution 1
After mixing 0 μL and incubating at 40 ° C. for 5 minutes, 50 μL of the reaction solution was added dropwise to a cup (manufactured by Toyobo) on which an anti-biotin antibody was immobilized. Then 1%
50 μL of PBS containing BSA was added dropwise to the cup, and 10 μL of the second aqueous solution prepared in (4) was added dropwise. Four
200 μL of 0.0 after incubation for 3 minutes at 0 ° C.
After washing with PBS containing 5% Tween 20, 30 μL of TMBZ solution containing 0.05% 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) and 0.01% hydrogen peroxide was added dropwise. And 5.5 more after dropping
The amount of change in the reflection intensity of the laser light having a wavelength of 650 nm was measured for about 7.5 seconds and for 40.5 to 42.5 seconds. The measurement results are shown in Table 1. As shown in Table 1,
The HBs antigen measuring reagent was highly sensitive, and the HBs antigen contained in the sample solution was measured without causing nonspecific reaction. It was also possible to detect the HBs antigen in the test solution containing the HBV escape mutant. Further, in the above measurement, the total time required to obtain the measurement result was 12 minutes as shown in Table 3. That is, the present invention enabled rapid measurement of the amount of HBs antigen.

Comparative Example 1 The concentrations of 1-B antibody and 2-B antibody were 39 μg / mL and 1 μg / mL, respectively, in this order, that is, the ratio of 1-B antibody to 2-B antibody was 1 to 0 in this order. The same procedure as in Example 1 was carried out except that the first aqueous solution was prepared so that the total concentration of 0.026, 1-B antibody and 2-B antibody was 40 μg / mL. The measurement results are shown in Table 1. The HBs antigen measuring reagent was highly sensitive, and the HBs antigen contained in the test solution was measured without causing nonspecific reaction. However, HBs antigen could not be detected in human serum containing the HBV escape mutant.

Comparative Example 2 The concentrations of 1-B antibody and 2-B antibody were 10 μg / mL and 30 μg / mL in this order, respectively, that is, the ratio of 1-B antibody and 2-B antibody was 1: 3 in this order. First to be
The same procedure as in Example 1 was carried out except that an aqueous solution was prepared.

The measurement results are shown in Table 1. The HBs antigen reagent was able to detect the HBs antigen contained in the analyte solution with high sensitivity, and the HBs antigen could be detected also in human serum containing the escape mutant of HBV, but the analyte containing the HBs antigen of known concentration was detected. There was a deviation in the HBs antigen concentration in the solution.

Comparative Example 3 The concentrations of 1-B antibody and 2-B antibody were 0.4 μg / mL and 0.4 μg / mL, respectively, in this order, that is, the total concentration of 1-B antibody and 2-B antibody was The same procedure as in Example 1 was carried out except that the first aqueous solution was prepared to have a concentration of 0.8 μg / mL. The measurement results are shown in Table 1. The HBs antigen reagent had low sensitivity, and the HBs antigen could not be detected in a test solution containing a low concentration of HBs antigen.

Comparative Example 4 The concentrations of 1-B antibody and 2-B antibody were 60 μg / mL and 60 μg / mL in this order, respectively, that is, the total concentration of 1-B antibody and 2-B antibody was 120 μg / mL. Example 1 was repeated except that the first aqueous solution was prepared as described above. The measurement results are shown in Table 1. The non-specific reaction increases with the HBs antigen measurement reagent, and HBs in the analyte solution
The antigen could not be measured specifically.

[0031]

[Table 1]

Comparative Example 5 The concentrations of 1-P antibody and 2-P antibody were 10 μg / mL and 0.2 μg / mL, respectively, in this order, that is, the ratio of 1-P antibody to 2-P antibody was 1 in this order. The same procedure as in Example 1 was carried out except that the second aqueous solution was prepared so that the ratio was 0.02. The measurement results are shown in Table 2. The HBs antigen measuring reagent was highly sensitive, and the HBs antigen contained in the test solution was measured without causing nonspecific reaction. However, HBs antigen could not be detected in human serum containing the HBV escape mutant.

Comparative Example 6 The concentrations of 1-P antibody and 2-P antibody were 2.5 μg / mL and 7.5 μg / mL, respectively, in this order, that is, the ratio of 1-P antibody to 2-P antibody was in this order. Example 1 was repeated except that the second aqueous solution was prepared so that the ratio became 1: 3.
The measurement results are shown in Table 2. The HBs antigen reagent highly sensitively detects the HBs antigen contained in the analyte solution, and
HBs in human serum containing V escape mutant
Although the antigen could be detected, there was a deviation in the HBs antigen concentration in the test solution containing the HBs antigen of known concentration.

Comparative Example 7 The concentrations of 1-P antibody and 2-P antibody were 0.2 μg / mL and 0.2 μg / mL, respectively, in this order, that is, the total concentration of 1-P antibody and 2-P antibody was The same procedure as in Example 1 was carried out except that the second aqueous solution was prepared so as to have a concentration of 0.4 μg / mL. The measurement results are shown in Table 2. The HBs antigen reagent had low sensitivity, and the HBs antigen could not be detected in a test solution containing a low concentration of HBs antigen.

Comparative Example 8 The concentrations of 1-P antibody and 2-P antibody were 30 μg / mL and 30 μg / mL in this order, respectively, that is, the total concentration of 1-P antibody and 2-P antibody was 60 μg / mL. The same procedure as in Example 1 was carried out except that the second aqueous solution was prepared as described above. The measurement results are shown in Table 2. With the HBs antigen measuring reagent, the non-specific reaction became large, and the HBs antigen in the sample solution could not be specifically measured.

[0036]

[Table 2]

Comparative Example 9 An example in which HBs antigen was measured by directly immobilizing the monoclonal antibody A and the monoclonal antibody B on a filter-like solid phase will be given below. (1) Preparation of cup with immobilized monoclonal antibody A and monoclonal antibody B First, a filter made of glass fiber (Nippon Sheet Glass Co., Ltd.) having an average fiber diameter of 1.0 μm and an average fiber length of 1 mm was dipped in distilled water, and then The filter was sandwiched between dot blot apparatuses (manufactured by Bio-Rad). Then, the inside of the dot blot apparatus was decompressed by a pump, and (a) 100
100 μL of mM citrate buffer (pH 3.0),
(B) 100 μL of PBS containing 4 μg / mL of monoclonal antibody A and monoclonal antibody B, (c) 1
100μm of 00mM citrate buffer (pH 3.0)
100 μL of PBS containing L, (d) 5% glycerol, and 1% bovine serum albumin was sequentially supplied in the above order after waiting for the liquid supplied immediately before to be aspirated. Then, remove the filter from the dot blot apparatus, remove excess water with a filter paper, and add 5% glycerol, and 1
Immersion in PBS containing% bovine serum albumin for 10 minutes. After removing excess water with a filter paper, the filter was dried under reduced pressure. Then, the filter was cut into a circle along the shape of the inlet and set on the opening side of the container having the opening.
Further, an absorption layer made of olefin-based non-woven fabric and cellulose was placed underneath, and finally fixed by a bottom plate so that the laminate would not fall off, and a cup on which the monoclonal antibody A and the monoclonal antibody B were fixed (hereinafter referred to as “direct fixation”). Sometimes referred to as "chemical cup") was prepared.

(2) Preparation of labeled antibody reagent A reagent having the same composition as the second aqueous solution of Example 1 was prepared and used as a labeled antibody reagent. (3) Measurement of test solution Human serum containing or not containing HBs antigen was used as a test solution in the following measurement. The following measurement was also performed using a commercially available analyzer ID-1000 (manufactured by Toyobo). First, 50 μL of the sample solution was dropped on the direct immobilization cup and incubated at 40 ° C. Next, 50 μL of PBS containing 1% BSA was dropped into the cup, and 10 μL of the labeled antibody solution prepared in (2) was further dropped. 200μ after incubation at 40 ℃ for 3 minutes
Rinse with PBS containing 0.05% Tween 20 and then TMBZ containing 0.05% 3,3 ', 5,5'-tetramethylbenzidine (TMBZ) and 0.01% hydrogen peroxide. 30 μL of the solution was dropped, and the amount of change in the reflection intensity of the laser beam having a wavelength of 650 nm was measured for 5.5 to 7.5 seconds and 40.5 to 42.5 seconds after the dropping. The measurement results are shown in Table 3. As shown in Table 3, the total time required to obtain the measurement result was as short as 7 minutes, but the HBs antigen reagent had low sensitivity, and the HBs antigen was low in the test solution containing a low concentration of HBs antigen. Could not be detected.

Comparative Example 10 An example of measuring the HBs antigen by the ordinary sandwich method in which the monoclonal antibody A and the monoclonal antibody B were immobilized on a microplate is shown below. (1) Preparation of Microplate Immobilized with Monoclonal Antibody A and Monoclonal Antibody B First, 100 μL of PBS containing 4 μg / mL of monoclonal antibody A and monoclonal antibody B was applied to a 96-well microplate (Nunc). And 40 ℃
It was left still for 1 hour. Then, after removing the solution, 30
The microplate was washed 3 times with 0 μL of PBS.
Then, 300 μL of P containing 1% bovine serum albumin
BS was applied and left at 40 ° C. for 1 hour. Then
After removing the solution, the microplate was washed with 300 μL of PBS three times to immobilize the monoclonal antibody A and the monoclonal antibody B (hereinafter, simply referred to as “immobilized microplate”). There is).

(2) Preparation of labeled antibody reagent 1-P antibody concentration prepared in (2) of Example 1, and 2
The labeled antibody reagent was added to PBS so that the P antibody concentration was 1 μg / mL, and the mouse serum concentration and fetal calf serum (FCS) concentration were 10% and 3%, respectively. (3) Measurement of test solution Human serum containing or not containing HBs antigen was used as the test solution in the following measurement. First, the sample solution 5
Apply 0 μL to the immobilized microplate and
It was left still at 0 ° C. for 5 minutes. The solution is then removed, 20
After washing three times with 0 μL of PBS containing 0.05% Tween 20, PB containing 300 μL of 1% BSA
S was applied and left at 40 ° C. for 5 minutes. The solution was then removed and 200 μL of 0.05% Tween was added.
After washing three times with PBS containing 20, 100 μL of the labeled antibody reagent prepared in (2) was applied, and the mixture was allowed to stand at 40 ° C. for 5 minutes. Then, the solution was removed, and after washing 3 times with 200 μL of PBS containing 0.05% Tween 20, 0.05% 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), and 0% were added. The reaction was stopped by applying 100 μL of a TMBZ solution containing 0.01% hydrogen peroxide, allowing to stand at room temperature for 3 minutes, and further applying 100 μL of 1M sulfuric acid. Then wavelength 600
The absorbance at nm was measured with a microplate reader (Bio-Rad). The measurement results are shown in Table 3. As shown in Table 3, the HBs antigen reagent had low sensitivity, and the HBs antigen could not be detected in the test solution containing a low concentration of HBs antigen. Further, the total time required to obtain the measurement result also requires 40 minutes, which is 3 times as compared with Example 1.
The measurement time was extended to a little less than twice.

Comparative Example 11 The procedure of Comparative Example 10 was repeated except that the test solution was applied to the immobilized microplate and then allowed to stand for 30 minutes, and the labeled antibody reagent was applied and then allowed to stand for 30 minutes. The results are shown in Table 3. As shown in Table 3, the HBs antigen measuring reagent was able to detect the HBs antigen even in a test solution containing a low concentration of HBs antigen.
However, the total required time for obtaining the measurement result required 90 minutes, and the measurement time was further extended as compared with Comparative Example 10.

[0042]

[Table 3]

From the above results, the ratio of 1-B antibody to 2-B antibody in the first aqueous solution was 1 to 2 in 1-B antibody.
It was revealed that when the -B antibody was less than 0.05, the HBs antigen could not be detected in the test solution containing the HBV escape mutant, and when it was more than 2, the HBs antigen concentration was deviated. Furthermore, when the total concentration of both 1-B antibody and 2-B antibody is less than 1 μg / mL, the detection sensitivity of HBs antigen is insufficient, and 100 μg / mL
It became clear that it is difficult to specifically measure the HBs antigen in the analyte solution because the non-specific reaction becomes larger when it is higher than mL. Similarly, with the second aqueous solution, 1
Ratio of -P antibody to 2-P antibody When 1-P antibody is less than 0.05 with respect to 2-P antibody, HBs antigen cannot be detected in a test solution containing HBV escape mutant, and 2 It was revealed that the larger the concentration, the more the concentration of HBs antigen varied. Furthermore, 1-
The total concentration of both P antibody and 2-P antibody is 0.5 μg / m
If it is less than L, the detection sensitivity of HBs antigen is insufficient, and
It was revealed that it is difficult to specifically measure the HBs antigen in the test solution because the non-specific reaction becomes large when the concentration is higher than 0 μg / mL. Further, it was revealed that when the monoclonal antibody A and the monoclonal antibody B were directly immobilized on the solid phase, the sensitivity was insufficient or the measurement time was extended.

[0044]

EFFECT OF THE INVENTION By using the combined reagent comprising the first aqueous solution and the second aqueous solution of the present invention, it becomes possible to qualitatively or quantitatively determine the HBs antigen with high sensitivity and speed. That is, the present invention has made it possible to provide a reagent for qualitatively or quantitatively determining the presence of HBs antigen having high sensitivity and high sample processing ability. Furthermore, the combination reagent for qualifying or quantifying the presence of the HBs antigen of the present invention can cope with the escape mutant of HBV by using a monoclonal antibody specific to the pre-S2 antigen.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hisahiko Iwamoto             320-11 Hino, Hino City, Tokyo             In and tea (72) Inventor Yoshinori Yoshimura             320-11 Hino, Hino City, Tokyo             In and tea F term (reference) 2G045 AA25 CA26 DA36 FB03 FB07                       GC15 HA16

Claims (3)

[Claims] (1) A first biotinylated monoclonal antibody against a common antigenic determinant a of HBs antigen and a second biotinylated monoclonal antibody against pre-S2 antigen of hepatitis B virus in a weight ratio of 1 in this order. Vs. 0.05
At a ratio of ~ 2, and a total of both of 1 to 100 µg / m
A first aqueous solution containing a concentration of L, and (2) a first enzyme-labeled monoclonal antibody against the common antigenic determinant a of the HBs antigen and a second enzyme-labeled monoclonal antibody against the hepatitis B virus pre-S2 antigen. The presence of the HBs antigen was qualitatively determined by the sandwich method, which consisted of the combination of the second aqueous solutions containing the order of weight ratio of 1: 0.05 to 2 and the total concentration of both of them was 0.5 to 50 μg / mL. Or a combination reagent for quantification. 2. The site in the common antigenic determinant a region recognized by the first biotinylated monoclonal antibody and the site in the common antigenic determinant a region recognized by the first enzyme-labeled monoclonal antibody are different from each other. Combination reagents. 3. The combination according to claim 1, wherein a site within the pre-S2 antigen region recognized by the second biotinylated monoclonal antibody and a site within the pre-S2 antigen region recognized by the second enzyme-labeled monoclonal antibody are different. reagent.