JPS6146784B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for measuring antigens or antibodies by enzyme immunoassay.

Using the substance to be measured, i.e., low-molecular and high-molecular antigenic active substances (including substances that can be haptens), experimental animals such as domestic rabbits are made to produce antibodies that bind to the antigenic active substances, and the antigenic active substances and antibodies A method for measuring antigens or antibodies using a specific reaction between them is known as an immunoassay method.

Substances that can be measured here include various polypeptide hormones, steroid hormones, vitamin B ₁₂ , folic acid, thyroxine, triiodothyronine, complement, α-fetoprotein, carcinoemprionitric antigen, and organ and various enzymes and proteins in the blood, various microbial antigens such as HBs antigen, plant hormones, antibiotics, etc. Particularly important measurement targets are pituitary hormones, HBs antigen, insulin, immunoglobulin, and liver enzymes.

Immunological assays conventionally used to measure these trace substances include precipitation reactions, agglutination reactions, and complement fixation reactions, which have been put to practical use. However, these methods visually detect the end of the reaction, and generally have low sensitivity or are semi-quantitative. To measure these hormones and various biologically active substances that exist only in trace amounts, it is desirable to establish highly sensitive and quantitative methods. ) have been developed and used.

However, this RIA method has various major drawbacks due to the use of radioactive isotopes, such as the risk of injury to the human body due to radiation, the need for special facilities to prevent the spread of radiation, the complicated operation, and the long time required for measurement. In the case of isotopes with short half-lives, labeled antigens cannot be stored for long periods of time, etc., and a major obstacle to their introduction into clinical testing is that they cannot be tested in regular clinical laboratories. It's summery. For this reason, there was a need for a more common method for clinical testing and a method that would not lead to environmental pollution.

In order to solve these drawbacks of RIA method, enzyme immunoassay method (hereinafter referred to as EIA method) was developed.
Its characteristics, reaction mechanism, and application examples have already been announced. (“Enzyme immunoassay”, edited by Eiji Ishikawa et al., Igaku Shoin, 1978, hereinafter referred to as reference (1))
The EIA method is labeled with an enzyme, whereas the RIA method is labeled with a radioisotope.
I don't see any shortcomings in the law.

However, the solid phase supports conventionally used in the EIA method are known to be inorganic materials such as polysaccharides such as cellulose and Cephalose, glass, and silicone rubber. It is often used with

In these cases, it may be difficult to remove free antigens, antibodies, enzyme-antibody conjugates, etc., or antibodies may not adhere or react sufficiently due to defects in the material of the carrier, or conversely, free antigens, antibodies, enzyme-antibody conjugates, etc. There are various problems such as non-specific adsorption of various components in serum and urine and interference with antigen-antibody reactions, and non-specific attachment of enzyme-antibody conjugates to the carrier itself.
Due to these factors, the blank value becomes abnormally high, the measurable region becomes extremely narrow, and the quantitative performance of the target object is particularly poor in the low concentration region. For this reason, glass other than those made by aminoalkylsilylation are not put into practical use.

Therefore, resin molded products made of various synthetic polymers have recently begun to be used as solid phase supports, but resin supports made of these synthetic polymers generally do not have active reactive groups on their surfaces, so they cannot be used for antibodies or Physical adsorption is used to immobilize the antigen.
Therefore, during washing during the measurement process, the immobilized antibody or antigen may be released, making accurate measurement impossible, or the antigen-binding region (Fab variable region) of the immobilized antibody may be released. may be sterically hindered.

The inventors aimed to improve the shortcomings of these EIA solid-phase carriers and develop useful EIA test reagents with a sensitivity equivalent to or higher than that of the RIA method. As a result of research and repeated experiments on the adsorption mechanism, a method was previously discovered that uses a carrier surface aminated using a specific reagent and then chemically bonded with antibodies or antigens to form a solid phase. We have discovered that this method is significantly superior to existing methods, and have completed the present invention.

That is, the gist of the present invention is to provide a resin into which active amino groups have been introduced by reacting an antibody or antigen corresponding to the antigen or antibody to be measured with ethyleneimine in a method for measuring an antigen or antibody by enzyme immunoassay. The present invention relates to a method for measuring an antigen or antibody, which is characterized in that the antigen or antibody is immobilized on a carrier having a surface by chemical bonding.

In the present invention, a carrier having a resin surface is used, and examples of the carrier include methyl acrylate,
A carrier formed from a homopolymer of monomers such as methyl methacrylate, acrylic acid, methacrylic acid, acrylonitrile, maleic anhydride, or a copolymer containing these monomers can be used, such as polymethyl methacrylate, Acrylonitrile-butadiene-styrene copolymer resin, styrene-
Preferably, a maleic anhydride copolymer or the like molded into an appropriate shape such as a sphere or a cylinder by a molding method such as an injection molding method can be used. In addition, active amino groups are introduced into the above-mentioned carrier. To introduce amino groups, the carrier is immersed in an aqueous solution containing hydrochloric acid or caustic soda, and left at 40 to 80°C for several hours until the surface of the carrier is absorbed. This can be carried out by hydrolyzing the carrier, then transferring the carrier to water containing a few percent of acetone, and adding ethyleneimine dropwise to cause a reaction.

As for the carrier for EIA, it is preferable that the amount of functional groups introduced per carrier is 10 ⁴ to 10 ⁶ positions.

In this way, a carrier having a resin surface into which active amino groups have been introduced can be prepared.

Next, in the present invention, an antibody or antigen corresponding to the antigen or antibody to be measured is immobilized by chemical bonding onto the carrier prepared above into which active amino groups have been introduced. As for antibodies for immobilization, monoclonal antibody-secreting hybridomas (hybridoma) are created by fusion of antibody-producing cells and mouse myeloma cells using the method introduced by Kohler and Milstein, and the antibodies secreted by these hybridomas are used. good. The mouse myeloma cell line name is P ₃ /
NS1/1-Ag4-1 (abbreviated as NS-1), SP2/0
-Ag14 (abbreviated as SP-2), and splenocytes of BALB/C mice immunized with Freund's complete adjuvant can be used as antibody-producing cells. Furthermore, as the cell fusion method, the method shown in Immunology Experimental Procedures, 2221 ('78) (edited by Takeshi Watanabe et al., Japanese Society of Immunology) can be adopted. Furthermore, as antibodies for immobilization, in addition to the antibodies themselves produced by hybridomas, F(ab') ₂ fragments obtained by decomposing antibodies with pepsin, and F(ab') ₂ fragments such as 2-mercaptoethanol, etc. Any Fab′ fragment obtained by treatment with a reducing agent can be used.

Further, on the carrier into which the active amino group is introduced,
To immobilize the above antibody or antigen for immobilization by chemical bonding, a commonly used binding agent is used. As the binding agent, glutaraldehyde, which is commonly used in immunohistochemistry, is preferably used. The reaction can crosslink the carrier and the IgG molecules of the antibody.

In addition, when a thiol group is introduced into an antibody, it is also possible to use maleimide as a binding agent to perform the bonding by crosslinking the thiol group and the amino group, which is particularly effective when the antibody is Fab'. .
As the maleimide, N-hydroxysucciniside ester of 4-(maleimidomethyl)-cyclohexane-1-carboxylic acid and metamaleimidobenzoyl-N-hydroxy-succiniside ester are suitable. The immobilized antibody thus obtained was subjected to an EIA test using either the competitive binding method or the sandwich method described in reference (1).
It can also be applied to law. Conversely, when immobilizing antigens, hybridoma-produced antibodies can be immobilized on Sepharose413.
An antigen purified by affinity chromatography and immobilized on a gel such as the above can be immobilized on a carrier in the same manner as described above.

In the present invention, a carrier having a resin surface into which active amino groups have been introduced, as described above, is immobilized by bonding antibodies or antigens through chemical bonds, and is used for measurement by enzyme immunoassay. When washing the carrier after reacting with an antigen or antibody, or after reacting with an enzyme-labeled antibody, it is possible to use a surfactant with strong detergency. Interference by various components inside can be prevented. As a result, it becomes possible to measure lower concentration regions.

Furthermore, when measuring antigens or antibodies by the EIA method, a labeling enzyme is used, and in the present invention, alkaline phosphatase, alkaline phosphatase,
It is preferable to use catalase, peroxidase, β-glucuronidase, β-D-darcosidase, β-D-galactosidase, urease, glucose oxidase, and galactose oxidase.
-D-galactosidase is preferably used.

The measurement method of the present invention is as described above,
An antibody or antigen corresponding to the antigen or antibody to be measured is immobilized on a carrier by chemical bonding,
Since measurements are carried out using the EIA method using the carrier in this state, it naturally does not have the drawbacks of the RIA method due to the use of radioactive isotopes;
Drawbacks in the method, i.e. free antigens, antibodies, enzymes -
It may be difficult to remove antibody-conjugated substances, etc., or the antibody may not adhere or react sufficiently due to defects in the material of the carrier, resulting in non-specific binding of the antigen to the surface of the immobilized antibody (carrier). , it was discovered that non-specific adsorption between enzymes and labeled antibodies caused abnormally high blank values and poor quantitative performance of target substances, especially in the low concentration range. Tests can be easily and accurately performed in testing rooms and testing centers using measuring instruments and devices that are almost the same as conventional ones.

The present invention will be explained below based on examples.

Example: Measurement of hepatitis B virus surface antigen (HBs Ag) by the Sand-Deutsch method (A) Manufacture of carrier Polymethyl methacrylate molded into a sphere (D = 6 mm) by injection molding was immersed in 1N-NaCl. Hydrolysis reaction was carried out at ℃ for 5 hours.
Next, the mixture was transferred to water containing 5% acetone, and ethyleneimine was added dropwise. In this way, amino groups were introduced onto the surface of the polymethyl methacrylate spheres.

(B) Antigen and antibody HBs antigen (HBs Ag) is a subtype distributed by Meguro Institute Co., Ltd. (Ikeda City, Osaka Prefecture).
A purified antigen of adr was used. The antigen was emulsified with Freund's complete adjuvant (Difco Lad. Detroit, US) using conventional methods.
BALB/C mice were primarily sensitized subcutaneously with 10 μg of HBsAg per mouse. 1 month later HBs antigen 50μg
were subjected to secondary sensitization intravenously, and 3 days later, splenocytes were used as antibody-producing cells for fusion with myeloma NS-1. Hybridomas were grown using HAT medium, antibody activity in the culture supernatant was examined, and those with activity were used as anti-HBs antibodies (monoclonal antibodies).

(C) Binding of antibody to carrier The carrier prepared in (A) was immersed in carbonate buffer of pH 5.0, and 1/10 of 5% glutaraldehyde solution was added.
The mixture was reacted at 4°C for 16 hours and then washed with 0.03M phosphoric acid buffer (PH7.4). Adjust the antibody to a concentration of 1 μg/cc in 0.1M phosphate buffer and add to cover the carrier. Incubate at 35°C for 2 hours and leave at 4°C for 16 hours. Then A buffer (0.1%
Bovine serum albumin, 0.01% _NaN3 , 1.0mN
0.01M Na-phosphate buffer containing MgCl ₂ and 0.1M NaCl).

(D) Preparation of enzyme-labeled antibody The monoclonal antibody prepared in (B) was digested with pepsin and Fab' was used, which was further reduced with 2-mercaptoethanol.

β-D-galactosidase (β-Gal) derived from Escherichia coli was selected as the enzyme, and a commercially available product from Boehringer (Mannheim) was used. β-Gal and
The bonding of Fab′ is performed using excess N,N′-orthophenylene dimaleimide to connect the SH group of Fab′ to β-
A method of crosslinking the SH groups of Gal was adopted. Fab′ has an SH group far from the antigen binding site, and β-Gal does not affect activity and is highly reactive.
Due to the presence of SH groups, crosslinking of SH groups with dimaleimide is suitable.

(E) Measurement of enzyme activity The activity of the enzyme β-Gal used for labeling was determined by using 4-methylumperipheryl-β-D-galactoside (4MUG) as a substrate and releasing 4-methylumperipherone (4MUG). ) was measured using a fluorometer under conditions of an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. One unit was defined as the enzyme activity that decomposed 1 μmol of 4MUG into 4MU in 1 minute at 30°C.

(F) Preparation of quantitative curve Diluted to various concentrations in 200μ of A buffer
One monoclonal antibody-bound carrier prepared in (C) was added to 200μ of the HBsAg-containing solution, shaken at 37°C for 6 hours, and then left at 4°C for 16 hours.

Next, the carrier was taken out and washed with 0.01M Na-phosphate buffer (PH7.0), and then shaken at 37°C for 4 hours with β-Gal-Fab', which showed an activity of 100μ units in a 20-minute reaction at 30°C. After reacting while
After washing with buffer A, the solid phase carrier spheres were transferred to another test tube.

For this carrier sphere add 0.1mM 4MUG at 30°C.
React for 1 minute, then use a reaction terminator (0.1M glycine-
HCl buffer) was added and then the free 4MU was measured with a fluorometer. The EIA thus obtained
The quantitative curve is shown in FIG.

Next, one antibody-bound carrier was added to 200μ of serum from a normal person, and 4MU was measured in the same manner as described below using buffer A. The quantitative curve obtained was also shown in Figure 1. Shown in the figure.

(G) Results As shown in Figure 1, the conventional measurement limit of the EIA method using a resin carrier to which antibodies are chemically bonded is
It is clear that it is well below 0.5ng/cc to 1ng/cc.

If up to 2.1 times the blank value is considered negative, the measurement limit is approximately 0.01 ng/cc.

Comparative Example A quantitative curve for HBsAg was obtained under the same conditions as in the case of using buffer A in the above example, except that a monoclonal antibody was physically adsorbed using a polystyrene carrier, and is shown in FIG.

In this case, the lower limit of measurement is several ng/cc (preferably
5ng/cc).

[Brief explanation of the drawing]

FIG. 1 is a graph showing quantitative curves obtained in Examples and Comparative Examples of the present invention. 1... Quantification curve according to this example conducted using A buffer, 2... Quantification curve according to this example conducted using human serum, 3... Quantification curve according to a comparative example.

Claims (1)

[Claims] 1. In a method for measuring an antigen or antibody by oxygen immunoassay, a resin surface into which active amino groups are introduced by reacting an antibody or antigen corresponding to the antigen or antibody to be measured with ethyleneimine. 1. A method for measuring an antigen or antibody, which comprises immobilizing an antigen or antibody on a carrier having a chemical bond. 2 The first resin is polymethyl methacrylate
Measurement method described in section. 3. The measuring method according to item 1, wherein the resin is acrylonitrile-butadiene-styrene copolymer resin (ABS). 4. The measuring method according to item 1, wherein the resin is a styrene-maleic anhydride copolymer.