JPH0486558A - Method and reagent for detecting antibody - Google Patents

Abstract

PURPOSE:To enable clear detection of an antibody specific to component antigens by adding the antigens with mobility thereof close to one another and a small amount of antigens as such refined and concentrated in the course of an electrophoresis. CONSTITUTION:During an electrophoresis, antigens which are so close in electrophoretic mobility to one another or so limited in quantity to make it hard to detect corresponding antibodies among those as component antigens contained in mixed antigens are added to gel and the electrophoresis is continued. For example, the mixed antigens are HTLV-1 virus antigens and component antigens are HTLV-lenv antigens. Regarding a period of adding the component antigen, when added in the latter period of the electrophoresis the antigens added can be demarcated from other component antigens easily and clearly because a moving distance is limited with a shorter migrating time. The component antigens separated by the electrophoresis are transferred onto a carrier with a migration patter thereof being maintained as intact. The carrier (namely, reagent) is caused to react an antibody in a sample to be inspected (e.g. serum). Then, the antibody bonded specifically to the component antigens is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for detecting antibodies in a test sample and a reagent used in the method.

[Prior art] Western plot method (hereinafter sometimes referred to as WB method)
After fractionating antigen proteins by gel electrophoresis, the fractionated proteins are transferred onto a carrier such as a nitrocellulose membrane while maintaining the electrophoretic pattern, and then reacted with antibodies to transfer the fractionated proteins onto the carrier. This method detects antibodies bound to antigens and determines which antigens the antibodies in the test sample have specifically reacted to.As it is highly sensitive, it is useful for serological diagnosis of infectious diseases and for determining the specificity of the antibodies produced. This is a widely used technique for determination or identification of products produced by genetic engineering.

For example, the WB method has also been used to examine whether a patient is infected with acquired immunodeficiency syndrome virus (HIV) or human adult T-cell leukemia type 2 virus (HTLV-1+).

In these methods, first, a virus is partially decomposed using an ionic or nonionic surfactant, and then subjected to gel electrophoresis as an antigen. Since viruses contain multiple proteins such as core protein and envelope protein, the above antigens are mixed antigens, and each antigen is fractionated by electrophoresis based on its molecular weight and charge. Next, the antigen is transferred onto a carrier such as a nitrocellulose filter while maintaining the electrophoretic pattern of the antigen, and this is reacted with the antibody in the test sample to detect the antibody bound to the antigen, thereby identifying the antigen. Examine the presence or absence of antibodies that bind to the antigen. It is important to know which component antigen of the virus the antibody that binds to is present in the sample in order to eliminate false positives and make an accurate diagnosis.

Conventional WB methods like this are good when mixed antigens are clearly separated by electrophoresis, but when the mobilities of two or more antigens are close to each other, the electrophoretic bands tend to overlap. In such cases, it is difficult to determine which antigen the antibody binds to. The same applies when the amount of a certain component antigen in a mixed antigen is smaller than that of other antigens. For example, gp46, one of the envelope (envl) proteins, is an important diagnostic antigen in diagnosing whether or not you are infected with the HTLV-I virus, but its electrophoretic mobility makes it a core protein. P
Because the mobility is close to that of P53, it cannot be clearly separated from P53 depending on electrical interlocking. Therefore, when examining HTLV-I virus infection using the conventional WB method, it is difficult to clearly determine the presence or absence of anti-gp46 antibodies, and furthermore, it is difficult to determine whether or not you are infected with HTL-I virus. It has become.

[Problems to be Solved by the Invention, Tun] Therefore, the purpose of the present invention is to solve the problem when the electrophoretic mobilities of two or more component antigens in a mixed antigen are close to each other, or when the electrophoretic mobility of two or more component antigens in a mixed antigen is Even when the amount of antigen is small compared to other antigens, it is possible to clearly determine whether antibodies specific to such component antigens are present in the test sample. An object of the present invention is to provide a detection method and a reagent for the same.

[Means for Solving the Problems] As a result of intensive research, the present inventors have discovered that antigens whose electrophoretic mobility is close to those of other component antigens (hereinafter sometimes referred to as mobility-proximal antigens) ) and small amounts of antigens (hereinafter sometimes referred to as small amount antigens) are purified and concentrated.
The present invention was completed by discovering that the above-mentioned problems could be solved by adding electricity during the water movement.

That is, the present invention comprises a step of subjecting a mixed antigen to gel electrophoresis, a step of adding a component antigen, which is a component of the mixed antigen, to the gel and continuing electrophoresis, and a step of performing electrophoresis while maintaining the electrophoresis pattern. a step of transferring the separated antigen onto a carrier, a step of reacting the antigen transferred onto the carrier with an antibody against the mixed antigen contained in the test sample, and a step of detecting the antibody bound to the antigen. Provided is a method for detecting an antibody in a test sample, the method comprising:

Furthermore, in the present invention, a mixed antigen is subjected to gel electrophoresis, and then a component antigen, which is a component in the mixed antigen, is added to the gel, and the electrophoresis is continued, and the antigen is separated by electrophoresis while maintaining the electrophoretic pattern. The present invention provides a reagent for detecting antibodies against the mixed antigen in a test sample, which is composed of an antigen transferred to a carrier.

[Effects of the Invention] According to the present invention, when the electrophoretic mobilities of two or more component antigens in a mixed antigen are close to each other, or when the amount of at least one component antigen in the mixed antigen is smaller than that of other antigens, Provided is a method for detecting antibodies and a reagent for the same, which makes it possible to clearly determine whether or not antibodies specific to such component antigens exist in a test sample, even if the antibodies are small. .

According to the method of the present invention, purified mobility proximal antigens and small amounts of antigens are separately added to the gel during electrophoresis, so antigen bands are formed at positions different from the original bands, so other antigens are Since the purified and concentrated component antigen is added separately, a clear band can be formed even with a small amount of antigen.

Therefore, according to the method of the present invention, it is possible to clearly detect antibodies specific to antigens with close mobility or antigens with low abundance.

[Detailed Description of the Invention] The mixed antigen to be subjected to the method of the present invention may be any antigen containing multiple component antigens (for example, HI
Viruses such as V and HTLV-1 can be treated with anionic detergents (e.g. sodium dodecyl sulfate (SDS)) or nonionic detergents (e.g. Nonidet P-40).
(trade name) and Triton X-100 (trade name)) in a conventional manner.

Gel electrophoresis of mixed antigens can be performed in a manner similar to the conventional WB method. For example, based on the Laemmli method, electrophoresis in a polyacrylamide gel after treatment with SDS (5DSPAGE) is performed. can be done.

Next, during electrophoresis, component antigens contained in the mixed antigen that have electrophoretic mobility close to that of the component antigen to be used (i.e., antigens with similar mobility) or because the amount of antigen is small are detected. An antigen that makes it difficult to detect the corresponding antibody (ie, the low-abundance antigen described above) is added to the gel, and electrophoresis is continued. These component antigens are antigens that almost overlap with bands of other antigens or are so small in amount that detection of their corresponding antibodies may be difficult or inaccurate using conventional WB methods. The timing of adding these component antigens is not particularly limited, but conditions are selected so that the bands of the component antigens do not overlap after electrophoresis. Such conditions can be easily selected through routine preliminary experiments, but usually, if added at the final stage of electrophoresis, the added component antigen will migrate less distance due to short body movement time. It is often easily and clearly separated from other component antigens. Although not necessarily required, it is preferable that the component antigen added be purified. However, it is also possible to add a mixture of two or more types of component antigens, as long as they can be clearly fractionated by electrophoresis after addition of the component antigens. In the examples described below, HTLV-1
In the analysis of antibodies against the virus, the env antigen of the HTLV-1 virus is added.

In this way, if a mobility proximate antigen or a small amount of antigen is added to the gel during electrophoresis and electrophoresis is continued, the added component antigen will remain electrophoresed from the beginning during the subsequent electrophoresis. Since it is shorter than other component antigens, it migrates a short distance, and therefore, after electrophoresis, a band is formed at a different position from its original position. Therefore, even if the original band almost overlaps with other bands, a clear band can be formed at a completely different position, and the corresponding antibody can be clearly detected.

In addition, even in the case of a small amount of antigen, since the antigen is added separately later, a sufficient amount of antigen can be added for detection of the corresponding antigen, and therefore, the detection of the corresponding antibody can also be carried out clearly. can.

Next, the component antigens separated by electrophoresis are transferred onto a carrier while maintaining the electrophoretic migration pattern described above. This step can be performed in exactly the same way as the normal WB method. That is, the fractionated component antigens are transferred onto a conventionally commonly used carrier such as a nitrocellulose filter or a nylon membrane filter, and the component antigens in the gel are electrohydrodynamically transferred onto the carrier according to a conventional method. This can be done by Note that the material transferred onto the carrier in this manner constitutes the reagent of the present invention.

Next, the carrier onto which the antigen has been transferred is reacted with the antibody in the test sample. Samples to be tested include, for example, body fluids such as serum when investigating infection with a virus, etc. In addition, when examining the corresponding antigen of an antibody produced by a hybridoma, or using genetically engineered animal cells, etc. In cases where the protein produced is to be identified, culture supernatants or cell lysates of hybridomas, animal cells, etc. can be used. However, the test sample is not limited to these (it may be any sample in which antibodies against component antigens are detected).The reaction between the antigen and antibody on the carrier is exactly the same as in the conventional WB method. It can be done by

Antibodies that specifically bind to the component antigens are then detected.

This can also be carried out in exactly the same manner as the conventional WB method. For example, after washing away antibodies non-specifically adsorbed to the carrier, a second antibody labeled with an enzyme such as biotin or peroxidase is reacted. and can be detected by the usual biotin-avidin method or enzyme immunoassay method.

The method of the present invention is useful not only for diagnosing infectious diseases such as viruses such as HTLII and HIV, but also for investigating the corresponding antigen of antibodies produced by hybridomas, and for investigating proteins produced by genetically engineered animal cells. It can also be applied to cases such as identification, and is a basic technique with an extremely wide range of applications.

Hereinafter, the present invention will be explained in more detail based on Examples.

However, the present invention is not limited to the following examples.

[Example 1] Preparation of 11 HTLV-I viral antigen TCL-Kan cells (IJ), an HTLV-1 producing cell line.

Kannagi et al., J. Immunol.
130 f6) 2942-2946f1983+
RPMI-16 containing 10% fetal bovine serum
40 medium and incubated at 37°C in air containing 5% carbon dioxide.
It was cultured in This culture solution was centrifuged using a continuous rotor to remove cells and obtain a supernatant. Viruses were recovered from this supernatant by isopycnic gradient ultracentrifugation using a continuous rotor. Add to this a nonionic surfactant (final concentration of 05%).
Nonidet P-40 (trade name) was added to obtain the HTLV-I virus antigen.

(2) HTL by affinity chromatography
Purification of V-1 env antigen Mice were immunized with the HTLV-I virus antigen obtained in the above il+ procedure, mouse tile cells and myeloma cells were fused according to the conventional method of Köhler and Milstein, and HTLV-T
Hybridomas that produce antibodies specific to the env antigen are selected and cloned, and the hybridomas are inoculated intraperitoneally into mice and subjected to ammonium sulfate fractionation and DEAE from mouse ascites.
anti-HTLV-I by ion-exchange chromatography method.
A monoclonal antibody against the env antigen was obtained. This monoclonal antibody reacts with HTLV-1 infected cells, but
that it does not react with uninfected cells, and that the molecular weights of the corresponding antigens are 68,000 and 46,000 Daltons;
In addition, genetically engineered or combinant HTLV
-It has been confirmed that it strongly reacts with the env protein.

This monoclonal antibody was transferred to Sepharose 4B gel (manufactured by Pharmacia) activated with cyanogen bromide in a conventional manner.
An affinity column was prepared by binding at a ratio of mg/ml gel. pH 7, containing 0.5M sodium chloride
The culture supernatant was poured onto a column equilibrated with 5ml of 1J Tris-HCl buffer to allow sufficient binding of HTLV-Ienv, and then washed with 5mM Tris-HCl buffer containing 0.5M sodium chloride. After unbound components are no longer observed, 0.1M lactate buffer at pl+2.5 is poured, bound components are collected, and immediately added to 0.1M phosphate buffer M (pH 75) containing 0.15M sodium chloride. Dialysis was performed against Purified HTLV-I env from the obtained binding component
I got the antigen.

(3) Preparation of antigen transfer nitrocellulose membrane (NC membrane)
- Gel concentration 125% (
5DS-polyacrylamide gel electrophoresis (5O5-PAGE) was performed using 1 cm of the concentrating gel and 5.5 cm of the separating gel until the leading edge of the electrophoresis reached 2/3 of the top (about 2 hours). Then, HTLV-1en purified in (2)
v antigen was added, and electrophoresis was continued until the top of the electrophoresis reached 1 cm from the bottom (1 hour). After the electrophoresis, the polyacrylamide gel was prepared using the method of Tobin et al.
44612), and then blocked in 3% bovine serum albumin at 4°C overnight to obtain an HTLV-I antigen-transferred NC membrane (hereinafter referred to as the NC membrane of the present invention). . On the other hand, for control, purified HTLV
An NC membrane (hereinafter referred to as conventional NC membrane) was prepared in the same manner as above except that the 4env antigen was not added.

(4) Measurement of HTLV-I antibodies in serum A comparison between the NC membrane of the present invention prepared in (3) above and the conventional NC membrane was performed using HTLV-I antibodies.
HTLV-I using LV-I antibody positive and negative sera.
I antibody was measured. For measurement, each serum diluted 30 times was used, reacted for 1 hour at room temperature, then reacted with biotin-labeled anti-human antibody for 1 hour, further reacted with peroxidase-labeled avidin for 1 hour, and developed color using 4-chloro-1-naphthol. I let him do it. 0.05% Twee in cleaning solution
0.01 M PBS containing n-20 (pH 7,4)
It was used. The results are shown in Figure 1.

As is clear from FIG. 1, when analysis was performed using the NC membrane of the present invention, gp4
A band corresponding to 6 antigens was clearly observed at a position corresponding to the upper end of the position with low mobility. On the other hand, when using the conventional NC membrane, the band corresponding to gp46 antigen almost overlaps with the band corresponding to p53 antigen in the analysis of positive serum, and it is difficult to determine whether anti-gp46 antibodies are present in the serum. could not be determined clearly.

[Brief explanation of drawings]

Figure 1 shows the results of HT using the inventive NC film and the conventional NC film.
FIG. 2 is a diagram showing the results of antibody detection using LV-I antibody-positive serum. Patent applicant Fujirebio Co., Ltd.

Claims (4)

[Claims] (1) A step in which the mixed antigen is subjected to gel electrophoresis, a step in which the component antigens in the mixed antigen are added to the gel, and further electrophoresis is continued, and the antigen is separated by electrophoresis while maintaining the electrophoretic pattern. a step of transferring the antigen transferred onto the carrier onto a carrier; a step of reacting the antigen transferred onto the carrier with an antibody against the mixed antigen contained in a test sample; and a step of detecting the antibody bound to the antigen. , a method for detecting antibodies in a test sample. (2) The mixed antigen is an HTLV-I virus antigen, and the component antigen added to the gel is an HTLV-Ienv antigen.
The method according to claim 1, wherein the antigen is an antigen. (3) Subject the mixed antigen to gel electrophoresis, then add the component antigens in the mixed antigen to the above gel, continue electrophoresis, and use the electrophoretically separated antigen as a carrier while maintaining the electrophoretic pattern. Consisting of transcribed
A reagent for detecting antibodies against the mixed antigen in a test sample. (4) The mixed antigen is an HTLV-I virus antigen, and the component antigen added to the gel is an HTLV-Ienv antigen.
The reagent according to claim 3, which is an antigen.