JPS63234160A - Enzyme immunoassay - Google Patents

Abstract

PURPOSE:To simplify measurement by adding hydrogen peroxide and o-phenylene diamine to a specimen soln. contg. an antigen or antibody which is a specimen and contg. a peroxidase labeled antibody or antigen, thereby coloring a fibroin film. CONSTITUTION:The fibroin film to which the antibody (or antigen) to the specimen is included and immobilized is immersed in the specimen soln. contg. the antigen (or antibody) which is the specimen and contg. the peroxidase labeled antibody or antigen to effect an immune reaction. The substrate soln. contg. the hydrogen peroxide and o-phenylene diamine is then added to the soln. to oxidize the o-phenylene diamine by the peroxidase and to color the film to brown. The fibroin film is then taken out of the reaction liquid and the degree of coloring of the film is measured, by which the antigen or antibody quantity is measured. The easy measurement of the specimen quantity is thereby permitted.

Description

Detailed Description of the Invention (Industrial Utilization) The present invention relates to an enzyme immunoassay method, and more specifically, the present invention relates to an enzyme immunoassay method. In the enzyme immunoassay method used, the degree of coloring of the fibroin membrane by a dye generated from hydrogen peroxide and O-phenylene cylindrical enzyme using peroxidase is measured, and the antibody or antigen sample is easily measured. Pause.

(Prior Art) It is widely practiced to detect and quantify specific antigens or antibodies contained in a specimen by utilizing the high specificity of antigen-antibody reactions, and to utilize this in the diagnosis or treatment of diseases. In particular, radioimmunoassay and enzyme immunoassay are becoming increasingly useful as trace analysis techniques in clinical tests.

When implementing this immunoassay method, the antigen to be measured (
An antibody (or antigen) corresponding to the antigen (or antibody) to be measured is immobilized in advance on a suitable insoluble carrier by a chemical bonding method, an adsorption method, or an entrapment method, and the immobilized antibody (or antigen) is attached to the antigen (or antigen) to be measured. The so-called phase method is used to react with antibodies (antibodies).

However, in immunoassay using the face phase method, after the immune reaction, the immobilized antibody or antigen must be removed from the reaction solution and washed to separate the unreacted labeled antibody or antigen.
This makes the operation in immunoassay extremely complicated.

(Problems to be Solved by the Invention) The present inventors have completed the present invention as a result of diligent research in view of the problems of the above-mentioned existing methods. Another object of the present invention is to provide an immunoassay method that can easily measure an antigen or antibody as a specimen without removing the antigen from a specimen solution and washing away unreacted labeled antibodies or antigens. Other objects and effects of the present invention will become apparent from the following description.

(Means for Solving the Problems) The above purpose is to use an enzyme immunoassay method in which an antigen or antibody as a specimen is added to a specimen solution containing an antigen or antibody as a specimen and a peroxidase-labeled antibody or antigen. After immersing a fibroin membrane entrapping and immobilizing the corresponding antibody or antigen and performing an immune reaction, the fibroin membrane has no choice but to be removed, and subsequently, hydrogen peroxide and O-F22 are added to the sample solution after the immune reaction. An important feature in the present invention is the use of 0-phenylenecyatron, labeled peroxidase, and hydrogen peroxide as a coloring system. When this system is applied, the dye produced by labeled peroxidase bound to the immobilized antibody or antigen during the immune reaction is more easily adsorbed to the fibroin membrane than the dye produced by labeled peroxidase that remains unreacted and present in the reaction solution. It is now possible to measure the antigen or antibody as a specimen without removing unreacted peroxidase-labeled antibodies or antigens by washing.

The antibody or antigen-immobilized fibroin membrane used in the present invention is JP-A No. 60-142259 or JP-A No. 60-156.
It can be produced by the method described in Japanese Patent No. 129.

The above-mentioned immobilized antibody or antigen is a film of fibroin, and a film of fibroin alone or a film coated or adhered to another substrate can be used.

Specific examples of substances that can be measured by the method of the present invention include the following.

■ 4-lipeptide hormones such as insulin, chorionic gonadotropin, placental lactogen, and luteinizing hormone, and antibodies thereof.

(2) Serum proteins such as IgG, IgM, IgM, IgE, α-fetoprotein, carcinogen, haptoglobin, and antibodies thereof.

■ Toxins or viruses such as Escherichia coli toxin, cholera toxin, hepatitis virus, rubella virus, influenza virus, and their antibodies.

■ Steroid hormones or drugs such as estradiol, progesterone, testosterone 1, phenytoin, brocainamide, kanamycin, penicillin, barbituric acid, and antibodies thereof.

When measuring the above-mentioned substances, immobilized antibodies or antigens in which the corresponding antibodies or antigens are comprehensively immobilized on fibroin are used.

The measurement can be carried out by either a competitive reaction method or a non-competitive reaction method such as the Sand-Deutsch method.

The peroxidase for labeling antibodies or antigens may be derived from plants, animals, microorganisms, etc., but horseradish peroxidase is particularly preferred due to its ease of availability and activity.

The method of binding and labeling peroxidase to an antibody or antigen is not particularly limited, and for example, a method using a bifunctional reagent such as glutaraldehyde or diisocyanate, or after introducing a functional group to a peroxidase antibody or antigen. This may be carried out by appropriately selecting from known methods such as bonding methods.

When performing an immunoassay, a fibroin membrane on which an antibody (or antigen) corresponding to the specimen is immobilized is immersed in a specimen solution containing an antigen (or antibody) as a specimen and a peroxidase-labeled antibody or antigen; Perform an immune reaction. The time and temperature required for the immune reaction lζ may be selected as appropriate, but it is usually carried out at 4 to 46°C for 6 minutes to 24 hours. Further, the antigen (or antibody) as a specimen and the peroxidase-labeled antibody or antigen may be added at the same time, or may be added sequentially at appropriate time intervals.

The concentration of the peroxidase m-recognizing antibody or antigen depends on the concentration of the specimen, but it is usually sufficient to use 0.05 to 10μm7tnl, and the amount of the specimen solution also depends on the size of the fibroin membrane on which the antibody (or antigen) is entrappingly immobilized. It depends, i always o, i~
It is sufficient if the fibroin membrane can be completely immersed at 5 m/'.

A substrate solution containing hydrogen peroxide and 0-phenylenediamine is then added. The concentrations of persolubilized hydrogen and 0-phenylenediamine were each 0.1 to 20 mM.

0.5-100 mM is preferred. When the substrate solution is added, 0-phenylenediamine is oxidized by peroxygen and sidase and colored brown. 80 seconds after adding substrate solution
The fibroin membrane is removed from the reaction solution after 10 minutes, preferably 1 to 5 minutes, and the degree of coloration of the membrane is measured with the naked eye or by reflection, thereby measuring the amount of the antigen or antibody that is the specimen in the specimen solution.

A suitable support material can also be used to remove the fibroin membrane from the reaction solution.

In order to stop the enzymatic reaction, a peroxidase inactivator such as sulfuric acid may be added to the reaction solution, or the removed fibroin membrane may be immersed in a solution of the inactivator before measurement.

The amount of specimen may be measured by comparison with a standard specimen containing a known amount of specimen.

(Effects of the Invention) According to the method of the present invention, it is possible to measure the amount of a sample in a porcelain bag without removing the immobilized antibody or antigen from the sample solution and washing and removing unreacted labeled antibodies or antigens, and in addition, it is possible to measure the amount of the sample in a colored sample The fibroin membrane that entrappingly immobilizes antibodies or antigens has the advantage of being able to stably preserve its colored state.

The method of the present invention will be specifically described below with reference to Examples.

Example 1 (1) Preparation of fibroin aqueous solution: 100I of raw silk was mixed with 1.0% by weight Marcel soap aqueous solution 5I! and scoured at 80° C. for 8 hours.

After washing with water, add 0.6% Marcel soap aqueous solution 61
Fibroin raw material '1211 from which sericin and the like were substantially removed was obtained by immersion in water and scouring at 80°C for 8 hours.

Kneader containing 100 I of water and 80 I of ethyl alcohol
After dissolving 160I of calcium chloride in the solution and raising the temperature to 76°C, add 70JF of the above 71 proine raw material, and add 160I of calcium chloride while stirring.
Dissolved for hours. Then, 180I warm water (76°C) was added to dilute and mix. After cooling the fibroin solution,
A 6.7% by weight aqueous fibroin solution was desalinated by dialysis against running water using a hollow fiber dialyzer.
m/ was obtained. The residual amount of calcium chloride is 0.08 weight ft.
It was N.

(2) Production of monoclonal anti-human FP antibody-immobilized fibroin film: Monoclonal anti-human FP antibody (immunized animal mouse) was dissolved in physiological saline to prepare an antibody solution of 250 μm and 17 ml. Next, this solution was placed on a glass plate partitioned on all sides so that the amount of antibody was 10μI/Cm! 15
It was dried at ℃ for 8 hours. A solution prepared by adding glycerin to the fibroin aqueous solution at a concentration of 80% by weight relative to the fibroin was cast on top of the aqueous fibroin solution, and the mixture was dried at 20°C for 10 hours to cause a rebound, resulting in a monoclonal antihyde FP with a thickness of 60 μm. An antibody-immobilized fibroin film was obtained.

The obtained fibroin film was cut into a size of 7 x 12 mm, and the tip of a 200 μm thick polyester sheet (width 2 mm, length 10 cm) was glued to one end of the short side of the film using a cyanoacrylate adhesive. ,
It was used as an in-phase antibody.

(3) Measurement of human FP Dispense 0 and s mt of physiological saline containing 0.6 wt% bovine serum albumin into test tubes with an inner diameter of IQ mm, and completely immerse each film (n = 2 each). Insert it as shown.
It was left at room temperature for 1 hour. Next, after removing the physiological saline solution containing 0.6% by weight bovine serum albumin by suction,
, 20, 40, 80° 160 nJ//ml standard fluid
P, 10% by weight of horse serum and monoclonal anti-TomFP antibody (immunized animal mouse, however, the recognition site is different from the immobilized monoclonal antibody) labeled with peroxidase [1,2 μl/ml s enzyme labeling] Journal of Histochemistry, Endocytochemistry
chemistry and oytochemistry)
The periodic acid oxidation method was used in accordance with the method described in Vol. 22, p. 1084 (1974). ] 0, 1
0.5 m of physiological saline containing wt% bovine serum albumin
/ and left at 22°C for 1 hour.

Next, each test tube was filled with 19.1 ml (0.5 mI of o-phenylenecyatron, citric acid-phosphoric acid containing 2.45 mM hydrogen peroxide, 2 sodium buffer solution (pH 5,0)).
! A portion of the mixture was added and allowed to stand at room temperature for 2 minutes. After taking out the film, it was immersed in IN sulfuric acid for 6 seconds to stop the enzyme reaction.

When the degree of coloring of the film was observed, it was found that FPO11O1
The reddish-brown color clearly became darker in the order of 20, 40, 80, and 160 nl/ml. Also, glue the film onto a blank sheet of paper.
When the reflected light intensity at 490 nm was measured, it was possible to quantify MuFP as shown in Table 1.
”:'T7-:' (Hereinafter, the remainder is white) Table 1 Example 2 (a) Production of monoclonal anti-hOG antibody-immobilized fibroin film Monoclonal anti-hcG antibody (immunized animal mouse) was dissolved in physiological saline, An antibody solution of 260μl/ml was prepared.Next, the solution was applied onto a glass plate partitioned on all sides so that the amount of antibody was 10μ#/Cm, and the solution was incubated at 20°C.
It was dried for 3 hours. Next, the fibroin aqueous solution obtained in the same manner as in Production Example 10) was made into a wire to obtain a 16.7% by weight fibroin aqueous solution, and a solution containing glycerin added to the fibroin at 80 Mλ% was cast on top of the wire. The film was dried at 20° C. for 8 hours to form a film, thereby obtaining a monoclonal anti-hOG antibody-immobilized fibroin film having a thickness of 90 μm.

As in Example 1, it was cut into sizes of sl, 2XO, and Tcm, and used by adhering to a polyester sheet.

(2) Measurement standard for hcG 0.5 ml each of 0 and 1 It% bovine serum albumin physiological saline solutions containing hOG at moderate concentrations as shown in Table 2! The above films (n=10 each) were immersed in the solution and reacted at 26° C. for 80 minutes. After washing with water, anti-h
CG antibody (IgG fraction of rabbit serum) labeled with horseradish peroxidase [1.0 μI/m! ,enzyme! 5. The test was carried out in the same manner as in Test Example 1. ] Containing 0,1% in heavy state
Bovine serum albumin physiological saline solution 0.5ml! and reacted at 25° C. for 80 minutes.

Next, an enzymatic reaction was carried out in the same manner as in Example 1, and the degree of coloring of the fibroin film was determined by hOG500 mI.
U/mI! Based on the standard, dark ones were judged as positive, and pale ones were judged as negative with the naked eye. hOG4 as shown in Table 2
2 S mIU/m/ or less, all negative, 62E
All samples with mIU/m/ or higher were determined to be positive, and hOG could be accurately measured in the container.

Table 2

Claims (3)

[Claims] (1) In enzyme-linked immunosorbent assay, a fibroin membrane on which an antibody or antigen corresponding to the antigen or antibody as a specimen is immobilized is placed in a specimen solution containing an antigen or antibody as a specimen and a peroxidase-labeled antibody or antigen. After immersion and immunoreaction, without removing the fibroin membrane, a substrate solution containing hydrogen peroxide and o-phenylenediamine is added to the sample solution after the immunoreaction to color the fibroin membrane. An enzyme immunoassay method characterized by measuring the amount of o-phenylenediamine oxide. (2) The enzyme immunoassay method according to claim (1), wherein the peroxidase is derived from Western wasabi. (3) The substrate solution contains 0.1-20mM hydrogen peroxide and 0.
．． The enzyme immunoassay method according to claim (1) or (2), which contains 6 to 100 mM o-phenylenediamine.