JPH05142227A - Detecting method - Google Patents

Abstract

PURPOSE:To establish the method for screening more antibodies simply and efficiently by performing the reaction of antigens and antibodies in a liquid phase, and detecting the formed immune complex or the reaction material by using label material. CONSTITUTION:Antibody-producing cell culture medium, fixed-phase antibodies for recognizing the antibodies produced with cells and labeled antigens, which are recognized with the antibodies produced with the cell, are made to react. The fixed phase and the liquid phase are separated, and the labels of the fixed phase and the liquid phase are measured. Namely, the cells are selected by measuring the antibodies, which are secreted from the antibody-producing cells into the culture medium. As the antibody-producing cells, e.g. the culture medium of hybridoma is used, and the hybridoma, which is producing monoclonal antibodies can be selected. As the fixed-phase antibodies, e.g. anti-IgG, anti-IgM and the like can be listed. The fixed-phase material can be the material, which adsorbs protein, such as polyethylene and polycarbonate. The label material can be the material which can detect enzyme, fluorescernce material and the like.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting antibody-producing cells by carrying out an immune reaction using a labeled antigen and a solid-phased antibody.

[0002]

2. Description of the Related Art Antigen measurement using an antibody is an essential method in the field of clinical examination today. 197
5 years G. Keller and C.I. Milstein (Natur
e, 256 , 495, (1975)), the establishment of cell fusion technology has enabled the production of monoclonal antibodies against specific antigens. That is, an animal such as a mouse is sensitized with a specific antigen as an immunogen to activate and increase antibody-producing B cells. The antibody-producing B cells and myeloma cells (myelo-
Cells are fused with polyethylene glycol to establish antibody-producing hybridomas. From these hybridomas, it became possible to obtain the required amount of the monoclonal antibody when required.

The assay system is the most important factor in obtaining excellent monoclonal antibodies. For that purpose, it is important to find and clone antibody-positive wells early after cell fusion. The assay method for finding antibody-positive wells is roughly divided into a liquid phase method and a solid phase method, but recently, the solid phase method is generally used.

The solid phase method is Engvall and Perlman.
Reported in 1971 by Immun (Immun
Chemistry, 8 , 871). In this assay method, the antigen is first adsorbed on a plate, a cell culture medium containing the antibody is added thereto, and a secondary antibody (antibody against the target antibody) labeled with an enzyme or a radioisotope is first used. Is a method of detecting the antibody of. The disadvantage of this method is that the antigen is adsorbed on the solid phase, so that the structure of the antigen is not in a natural state but in a denatured state.

That is, the antibody to be detected is not strictly against the antigen in the natural state, and the orientation of the antigens adsorbed on the solid phase is in the same direction. Therefore, the antibody obtained from the antigen recognizes the antigen in the same direction. It is pointed out that it has only parts. Moreover, it is possible that the antibody obtained by the solid phase method does not bind to the antigen in the assay in solution (Milleret.
al. , 59 , 277, J. Am. Immunol. Met
h. (1983)). In addition, in the solid phase method, nonspecific adsorption of the secondary antibody cannot be avoided, and as a result, there is a high risk of selecting nonspecific antibody-producing cells. Anti mouse I
The frequency is particularly high in the secondary antibody against gG. In addition, some antigens may not be adsorbed to the solid phase and may not be able to efficiently detect antibody-producing cells.

A system often used in the liquid phase method is a radioimmunoassay, which is a method of detecting an antibody in a cell culture medium by using a radiolabeled antigen. When using radioactive materials, it is necessary to install a radioactive material usage area and special equipment, and it is not always possible to use it as a general law in terms of safety and ease of management. Furthermore, the experiment is complicated and the burden on the parties is large.

[0007]

[0005] Today's clinical tests naturally use antibodies. However, increasing the sensitivity has often been given up as a property of an antibody if it is very difficult. Particularly in the sandwich enzyme immunoassay, it is desirable that the antigen recognition sites of the two types of antibodies are different and that their positions are separated in the molecule. An object of the present invention is to provide a method for conveniently and efficiently screening a large number of antibodies.

[0008]

Means for Solving the Problems The inventors of the present invention have made diligent studies on the above-mentioned problems. As a result, an antigen-antibody reaction is carried out in a liquid phase and the formed immune complex or unreacted substance is detected by a labeling substance. The inventors have found that these problems can be solved and completed the present invention. That is, the present invention comprises: a. Antibody-producing cell culture medium, immobilized antibody that recognizes the antibody produced by the cells,
And a labeled antigen recognized by an antibody produced by the cells, b. Separating the solid and liquid phases, c. A method for detecting antibody-producing cells, which comprises measuring a solid-phase or liquid-phase label. Hereinafter, the present invention will be described in detail.

The present invention is to select antibody-producing cells by secreting the antibody secreted into the culture medium by immunoreaction to select the cells. The antibody-producing cells at this time are not particularly limited, but a hybridoma producing a monoclonal antibody can be selected by using, for example, a culture medium of the hybridoma. The culture medium used for the measurement may be pretreated, if necessary, so that the conditions are suitable for the immune reaction.

The immobilized antibody that recognizes the antibody produced by the cells is not limited as long as it is an antibody against the antibody produced by the cells. For example, anti-IgG, anti-IgM
Etc. The material of the solid phase of the immobilized antibody is not particularly limited as long as it is a material capable of adsorbing proteins such as polystyrene, polycarbonate, nylon, glass, filter paper, cepharose, and ferrite resin.

The present invention also uses a labeled antigen recognized by the antibody produced by the cells. The labeling substance at this time is not limited as long as it is a labeling substance such as an enzyme or a fluorescent substance that can be detected. Examples of the enzyme include peroxidase, β-D-galactosidase, alkaline phosphatase, urease, catalase, β-glucuronidase and the like, and examples of the fluorescent substance include fluorescamine, fluorescein cyanate, Examples thereof include tetralo-damine isothiocyanate.

The method for binding the label and the antigen is not particularly limited, and a usual method can be used. Further, an avidin-biotin bond may be interposed between the label and the antigen. In particular, the labeled antigen due to the bond of antigen-biotin-avidin-labeled substance is less likely to be sterically hindered by the labeling substance or the three-dimensional structure of the antigen is changed, and the antigen has a structure closer to its natural state. Become. Therefore, by using such an antigen, an antibody having more orientation can be selected.

The antibody-producing cell culture medium as described above, a solid-phased antibody that recognizes the antibody produced by the cells, and a labeled antigen recognized by the antibody produced by the cells are reacted. The order of the reactions at this time is not particularly limited, and all may be reacted at the same time, or may be reacted sequentially.

After the reaction, the solid phase and the liquid phase are separated, and the labeling of the solid phase or the liquid phase is measured. The method for separation and the method for measuring the label are not particularly limited, and a commonly used method may be used. Then, antibody-producing cells may be selected from the measurement results of the label.

[0015]

According to the present invention, it is possible to obtain a large number of cells secreting the antibody. It is intended to increase the specificity of antibodies used in EIA and RIA as very effective means in today's clinical tests, and to easily solve many demands for increasing or increasing the measurement sensitivity, Its application range is wide. The acquisition of more types of antibodies responds to clinical demands from their application in immunoassays. Further, since it is a liquid phase method, there is little risk of denaturing the antigen, and it is possible to efficiently obtain an antibody having many orientations with respect to the antigen.

[0016]

EXAMPLES Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 (1) Biotin binding of human myoglobin 1 mg / mL human myoglobin (manufactured by Scripps)
1 mL was dialyzed against 0.1 M carbonate buffer (pH 8.0) for 4 hours. After dialysis, 1mg / with dimethyl sulfoxide
100 μL of biotinyl-N-hydroxysuccinimide ester dissolved in mL was added and reacted for 6 hours. Phosphate buffered saline (0.01% phosphate buffer containing 0.85% NaCl, pH 7.4: PBS below) was used as the reaction product.
After dialysis for 5 hours, the target biotinylated human myoglobin was obtained. (2) Detection of antibody by liquid phase method Affinity-purified anti-mouse IgG polyclonal antibody (manufactured by TAGO) and anti-mouse IgM polyclonal antibody (manufactured by Cappel) were diluted 2000 times with PBS, and diluted to 9 times.
6-well plate (Manufactured by Nunc, Maxi Sorp)
100 μL / well was added to each of the above samples) and immobilized. The immobilization time was 1.5 hours at 37 ° C. Next 0.04%
Phosphate buffered saline containing Tween 20 (0.8
0.01% phosphate buffer containing 5% NaCl, pH 7.
4: Washed 3 times with PBS tween hereinafter, and blocked with PBS in which 0.2% bovine serum albumin was dissolved. 300 μL / well each was performed at 37 ° C. for 1.5 hours.

After washing, hybridoma culture medium No. 1 in which antibody-producing cells of a mouse sensitized with human myoglobin and myeloma cells were cell-fused. 1-17 was added at 50 μL / well. Simultaneously, biotinylated human myoglobin diluted 2000 times with PBS containing 0.5% bovine serum albumin was dispensed at 50 μL / well and reacted at 37 ° C. for 1.5 hours. After removing the solution and washing three times with PBStween, 50 μL / well of streptavidinylated horseradish peroxidase (manufactured by Bethesda Research) was added. React at 37 ° C for 1.5 hours, remove the solution, and remove P
Washed 3 times with BStween.

0.3 mg / mL 2,2-azinodi-
A substrate solution consisting of (3-ethylbenzthiazoline sulfate) -diammonium salt and a 0.1 M citrate buffer solution (pH 4.1) containing 0.01% hydrogen peroxide was added to each well in an amount of 100 μL and allowed to stand at room temperature for 5 minutes. After enzymatic reaction for a minute,
The enzyme reaction was stopped by adding 100 μL of 100 mM oxalic acid solution. For each well of the above microtiter plate, the absorbance at a wavelength of 415 nm and a control wavelength of 492 nm was measured by an automatic microtiter plate reader (manufactured by Toso Co., Ltd., MPR-A4, trade name). The results are shown in Figure 1.

Comparative Example 1 Detection of Antibody by Solid Phase Method Human myoglobin was added at a concentration of 2 μg / mL.
After diluting with BS, 100 μL / well was added to a MaxiSorp plate (manufactured by Nunc) for immobilization. The immobilization time was 1.5 hours at 37 ° C. Next, PBSt
The cells were washed 3 times with ween and blocked with PBS containing 0.2% bovine serum albumin at 300 μL / well. Incubate at 37 ° C for 1.5 hours and then wash,
100 μL of the above hybridoma culture solution was added to each well.
/ Well was added. The reaction was carried out at 37 ° C for 1.5 hours, and the solution was removed.

After washing 3 times with PBS, horseradish peroxidase-goat anti-mouse IgG antibody was added to PBS 5 times.
It was diluted 1,000 times and dispensed at 100 μL / well. 37
After incubating for 1.5 hours at 0.degree. C., 0.1 mg citric acid containing 0.3 mg / mL 2,2-azinodi- (3-ethylbenzthiazoline sulfate) -diammonium salt and 0.01% hydrogen peroxide. A substrate solution consisting of an acid buffer solution (pH 4.1) was added to each well in an amount of 100 μL, and the enzyme reaction was carried out at room temperature for 5 minutes.
μL was added to stop the enzyme reaction. The above microtiter
For each well of the plate, the absorbance at a wavelength of 415 nm and a control wavelength of 492 nm was measured using an automatic microtiter plate reader (manufactured by Toso Co., Ltd., MPR-A4, trade name).
It was measured at. The results are shown in Figure 1.

Comparative Example 2 Determination of Non-Specific Adsorption The same experiment as in Comparative Example 1 was carried out by using a plate having a solid phase which had been subjected to a blocking operation without adsorbing human myoglobin. The non-specific adsorption of the secondary antibody was examined. In other words, the wells that were observed to be positive in the system in the absence of antigen were not the cause of the antibody against the target antigen, but the color developed due to the nonspecific adsorption of the secondary antibody. is there.

From the results shown in FIG. 1, culture solution No. 14 was determined to be negative by the solid phase method, but positive by the liquid phase method of the present invention, and hybridomas not obtained by the solid phase method were obtained. I was able to. Further, as shown by culture solutions Nos. 1, 4, 13, and 15, the liquid phase method of the present invention had a significantly lower false positive rate due to nonspecific adsorption than the solid phase method. Furthermore, since the culture solutions Nos. 6 and 17 were both determined to be positive by the solid phase method and the liquid phase method of the present invention and were not nonspecific adsorption by the secondary antibody, the method of the present invention produced true antibodies. You can pick up all the existing cells.

[Brief description of drawings]

FIG. 1 is a diagram showing the absorbance of a color reaction at 415 nm of a culture solution No. 1-17 of a hybridoma used in Example 1 and Comparative Examples 1 and 2.

Claims (3)

[Claims] 1. A. Antibody-producing cell culture medium, immobilized antibody that recognizes the antibody produced by the cells,
And a labeled antigen recognized by an antibody produced by the cells, b. Separating the solid and liquid phases, c. A method for detecting antibody-producing cells, which comprises measuring a solid-phase or liquid-phase label. 2. The method according to claim 1, wherein the label is an enzyme or a fluorescent substance. 3. The method according to claim 1, wherein the label and the antigen are mediated by an avidin-biotin bond.