JPH02216461A - Method for measuring anti-pre-s antibody - Google Patents

Abstract

PURPOSE:To make quantitative measurement with lessened laboriousness and improved accuracy by using the S protein particles, M protein particles and L protein particles obtd. from single-cell living things. CONSTITUTION:Serum to be tested and the hepatitis B virus surface antigen S protein or M protein produced from the single-cell living things are mixed. The liquid mixture is brought into contact with the hepatitis B virus surface antigen M protein or L protein combined with a base produced from the single- cell living things. The antibodies to the Pre-S2 or S1, S regions of the hepatitis B virus surface antigen combined with the base are immunologically measured.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring anti-pre-S antibodies. More specifically, the present invention relates to hepatitis B virus (Flepati virus).
tis B virus; pr contained in the protein and M protein in the surface antigen (env protein)
The present invention relates to a method for quantifying antibodies against the e-S region by immunoassay.

Prior art and its problems In the serum of patients with acute hepatitis infected with HBV,
Prior to HBs antigen-anti-HBs antibody system, HBc antigen-anti-HBc antibody system, HBe antigen-anti-HBe antibody system, pre-
It is known that the s antigen-anti-pre-S antibody system is induced [A, Robert Neurath et al.
Nature (Nichi+-), 315.154 (1
985)]. Attention has been focused on the role that pre-3 antigen and anti-pre-S antibody play in the establishment of HBV infection, the onset mechanism and prevention of hepatitis B, and the like. In particular, antisera against the pre-S2 peptide within the pre-3 region neutralize HBV [A.

Robert Neurath et al., Vacci
ne (vaccine) 4.35 (1986)] and pre-S
Experiments using chimpanzees have recently demonstrated that a two-peptide vaccine can prevent the onset of acute hepatitis caused by HBV [Y, Itoh et al., Proc, N.
ATL, Acad, Sci, (Processing
of the National Academy of Sciences) USA, 83.9174 (1986. Also, pr
It has been reported that antibodies against the pre-sl peptide in the e-S region block the binding of HBV to hepatoma-derived cells [A.

Robert Neurath et al., Ce1l (
Cell), 46, 429 (1986)]. In this way anti-p
As the importance of re-s antibodies is recognized, anti-p
A system for quantifying re-3 antibodies easily and with high sensitivity is desired.

When antibodies in human serum are quantified by conventional enzyme immunoassay, this is very different from when serum from experimental animals is used, and significant non-specific reactions are observed. Therefore, when human serum is used, it must be diluted to a high degree for measurement, and the sensitivity of the enzyme immunoassay method is often insufficient. Therefore, more sensitive radioimmunoassays have been utilized to quantify low levels of antibody titers in human blood. Quantification of anti-pre-S antibodies is no exception.
Currently, it relies on radioimmunoassay.

For example, anti-pre-
3 antibodies have been measured by antiglobulin radioimmunoassay using antibodies bound to pre-s peptide on a solid phase [A, Robert Neurath et al.
Nature, 315.154 (1985)]
.

In addition, human serum was added to HB with a certain amount of pre-S antigen.
After reacting with 6 antigen solution, unreacted pre-8 antigen-containing HBs antigen is bound to beads coated with anti-pre-3 antibody and detected with 1''5I-anti-HBs antibody. Anti-pre-3 antibody titers have been determined by the inhibition method [A, Budkovska et al.

tlepatology, 6, 360 (198
6)]. Furthermore, the polymerized albumin receptor (Polymerized albumin receptor) located within the pre-32 region
Human antibodies against umin receptor;
Anti-PAR activity has been determined by inhibition method by detection with I-monoclonal anti-poly-H3A antibody [
H. Kasoto et al., Flepatology (
Hepatology), 6, 354 (1986)].

As described above, as a system for quantifying anti-pre-s antibodies in human serum with high sensitivity, Radioimmunoa 1. However, synthetic peptides are still widely used as antigens. This current situation has many limitations in terms of safety and operability caused by radioactive substances, as well as the disadvantage that all anti-pre-3 antibodies caused by synthetic peptides cannot be detected. A highly sensitive measurement system capable of detecting induced anti-pre-3 antibodies has been awaited.

Means for Solving the Problems Against this background, the present inventors have developed a method for easily and highly sensitively determining all anti-pre-8 antibodies contained in human serum and laboratory animal serum without using radioactive substances. We started research to develop a system that can do this. Enzyme immunoassay (
EnzyII+e in+muo+.

assay; EIA and EnzyIlle
1 inked immun.

5orbent assay;
). When anti-pre-S antibodies in human serum are measured by conventional E1/L ELISA, non-specific reactions are observed, and to eliminate them, it is necessary to use serum diluted 100-400 times, and synthetic It was necessary to use peptides as antigens.

However, it has been found that non-specific reactions can be significantly suppressed by pretreatment of serum by adding small plasma-derived particles containing S protein and M protein consisting of "pre-32+S" to serum.
First, small spherical particles containing pre-82 are added to serum to absorb anti-S antibody and anti-pre-32 antibody, and then the next reaction between this mixture and tubular spherule-derived polypeptide (s, M, or L protein) is carried out. There is also a method for quantifying anti-preS1 antibodies by reacting with a labeled secondary antibody, or by adding small spherical particles containing only S protein to serum and absorbing anti-S antibodies.・Carry out the following reaction with polypeptide-rich polypeptide (M1S),
Furthermore, a method has been proposed for quantifying anti-pre-32 antibodies by carrying out a secondary reaction in the same manner as above [Journal of Immunological Methods].
Immunological Methods,
95 + (1986) 23-303゜However, this method separates small spherical particles, small spherical particles containing pre-52, and tubular spherules containing pre-S l and pre-32 from serum of HBs antigen-positive patients. Since they are separated from each other, it is difficult to purify them, and this measurement method itself is also complicated or has a drawback of low accuracy.

Therefore, in order to improve the above measurement method, the present inventors replaced polypeptides rich in small spherical particles obtained from serum, polypeptides derived from tubular spherules, and middle-sized polypeptides derived from small spherical particles. By using S protein particles, M protein particles, and L protein particles obtained from unicellular organisms by genetic engineering techniques, we improved the complexity and low accuracy of these particles, and completed a system that allows further quantification.

That is, the present invention provides (1) mixing a test serum and hepatitis B virus surface antigen S protein produced from a unicellular organism, and using the mixture as a hepatitis B virus produced from a unicellular organism and bound to a support. The pre-82 region of the hepatitis B virus surface antigen in the test serum is characterized by contacting with the surface antigen M protein and measuring antibodies against the pre-S2 region of the hepatitis B virus surface antigen bound to a support. Immunological assay for antibodies against.

(2) Mix the test serum and hepatitis B virus surface antigen M protein produced from a unicellular organism, and mix the mixture with hepatitis B virus surface antigen protein produced from a unicellular organism and bound to a support. Immunology of antibodies against the pre-S1 region of hepatitis B virus surface antigen in test serum, characterized by contacting and measuring antibodies against the pre-S1 region of hepatitis B virus surface antigen bound to a support. and (3) mixing the test serum and hepatitis B virus surface antigen S protein produced from a unicellular organism, and using the mixture as a hepatitis B virus produced from a unicellular organism and binding it to a support. The pre-S region of the hepatitis B virus surface antigen in a test serum is characterized by contacting with the surface antigen protein and measuring the antibody against the pre-3 region of the hepatitis B virus surface antigen bound to a support. provides an immunoassay method for antibodies against.

S protein, M protein, and L protein may be isolated from the serum of HBs antigen-positive individuals, but their purification is difficult, so those obtained by genetic engineering methods [P, Va
Lenzuela et al., Nature,
λdan 8.347 (1982): A, Miyano
Hara et al., Proc, Natl, Acad
, Sci, (Procedures of the National Academy of Sciences) USA, 8
0.1 (1983); M, L.

Michel et al., Proc. Natl.
Acad, Sci, (Procedures of the National Academy of Sciences)
USA, 81. ．． 7708 (1984); Y, P
ujisawa et al., Gene.

Sat 0.23 (1985); Y, 1toh &
Y, Pujisawa.

Biochem, Biophys, Res, C
ommun, (Biochemical and Biophysical Research Communications), 141
．． 942 (1986): P. Dehoux et al.
Gene, 48. l 55 (1986); Japanese Patent Publication No. 109795/1986; M, Kobayash
J. Biotech, (Journal Biotechnology), 8, 1 (1988)] may be used. Further, as the S protein, L protein or M protein may be degraded with protease to remove the pre-s region. As a protease, Staphylococcus aureus
eus) V 3 strain derived ■8 protease (Seikagaku Corporation)
is suitable.

When L protein 1M protein and S protein are obtained by genetic engineering techniques, animal cells or yeast are preferably used as hosts in order to maintain S antigenicity.

Anti-pre-3 antibody (anti-pre-3l antibody + anti-pr
eS2 antibody) or anti-pre-32 antibody, which is used to absorb anti-S antibody contained in serum, and anti-pre-32 contained in serum, which is used to measure anti-pre-3L antibody. As the M protein used for absorption of antibodies and anti-S antibodies, S proteins and M proteins produced from animal cells or yeast, and mixed proteins thereof are advantageously used.

As the L protein and M protein bound to the support, proteins with lower S antigenicity than the proteins used for absorption may be used (in addition to the L protein and M protein produced from yeast, Proteins produced by prokaryotes such as Escherichia coli (which have extremely low S antigenicity because they do not exhibit a particle structure) can also be used.

When M protein is produced in animal cells, the protein obtained is usually a particle consisting of S protein and M protein, so there is a drawback that the amount of pre-32 region bound to the support is small. Furthermore, since I7 protein can hardly be secreted and produced in animal cells, it is inappropriate to prepare L protein using animal cells as hosts.

The S protein and M protein used for absorption of the anti-S antibody, etc., and the M protein and L protein bound to the support are both yeast (e.g., Saccharomyces
Preferably, the protein produced by S. revisiae is used.

When using yeast producing trypsin-like protease as a host, L protein and M protein may be degraded by protease, so arginine at position 48 from the N terminus of M protein or a peptide containing it, preferably M
Peptides from the 44th to 49th positions from the N-terminus of the protein [(152nd to 157th from the N-terminus of the L protein (in the case of subtypes adr and adw), or peptides from 163 to 168th (in the case of the subtype ayw) ] It is preferable to mutate it so that it is deleted.

Examples of the support include particles made of materials such as polystyrene, polycarbonate, agarose (Sepharose), and microplates.

Binding of S protein, M protein or L protein to a support is carried out by bringing the support into contact with an aqueous solution or buffer solution containing the protein.

As a method for measuring antibodies bound to a support, enzyme-labeled protein A, protein G, secondary antibodies (e.g.
Anti-human (human) IgG antibodies, anti-heron IgG antibodies, anti-mouse IgG antibodies), etc. can be used. Enzyme labeling was performed using the maleimide method [M, Imamura et al., JAppl.
Biochem, (Journal of 7 Pride Biochemistry), 4.41 (1982)]
Alternatively, a biotinylation reagent [e.g., NH3-biotin (biotinyl-N-hydroxy succinimide).

Sulfo-NH3-biotin, NH3-iminobiotin (
Vector Laboratories, In
c,)] and an avidinylated enzyme in combination. Examples of enzymes used include horseradish verokindase, alkaline phosphatase beta-galactosidase, and glucose oxidase.

To create a calibration curve for quantitative determination, use anti-pre-8
It can be carried out using serum containing 1 antibody, anti-pre-32 antibody, or anti-preS antibody (anti-ρre-8l antibody + anti-pre-S 2 antibody), or commercially available immunoglobulin preparations can be used. . Examples include Nichaku HB globulin manufactured by Nippon Pharmaceutical Co., Ltd.

The operating method of the method for measuring anti-pre-3 antibodies of the present invention will be described below.

(1) Method for measuring anti-pre-32 antibody Step 1 Add S protein to the test serum (or its diluted solution) and react in advance. Through this operation, anti-S antibodies contained in serum and antibodies that nonspecifically adsorb to HB V env protein are absorbed by S protein.

The serum used is prepared from collected blood by a conventional method. Alternatively, an IgG fraction prepared from serum may be used. The reaction between serum and S protein is usually carried out at 4-37°C for 1-20 hours.

Step 2 Binding of M protein to the support is performed by bringing the support into contact with an aqueous solution or buffer solution containing M protein. The reaction is usually carried out at about 4°C for about 20 hours. Next, it is preferable to bring an aqueous solution or buffer containing casein into contact with the support in order to suppress non-specific antibody adsorption. The reaction is usually carried out at about 4°C for about 20 hours.

Step 3 The mixture prepared in Step 1 is brought into contact with the M protein-binding support prepared in Step 2. At this time, only the anti-pre-82 antibody binds to M protein. The reaction is usually carried out at 4-37°C for 1-20 hours.

Step 4 Separate the support and the mixed solution, contact the support with an aqueous solution or buffer containing an enzyme-labeled secondary antibody (e.g., anti-human IgG, anti-human IgM), protein A or protein G, and react with anti-pre-8 antibody bound to. The reaction is usually carried out at 4 to 370C for 1 to 20 hours.

Separation of the support and the mixed liquid can be easily performed by filtration or centrifugation if the support is a particle, or by washing away the mixed liquid together with a washing liquid if the support is a plate. The washing solution used is water, sodium chloride solution, phosphate buffer, etc., which do not affect enzyme activity.

Step 5 Separate the support and the mixed solution in the same manner as in Step 4, and allow the enzyme bound to the support to react with the substrate.

Examples of enzyme substrates include the following.

Horseradish peroxidase (HRP) (1)
5-aminosalicylic acid-H2C.

pH during reaction: 5.6 Measurement method: Absorption photometry (A45 [1) (2) o-phenylenediamine-H,O.

pH during reaction + 4.0 Measurement method: Absorption photometry (A=e*) (3) Tyramine-H, O.

pH during reaction: 8 Measurement method: Fluorometry (λex 320nm; 18m
405 nm) (4) ABTS [2,2'-azinodi(3-ethylbenzthiazoline)-6'-sulfonic acid] -Htot reaction pH: 4.0 Measurement method: Absorption photometry (A-as, A 5711 )
Alkaline phosphatase (1) pH during p-ditrophenol phosphate reaction: 8.2 Measurement method: Spectrophotometry (A410) (2') Phenyl phosphate-4-aminoantipyrine reaction pH: 10.5 Measurement method : Absorption photometry (As..) Beta-galactosidase (1) pH during o-nitrophenol-β-D-galactoside reaction: 7.8 Measurement method: Absorption photometry (A41°) (2) 4-Methylumbeli pH during feryl-β-D-galactoside reaction: 7.8 Measurement method: Fluorometry (λex 330 nm; 18 m
453 nm: pH at the time of measurement 10, 3) Glucose oxidase Glucose Using glucose as a substrate, the generated H, 02 is made to emit light by luminol-perokindase (pH 8, 5),
Measurement by chemiluminescence method All of the above enzyme activity measurement methods can be performed according to known methods. Moreover, sodium nitride, sulfuric acid, etc. are used as a reaction terminator.

At the same time, the amount of anti-pre-s2 antibody contained in the test serum can be calculated from a calibration curve prepared using a standard anti-pre-82 antibody solution.

(It) Measuring method for anti-pre-3l antibody The basic procedure is the same as in (1) Measuring method for anti-pre-52 antibody above, except that the S protein used in step 1 is changed to M protein and used in step 2 and onwards. The M protein to be bound to the support is referred to as L protein.

(III) Measuring method for anti-pre-3 antibody (anti-pre-3l antibody and anti-pre-S2 antibody) The basic procedure is the same as in (1) above for measuring anti-pre-52 antibody, but it is used from step 2 onwards. The M protein to be bound to the support is referred to as L protein.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention should not be limited thereto.

[Example 1] Method for measuring human anti-pre-32 antibody l) Preparation of solid phase J, Biotechnology, 8. 1 (
20 μg/d of purified modified P31 (M-P3ic) particles described in
10mM Na,)-IPO** at a concentration of 10mM
After adding M NaCQro, 005% (w/v) thimerosal (pH 8,0) to a buffer solution, 100 μσ was dispensed into each well of a microtiter plate (Nunc 1 Maxisorp) and incubated at 4°C. I left it for the night. 3. Ifi, (140mM NaC (!, 0.05% (w
/v) Tween 20) l After washing three times with 50 μg, 120 μg of blocking buffer (2% (w)
/v) Casein, 10mM Na, HPO, /N
aH, PO, (pH 7,1), 0. '005% (w/v
) Thimerosal) was added and left at 4°C overnight.

2) Pre-treatment and purification of serum 200 ml of serum collected from a human vaccinated with a vaccine containing modified M protein (M-P31c) particles as an immunogen
μg was placed in an Eppendorf tube and immobilized at 56°C for 30 minutes. This operation breaks down complement and reduces nonspecific reactions. After centrifugation at 15°000 rpm for 5 minutes, 5 μg of the supernatant was taken and mixed with ■8-absorbed antigen solution (60 μg/d V8 protease-treated M-P31c particles, 0.1% (w/v)), which corresponds to the S protein solution. Tween 2
0, 25% (V/V) fetal bovine serum, I 4.
OmM NaCN, 10mM Na,)-I
P.O.

/NaHtPO- (pH 7,1), 0.005% (w/
v) In addition to OOμg of thimerosal, the mixture was left standing at 37°C for 1 hour or at 4°C for 17 hours.

3) After washing the same phase three times with 150 µg of the primary reaction washing solution, the mixed solution prepared in 2) was dispensed in duplicate at 45 µρ per serum. After sealing and sealing, it was shaken vigorously at room temperature for 1 hour.

By this operation, S protein (V8 protease treated M-
The anti-pre-32 antibody that was not adsorbed to the P31c particles is adsorbed to the solid phase.

4) After washing the solid phase 5 times with 300 μC of the secondary reaction washing solution, add the secondary antibody solution (1/20000 (v/v) HRP-labeled goat-derived anti-human 1 gG (70 Power Zuppel), 0.1% (y/v)
Tween 20.25% (v/v) fetal bovine serum, 140
mM NaCC, 10mM NatHPO4/NaH
! PO4 (PH 7,1), 0.005% (w/v) thimerosal>50tt (1 aliquot and shaken vigorously for 1 hour at room temperature).

5) After washing the solid phase 5 times with 300 μg of color development washing solution, drain the water well, and HRP substrate solution (24,3n+M citric acid).

514 mM Na, HPO4 (pH 5,0), 0
．． 02% (w/v) o (ortho)-phenylenediamine, 0.0013% (v/v E to J) was added and reacted for 5 minutes at room temperature in the dark.
o, to stop the reaction, and add A and H to Titert.
eck MultiskanMC (Flow La
boratories).

6) Creation of calibration curve Dilute HB globulin manufactured by Nippon Pharmaceutical Co., Ltd. (0.1% (w/
v) Tween 20. 25% (v/v) fetal bovine serum, 140mM NaCQ + 10mM 'Nat
HPO4/NaHzPO, (pH 7,1), 0.005
% (w/v) thimerosal) using J/8, l
A standard anti-pre-32 antibody solution was prepared by diluting it stepwise to 1/16, 1/32, and 1/64 (v/v). Add 5 μQ of the antibody solution to 100 μQ of the 8-absorbed antigen solution,
Color development was performed using the same procedure as for serum. The measured values thus obtained are shown in Table 1.

Q O, 103010, 1950
,092 20,2810,178 40,4530,350'') l Unit corresponds to the amount of anti-pre-32 antibody contained in 5 μQ of eye drop HB globulin diluted with 1764.

In addition, the calibration curve obtained from Table 1 is Y(△A4l1riX
(Unit/5 ・tl Q sample), then Y=
0.004 + 0.086X correlation coefficient 0.9
It becomes 999.

Also, using the above calibration curve, purified modified M protein (M-
Table 2 shows the values measured in human serum before and after vaccination using P31c) particles as an immunogen.

Table 2 Before and after vaccination using purified modified M protein (M-P31C) particles as an immunogen 9,97 1.31 3.38 6.40 7.62 1) Vaccination schedule is week 0 and week 4 The mice were inoculated on the 24th week, and blood was collected in a conventional manner on the 28th week.

7) Setting the cutoff value Serum from 5 healthy people (anti-HBs antibody (anti-S antibody) negative anti-p
A total of 12 sets of experiments were conducted, each consisting of 1 set of 16 measurements of re-32 antibody (negative) per person, 4 sets per day for 3 days. As a result, the standard deviation of the measured value obtained from the total average was O,l 80 Units15a (l sample.From this value, the cutoff value was 2.58 SD 1
It was determined that the specimen was 0.460 Units 151LQ corresponding to m. In other words, serum exceeding this value is judged to be positive.

[Example 2] Method for measuring chimpanzee anti-pre-32 antibody 1) Preparation of solid phase A solid phase was prepared in the same manner as 1) of [Example 1].

2) Except that the animals vaccinated with the serum pretreatment vaccine were chimpanzees.
Pretreatment of serum was performed in the same manner as in 2) of [Example 1].

3) Primary reaction The reaction was carried out in the same manner as in 3) of [Example 1].

4) Secondary reaction secondary antibody solution (1/1000G (v/v) HRP-labeled Protea A (Bio-Rad), O, 1% (w/v)
Tween 20.25% (v/v) fetal bovine serum, 140
1M NaCQ= 105M Na*HPO+/
NaH*PO4 (pH 7,1), 0.005% (w/v
) Thimerosal) [Example 1]
The reaction was carried out in the same manner as in 4).

5) Color development Color development was performed in the same manner as in 5) of [Example 1].

6) Measured values In the same way as [Example 1], a calibration curve was obtained using the standard anti-pre-32 antibody solution, and Y(ΔA4s,), X(U
nits”/5 μQ sample), then Y=0.08
3+0.022X, the correlation coefficient is 0.999.”
) Nippon Pharmaceutical Co., Ltd. HB globulin 5 diluted 1/64
The amount corresponds to the amount of anti-pre-32 antibody contained in μg, and the amount of antibody was calculated from the standard curve. The results are shown in Table 3.

Table 3 Purified modified M protein in chimpanzees (MP31
c) Vaccination using particles as an immunogen 6.21 21.83 52.79 49.23 (υn1Ls 15 μg sample) The vaccination schedule is to inoculate at 0, 4, and 8 weeks, and then at 122 weeks. Blood was drawn from the eye.

[Example 3] Method for measuring mouse anti-pre-3l antibody 1)
Preparation of solid phase Purified protein produced by genetic engineering (Japanese Patent Application No. 63-9533.5) was coated on a microtiter plate according to 1) of [Example 2].

2) Pretreatment of serum The serum of BΔL B/c mice immunized with purified purified protein particles was pretreated according to 2) of [Example 1], but instead of the v8 absorption antigen solution, MP31c absorption antigen solution was used. (60μg/m M-P31c particles, 0.1%
(w/v) Tween 20. 25% (v/v) beef tallow round surface FJ, 140mM NaCR, 10mM
NatHP○4/ Na H2PO, (pH7,'l
), 0.005% (w/v) thimerosal) were used.

3) Primary reaction [The reaction was carried out in the same manner as in Example 11-3).

4) Secondary reaction secondary antibody solution (1/1.0000 (v/v) HRP
Labeled antibody mouse IgG (H+L) antibody (Bio-Rad).

0.1% (iv/v) Tween 20. 25% (V
/V) Beef tallow round surface, blue, 140mM NaC
Q, 10n+M Na, HPO.

/NaH2PO4 (pH 7,1), 0.005% (W/
V) Thimerosal) [Example 1]
] The reaction was carried out in the same manner as in 4).

5) Color development Color development was performed in the same manner as in 5) of [Example 1].

6) Measurement of anti-p in mice before and after inoculation with purified LH protein particles
Table 4 shows changes in re-3t antibody titer.

Table 4 Mouse anti-pre-SA before and after vaccination with purified LII protein particles as immunogen
0.465 B O,539 CO,343 D 0.680 Control 0.349 2, OO0 2,000 1,185 2,000 0,359 (A,,,) ″) Vaccination schedule is as follows: 5 weeks later, blood was collected.

The M-P31c particles and L■ particles used in the examples were each 5accharonyces cerevi.
siae AH22R-/pGLD P31-RcT
[FERMB was submitted to the Fermentation Research Institute (IFO) as IFO10206 and to the Institute of Microbial Technology (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan, pursuant to the Budapest Treaty.
M protein and S protein obtained from deposited as P-1059
accharomyces cerevisiae
AH22R/pGLD LIIP39-RcT[I
The protein was deposited with FO as IFo 10426 and with FRI as FERM BP-1747 under the Budapest Treaty.

Claims (3)

[Claims] (1) Mix the test serum and hepatitis B virus surface antigen S protein produced from a unicellular organism, and mix the mixture with hepatitis B virus surface antigen M protein produced from a unicellular organism and bound to a support. Immunology of antibodies against the pre-S2 region of hepatitis B virus surface antigen in test serum, which comprises contacting and measuring antibodies against the pre-S2 region of hepatitis B virus surface antigen bound to a support. measurement method. (2) Test serum and hepatitis B virus surface antigen M protein produced from a unicellular organism are mixed, and the mixture is mixed with hepatitis B virus surface antigen L protein produced from a unicellular organism and bound to a support. Immunization of antibodies against the pre-S1 region of hepatitis B virus surface antigen in test serum, characterized by contacting with the pre-S1 region of hepatitis B virus surface antigen bound to a support and measuring antibodies against the pre-S1 region of hepatitis B virus surface antigen bound to a support. scientific measurement method. (3) Mix the test serum and hepatitis B virus surface antigen S protein produced from a unicellular organism, and mix the mixture with hepatitis B virus surface antigen L protein produced from a unicellular organism and bound to a support. Immunology of antibodies against the pre-S region of hepatitis B virus surface antigen in test serum, characterized by contacting and measuring antibodies against the pre-S region of hepatitis B virus surface antigen bound to a support. measurement method.