JPS6082966A - Assay of antigen - Google Patents

Abstract

PURPOSE:To enable assay of several antigens by using the same water-insoluble carrier insolubilizing the second antibody by binding a complex obtd. by the reaction of antigen with antibody and labelled antibody to a water-insoluble carrier for insolubilizing the second antibody. CONSTITUTION:The assay of antigen in accordance with this invention comprises determination of the amt. of antigen by binding complex obtd. by the reaction between the antigen, antibody (a) and labelled antibody (b) to a water-insoluble carrier for insolubilizing the second antibody, and obtg. the amt. of antigen from the amt. of labelled antibody (b) bonded to the carrier. In this case, in order to permit bonding of the second antibody to the antibody (a) but to prevent from being bonded by the labelled antibody (b), a fact is utilized as one method that separation into bonded part with an antigen, i.e. Fab', Fab from other fragment Fc is possible when an antibody (i.e. immunoglobulin Ig) is decomposed restrictedly using protease such as pepsin or papain, etc. Namely, as antibody, Ig contg. Fc which has not been treated with protease is used; as labelled antiody (b), labelled Fab' or Fab contg. no Fc is used; and as the second antibody, a specific antibody for Fc is used.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying an antigen, and more specifically, the present invention relates to a method for quantifying an antigen. A water-insoluble carrier (hereinafter referred to as a second antibody-insolubilized water-insoluble carrier) in which an antibody (al-antigen-labeled antibody (bl) complex (hereinafter simply referred to as the complex)) is insolubilized (hereinafter referred to as the second antibody) after binding to the second antibody-insolubilized water-insoluble carrier, the amount of the labeled antibody (b) bound to the second antibody-insoluble water-insoluble carrier is measured by measuring the labeled substance, thereby determining the amount of the antigen. In recent years, with the development of clinical medicine, there has been an increasing demand for the determination of trace amounts of components in various biological fluids. Immunoassay methods are attracting attention as a method of measurement.In addition, the measurement targets are wide-ranging, including hormones such as insulin, growth hormone, thyroid-stimulating hormone (TSH), glucagon, gastrin, and calcitonin, and alpha-fe-1-protein (AFP). , carcinoembryonic antigen (CEA)
, biological proteins such as various immunoglobulins, enzymes such as creatine phosphokinase (CPK)' and enolase, and drugs such as various antiepileptic drugs and antibiotics are targets of immunoassay.

In recent years, a solid phase method using a water-insoluble carrier, the so-called Santoi-Fuchi method, has been developed in immunoassays, and this method has already been applied to clinical medicine because of its extremely high measurement sensitivity. For example, E, l5hikai++a,
Kato insolubilizes the antibody onto a piece of silicone resin, binds the antigen to it, and then reacts it with an antibody labeled with an enzyme (hereinafter referred to as enzyme-labeled antibody) to form a mixture of silicone resin piece - antibody - antigen - enzyme-labeled antibody. Antigens are measured with high sensitivity by forming complexes and measuring the activity of this enzyme. [Scandinavian Journal op Immunology (Scand, J. rmmunol,) No. 8
Volume, pages 43-55, (1978)].

However, although such conventional Santoi-Fuchi method has high sensitivity, it is necessary to prepare antibody-insoluble water-insoluble carriers corresponding to each antigen depending on the type of antigen to be measured, and it is difficult to measure various antigens. This was extremely inconvenient for quantitative determination. Particularly when this method is applied to an automatic measuring device, it is necessary to replace the antibody-insolubilized water-insoluble carrier depending on the antigen, which requires extremely complicated operations.

Therefore, the present inventors took the above into account, and as a result of intensive investigation into high-sensitivity measurement using a new solid-phase method to replace the conventional sandwich method, we determined that various antigens could be transferred to the same second antibody-insoluble water-insoluble carrier. They discovered a new method for quantifying antigens based on the sandwich method, which allows for quantitative determination.

That is, the present invention provides an antigen and an antibody (al and labeled antibody (b)
The complex obtained by reacting is bound to a second antibody-insolubilized water-insoluble carrier, and the amount of antigen is determined from the amount of labeling substance bound to the second antibody-insolubilized water-insoluble carrier. Here, it is necessary that the second antibody binds to the antibody (al) but not to the labeled antibody (b).The method described below is the most effective for making such a reaction possible. The present inventors have discovered.

The first method is that antibodies are a type of immunoglobulin, but when they are limitedly digested with proteases such as immunoglobulin (pepsin or pepsin), the antigen-binding portion (F a b ', F a b This method takes advantage of the fact that it can be separated into two parts with names such as n-hand LL') and other parts < F CRB and ■ hand bi-reru. That is, the antibody (al is an immunoglobulin containing talc that has not been treated with protease), the standard &h antibody (the bl is Fab' excluding pc), the standard Fab is 86
Use the one you made. Then use Fc+ as a second antibody.
One method is to use something specific for this purpose.

The second method uses different antibodies (a) and labeled antibodies (b).
This method uses antibodies obtained from heavy-weight animals and eight standard human antibodies. For example, use a rabbit antibody (G) as the antibody (al), and use a mouse antibody (B) as the labeled antibody (b).
Use votes that you are familiar with. Here, an antibody against two rabbit immunoglobulins can be used as the second antibody.

At this time, further apply the first method and the second method at the same time,
For example, an antibody (al may be an immunoglobulin containing rabbit FC, a labeled antibody (bl may be labeled mouse Fab or Fab', and a second antibody may be an antibody against rabbit FC). In addition, the method of the present invention quantifies multivalent antigens that have two or more antibody-binding sites, but antibody (a) and labeled antibody fbl compete for the same antibody-binding site on the antigen. It is also possible to combine them.

In this case, problems such as a decrease in measurement sensitivity arise, but in such a case, monoclonal antibodies that bind only to specific antibody binding sites are used as antibodies (al and/or labeled antibodies (bl)). Measurements can be carried out by using it for one side.

In the method of the present invention, if the antibody (a) is prepared in the same animal, the same second antibody-insoluble water-insoluble carrier can be used no matter what antigen is being measured.・Border method G Kona G) This is an excellent point.

As the water-insoluble carrier used for preparing the second antibody-insolubilized water-insoluble carrier, various synthetic polymers, glass, insoluble polysaccharides, etc. are used. The shape is spherical, rod-like, fibrous,
Fine particles are used. Furthermore, the inner wall of a synthetic resin test tube can also be used. In the case of fibrous or fine particulate materials, it is advantageous to use them by filling them in a column in consideration of operability and the like.

Antibodies used for antibody (a) and labeled antibodies fb and l include those contained in antisera obtained by immunizing various animals with antigens, or monoclonal antibodies produced by hybridomas such as mice. These antibodies are F, a b, f? al)' can be used as a fragment. However, the antibody (a) in the first method must be an Fc-containing immunoglobulin. As the second antibody, immunoglobulin itself or a Fab or Fab' fragment can be used.

Labeling substances used to produce labeled antibody (b) include radioisotopes, enzymes, and various fluorescent substances. In particular, enzymes are useful labeling substances because they have high detection sensitivity and do not have safety issues like radioisotopes.

In the labeled antibody (bl), conventionally known techniques can be applied to the method of binding the labeling substance and the antibody.

For example, techniques such as the chloramine T method have been established for radioisotopes. In the case of enzymes, various camping agents are used. Examples of camping agents include glucurfuldehyde, carbodiimide derivatives, and maleimide derivatives. Examples of radioisotopes suitable as labeling substances include (1), 1311, Nhc, and 3H1 enzymes such as β-D-galactosidase, peroxidase, acetylcholinesterase, lysozyme, glucose oxidase, and malate dehydrogenase.

In the case of fluorescent labeling, FITC-labeled antibodies can be obtained, for example, by conventional methods.

Physical adsorption or covalent bonding can be used as a method for preparing a second antibody-insolubilized water-insoluble carrier by insolubilizing the second antibody in the water-insoluble carrier. Physical adsorption on polystyrene spheres, silicone resin pieces, etc., cyanogen bromide on insoluble polysaccharides, etc.
A commonly used method is a covalent bonding method using epichlorohydrin, carbodiimidazole, or the like.

In the method of the present invention, various hormones, proteins, etc. in biological body fluids can be measured with high sensitivity by using the same second antibody-insoluble water-insoluble carrier.

This will be specifically explained below using Examples.

Example 1. Determination of thyroglobulin (1) Preparation of thyroglobulin antibody Purified thyroglobulin was injected into rabbits and anti-thyroglobulin serum was obtained by this method. Thyroglobulin antibody (rabbit) was obtained from this anti-thyroglobulin serum by salting out and DEAE-cellulose chromatography. This antibody was used as labeled antibody (b) manufactured by 8Itit.

On the other hand, BALB/C mice were immunized with the above-mentioned thyroglobulin, and the lung cells and myeloma cells were used in Kohler (
K6hler and Milster's method [Nature, Vol. 256, 49
5-497, (1975)], a thyroglobulin antibody-producing hybridoma was obtained. This hybridoma was cultured intraperitoneally in a mouse, and a thyroglobulin monoclonal antibody was obtained in the same manner as above the crotch. The immunoglobulin type of this antibody was determined to be immunoglobulin G. This antibody was used as it was as antibody ta+.

The second antibody was prepared in the same manner as above using anti-mouse immunoglobulin G serum obtained by injecting mouse immunoglobulin G into rabbits.

(2) Preparation of labeled antibody (b) The thyroglobulin antibody (rabbit) obtained in (1) was subjected to limited digestion with pepsin and fractionated with a Sephadex G-150 column to obtain F(ab')2. . This was reduced with mercaptoethylamine to F a b', and Fab'
SH group and β-D-galactosidase (derived from E. coli)
Labeled antibody (b) was prepared by bonding the SH group of with N,N''-0-phenylene dimaleimide.

(3) Preparation of second antibody-insolubilized water-insoluble carrier 50 μl of the second antibody obtained in (1) was covalently bonded to 10 ml of Sepharose 4B using cyanogen bromide. The second antibody-insolubilized Sepharose was packed into a 0.1-sized polystyrene mini-column.

(4) Creating a calibration curve In the following measurements, use 0.5% gelatin as a buffer solution.
p11 containing 0.1% bovine serum albumin, 0.3M salt, 1mM magnesium chloride, 0.1% sodium azide
7 of 10 mM phosphate buffer was used.

Thyroglobulin in buffer solution 0325.50.100.
Each of the solutions dissolved at a concentration of 200.400 ng/w
Mix 0.5ml of solution of 0.1me and antibody (al),
The reaction was carried out at 37°C for 1 hour. Add the labeled antibody (b) to this reaction solution.
After adding 0.5 l of the solution and further reacting at 37°C for 1 hour, the reaction solution was applied to a column of second antibody-insolubilized Sepharose. After washing the column with @ buffer solution, the column was filled with a solution of 0-nido-phenyl-β-D-galactoside, which is a substrate for β-D-galactosidase, and reacted overnight at 25°C. 1 ml of the produced 0-nitrophenol
When the column was washed out with an mM sodium carbonate solution and the absorbance of this washing solution at 420 nm was measured, the calibration curve shown in FIG. 1 was obtained.

(Quantification of thyroglobulin in group serum Thyroglobulin was quantified in the same manner as in (4) using 0.1-ml of human serum, and the concentration of thyroglobulin was determined from the calibration curve in Figure 1. When I checked the concentration of each, it was 9.5og/-12
It was 65 og/me and 24.5 og/-.

Example 2. Quantification of Thyroglobulin When monoclonal antibodies against thyroglobulin were produced using mice in the same manner as in Example 1, two types of immunoglobulin A type antibodies were obtained in addition to immunoglobulin G type antibodies. These immunoglobulin A type antibodies were reduced to prepare a labeled antibody (bl) bound to β-D-galactosidase according to Example 1 (2). The same one as in 1 was used.

0125.100, 200.400 ng of thyroglobulin in human serum excluding endogenous thyroglobulin
A calibration curve was created in the same manner as in Example 1 using the sample added at a concentration of /ml.

At the same time, the same operation was performed on 5 human serum samples with unknown thyroglobulin concentrations, and the thyroglobulin concentrations were determined from the calibration curve to be 11.2 og/m for each! ,59.5o
g/+t! , 8.5og/ml! , 158ng/mf
! , 98.3 og/me.

Example 3. Quantification of alpha-fetoprotein (AFP) Antibody to AFP obtained from rabbit (a)
It was used as Further, the same antibody was treated according to Example 1 (2) to obtain F(ab')2. This F(ab')2
12s r was introduced into the cells using the chloramine T method, and the labeled antibody (b
It was used as l.

The second antibody was prepared from anti-rabbit Fc serum obtained from goat. The second antibody-insolubilized water-insoluble carrier was prepared by insolubilizing the second antibody in Sepharose according to Example 1 (3).

AFP standard solution (0, 12, 5, 25, 5o, 100.2
00.400 ng/mR) and sample human serum at 2
0pI! Then, 0.5 - of a solution of labeled antibody (b) was added thereto and allowed to react at room temperature for 1 hour. Next, add antibody (a) to the reaction solution.
0.5 ml of the solution was added and reacted for 1 hour in the same manner. Add 0.5 of a suspension of second antibody-insolubilized Sepharose to this reaction solution.
rrd! was added, and after shaking for 1 hour, centrifugation was performed to remove the reaction solution. After washing the second antibody-insolubilized Sepharose with a buffer, measurement was performed using a gamma counter. When a standard curve was created and the AFPi degree of 5 human serum samples was determined from it, the results were 19.25, 15.321.57 ng/mf, respectively.
! Met.

Example 4. Determination of Enolase αγ Enolase is an enzyme with two subunits. There are three types of subunits, α, β, and γ, among which α
Enolase αγ having a subunit and a T subunit was quantified.

The purified γ subunit was injected into a rabbit, an antibody was prepared in the same manner as in Example 1, and this antibody was conjugated with β-D-galactosidase and used as a labeled antibody (bl). Antibody (al) was prepared from the antiserum obtained by injecting it into mice. The same water-insoluble carrier as in Example 1 was used as the second antibody-insoluble water-insoluble carrier.

Enolase αγ was dissolved in a buffer solution for 0.5.10.20.
A standard solution of 40.80.160.320 ng/mE was prepared.

To 20ml each of this standard solution and sample serum, add 0.5ml of antibody (al solution) and 0.5ml of labeled antibody (bl solution).
The reaction was carried out at 37°C for 2 hours. This reaction solution was passed through a second antibody-insolubilized Sepharose column (column content: 0.7), and after washing the column, the enzyme activity was measured according to Example 1.The calibration curve was calculated using the enzyme activity when using the standard solution. create and
The concentration of enolase αγ in 5 serum samples was calculated as follows: 1
4.3ng/mf, 12.5ng/ml, 98.5ng
/+++I', 1105 n/me, 25 ng/product.

Example 59 Quantification of secreted immunoglobulin A (SIgA) Secreted immunoglobulin A (SigA) is a combination of immunoglobulin A (IgA) and the secretory component (SigA).
It is a complex protein consisting of C). Purified IgA and SC
Each was injected into separate rabbits, and antibodies for each were prepared from the resulting antisera.

An antibody against IgA was used as antibody (a). The antibody against SC was F(ab')z as in Example 3, and 1
It was labeled with 25I and used as labeled antibody (b).

The second antibody-insolubilized water-insoluble carrier was the same as in Example 3.

SIgA standard solution (0, 10, 30, 60, 120 pg/
+++f') and 200 g of a 100-fold dilution of each sample serum, labeled antibody (0.5 + t! of bl solution, antibody (
0.5ml of al solution! were mixed and reacted at 37°C for 1 hour. Add 0.5mf of the second antibody-insolubilized Sepharose suspension to this reaction solution! was added, and the following procedure was performed in the same manner as in Example 3 to determine the SIgAI level of the sample serum. The S t g A concentration of 10 human serum samples was 15 pg/+++f! , 8.
5pJg/mR19, 4pg/-164pg/-124
pg/mn, 3.5pg/m, llpg/mf! ,
13Itg/ml, 987+g/mi! , 7.97
It was 1g/-.

Patent applicant Amano Pharmaceutical Co., Ltd. Figure 1 Thyroglobulin (nq/mT) Procedural amendment (method) % formula %] 1. Indication of the case 1982 Patent Application No. 192715 2. Name of the invention Method for quantifying antigen 3. Person making the amendment and relationship to the case Patent applicant address No. 2-7 Nishiki-Shimome, Naka-ku, Nagoya, Aichi Prefecture January 31, 1980 5. Specification subject to the amendment 6. Contents of the amendment (1) ) Add the following sentence to page 16, line 15 of the detailed statement.

! -4. Brief explanation of drawings

Claims (1)

[Scope of Claims] 1. A complex of antibody (a)-antigen-labeled antibody (b) obtained by reacting an antigen with an antibody (al and a labeled antibody fb labeled with a labeling substance) is reacted with an antibody (against al). A method of producing an antigen characterized by binding the antibody to an insolubilized water-insoluble carrier, and then measuring the amount of the labeled antibody (b) bound to the water-insoluble carrier by measuring a labeled substance to calculate the amount of the antigen. Assay method. 2. A method for quantifying an antigen according to claim 1, characterized in that both or one of the antibody (al and labeled antibody (bl) is a monoclonal antibody. 3. The method for quantifying an antigen according to claim 1, characterized in that the labeled substance is an enzyme 4. A method for quantifying an antigen according to claim 1 or 2. 4. Claims characterized in that a column is filled with a water-insoluble carrier in which an antibody against antibody (a) is insolubilized. The method for quantifying the antigen according to item 1, item 2, or item 3.