JPS6184560A - Method for measuring complement bindable antibody - Google Patents

Abstract

PURPOSE:To measure quickly a complement bindable antibody by fixing an antigen to a solid phase then bringing a complement constituting component bound with an antibody and labeling material into reaction with said antigen then determining quantitatively the labeling material. CONSTITUTION:The antigen such as, for example, simple herpes is fixed to the solid phase. The complement constituting component bound with the specific antibody and labeling material is then brought into reaction with such antigen. Serum is generally used for the antibody. The complement is constituted of 10-odd kinds of proteins contained in the fresh serum of an animal. The comple ment is activated mainly by an antigen-antibody complex. The complement constituting body and labeling material are bound with each other. The unadsorbed complement constituting component and reaction obstructing material are easily removed by washing from the antigen and antibody fixed to the solid phase and the complement constituting component bound with the labeling material. The labeling material of the conjugate adsorbed to the solid phase is then quantitatively determined.

Description

[Detailed Description of the Invention] (Field of Industrial Application) The present invention extends to human diseases such as infections caused by microorganisms such as bacteria and viruses, tumors, and glomerulonephritis, as well as animals and plants. The present invention relates to a method for measuring antibodies that bind to human bodies, which can be used to diagnose diseases and determine infection in a wide range of hosts.

(Problems to be Solved by the Invention) In clinical tests for infectious diseases, ulcers, glomerulonephritis, etc., the object of the test is whether antibodies specific to these diseases are present in blood Ca. There are various methods for measuring antibodies, and one of the most important is the complement fixation reaction. However, although the complement fixation reaction is a useful antibody testing method, it is not widely used, despite its importance, because the measurement operations are complicated and require extremely skilled techniques.

Therefore, if this measurement method were to be simplified and speeded up, and if it were possible to obtain measurement values with high sensitivity and reproducibility, it would greatly contribute to the industrial world.

(Prior Art) The conventional complement fixation reaction utilizes a reaction in which components 1 to 9 of complement sequentially bind to an antibody that specifically binds to an antigen. That is, an excess of complement is added to the generated antigen-antibody complex, the amount of remaining complement that is not bound to it is measured by a hemolysis method, and the complement wax subjected to binding is determined by the dilution method based on the degree of hemolysis. The amount of complement-fixing antibodies is thereby estimated. The hemolytic method utilizes the hemolytic reaction of complement against sensitized red blood cells made of sheep red blood cells, but the measurement operation is complex and requires skilled technique and knowledge.In addition, the sensitivity is low, and the measurement time is low. The drawback is that it takes two days.

Therefore, various methods have been discovered in order to eliminate the above-mentioned drawbacks. For example, Japanese Patent Application Laid-Open No. 55-45498 can be mentioned.

This invention is a so-called enzyme antibody method in which an antibody against a complement component is labeled with an enzyme and the amount of the antibody is measured from the enzyme activity. However, this invention has disadvantages in that, since it is necessary to use a labeled antibody against complement components, the reaction is carried out in two steps, a washing operation is required for each reaction, and several hours are required for measurement.

As a result of intensive research to improve these methods described above, the present inventors first added complement components to the antigen-antibody complex generated by the reaction of antigen and antibody. Focusing on the phenomenon that the C1 component binds and then sequentially binds and activates the complement C9 component, we tangentially label the complement component and determine the labeled substance as r, [llll. I learned that the binding antibody titer can be determined.

They have also discovered that by depleting complement C1q in the complement component, the efficiency of measuring the complement fixation reaction can be increased and complement fixing antibodies can be measured with high precision.

A technique for measuring antigen-antibody complexes in the serum of autoimmune diseases, Tadataka, etc. using enzyme-labeled complement C1q has been disclosed by Simpson et al. J.

Simpson J, Immunological M
ethods67.167-172.1984). However, this technology measures antigen-antibody complexes to test for autoimmune diseases, etc., and measures specific antibodies.
This invention is distinguished from the present invention, which is used for testing infectious diseases and the like.

(Means for Solving the Problems) The present invention involves immobilizing an antigen on a solid phase, reacting the antigen with a complement component to which an antibody and a labeled substance are bound, and then quantifying the labeled substance. This is a standard method for preparing complement-fixing antibodies, which is characterized by:

Next, the present invention will be explained in detail.

Various antigens of the present invention can be used. For example, Iri
Examples include antigens against viruses and bacteria such as rash, measles, rubella, influenza, herpes simplex, viral hepatitis, mumps and mycoplasma pneumonia, as well as antigens against drugs such as interferon and autoantibodies such as DNA. First, these antigens are immobilized on a solid phase. This makes operations such as cleaning easier, unlike in a liquid phase. The solid phase may be anything that does not allow the adsorbed antigen to be easily released; for example, synthetic polymers made of plastic materials such as polyvinyl chloride or polystyrene, natural polymers such as paper, or cells and tissues can be used. . As a representative example,
Examples include microtiter wells and polystyrene beads. Further, the antigen may be immobilized on the solid phase surface by physical adsorption, chemical covalent bonding, infection, or the like.

Next, an antibody specific to this antigen and a complement component bound to a label are reacted. Antibodies are the object of measurement in the present invention, and body fluids such as serum, cerebrospinal fluid, and 11+T can be used; however, since antibodies are contained in large amounts in serum, serum is generally used. do. Furthermore, the antibody used in the present invention must specifically bind to the antigen and must also be capable of binding to complement I4 components.

However, since most antibodies produced in vivo by antigens satisfy this condition, most antibodies can be measured in the present invention.

Complement is composed of several types of proteins contained in fresh animal serum, and is a protein complex that is involved in the body's defense against infection, immune response, inflammation, etc. 1. Copper bodies are mainly activated by antigen-antibody complexes, and are involved in various biological activities such as immune adhesion reactions, anti-phagocytosis reactions, adhesion reactions, killing and lysis reactions, and solubilization of immune complexes. shows. The complement component may be any component of the complement system, and for example, complements C4, C2, C3°C1q, etc. can be selected as appropriate. In particular, complement C1q is the component of the complement system that first binds to antigen-antibody complexes. Therefore, complement C1q can bind to antigen-antibody complexes independently, regardless of other complement components, so if complement C1q is labeled, antibody titer can be measured without using other complement components. Therefore, it is particularly preferable to use complement C1q because it simplifies the measurement. As a method for adjusting complement components, a general separation and purification method for proteins with physiological activity can be used. For example, "complementology"
The adjustment method described in Ishiyaku Publishing, 1981, pp. 141-158 can be used, but is not limited to this method at all.

Any substance that can be easily measured by common analytical methods can be used as the label that binds to complement components. For example, enzymes, fluorescent substances, dyes, enzyme substrates, etc. can be selected as appropriate. In particular, since enzymes act as catalysts, measurement sensitivity can be adjusted freely by changing the reaction temperature and reaction time.Enzymes are therefore more preferable labels because they can amplify and measure trace amounts of complement-fixing antibodies. . As the enzyme, for example, oxidase, alkaline phosphatase, β-galactosidase, alcohol dehydrogenase, etc. can be used.

Any binding method may be used to bind the label to complement components as long as the biological activity of complement and the measurement of the label are not inhibited. If a binder is used, periodontylamide, glutaraldehyde or maleimide derivatives can be used.

In the reaction of the complement component, which is the combination of an antigen immobilized on a solid phase, an antibody, and a labeled substance, when the three components are mixed, the reaction is completed and the dominant component binds. The reaction time and temperature differ depending on the type of antigen, etc., but those skilled in the art can easily set the reaction conditions.In short, as long as the conditions do not deactivate biological activities such as complement, they can be selected as appropriate. It is possible.

Since the conjugate consisting of antigen, antibody, and labeled complement components is immobilized on a solid phase, unadsorbed complement components and reaction inhibitors can be easily removed by washing.

The label of the conjugate adsorbed on the solid phase is then quantified. Quantitative methods can be used as appropriate, such as visual measurement, absorbance measurement of visible and ultraviolet light, fluorescence intensity or pulse count measurement, etc. For quantitative determination of labeled substances, directly measure the tray of labeled substances in the conjugate. Of course, it is also possible to use a fixed amount of label and then measure the amount of label that has not been subjected to binding.

By quantifying the labeled substance, the amount of complement-fixing antibodies against a specific antigen can be determined.

(Example of adjustment of complement components) New MAY rabbit straight run 100 go, 0. (326M ethylene glycol tetraacetic acid (EDTA) aqueous solution (pi-17)
, 5151 for 15 to 24 hours, and the resulting precipitate was collected by centrifugal separation (20,000 OOG, 20 minutes), and was added to a 0.75 M aqueous sodium chloride solution (1) containing 0.01 M EDTA. [Dissolved in (5,0)20-.

Insoluble substances are centrifuged <25. (]　OOc,s.

) and then add 0.063M EDTA water f
Dialyze against i* (pH 5,0) 5 t for 4 hours at 5°C and collect the precipitate by centrifugation (20,0OOG, 20 minutes). Approximately 5 μm of protein was obtained by this operation, of which more than 95% was complement C1q. The above protein was then treated with 0.05M tris(hydroquinemethyl)aminomethane, 1M sodium chloride, 0.005M E
DTA, an aqueous solution containing 10 tisucrose (pi(7,4
It can be dissolved in 11rnt and used in the present invention as complement C1q. Further repeating the above operation, complement C1
q may be further purified.

(Example 1 of preparation of complement components bound to labeled substance) 0.00
Dissolve in an aqueous solution (1) H7,4110- containing 5mM gDTA and 10% cyclose. 0.1 for this
Add 0.1 ml of M ditheothreitol solution and leave to react at very high temperature for 1 hour. Next, the reaction solution was transferred to a Sephadic G-25 column to collect +iaL and protein content. This protein fraction is concentrated to about 10 mg by ultrafiltration to obtain reduced complement C1q30.

Next, 20 μl of wasabi-derived peroxidase was dissolved in a phosphate buffer (pH 1416), and 4 ml of dimethylformamide was added. Furthermore, 2% 4-[maleimidomethyl]
Cyclohexa/1-carboxylic succinimide ester (CHMI dimethylformamide solution 0.2
, nl were added and allowed to stand at room temperature for 12 groups to react. This reaction solution was passed through a Sephadex G-25 column, and CH
Collect 20 pages of M-linked peroxidase.

Mix 20 liters of Goiso-type complement C1q 30W and C)
Collect fractions from 10,000 to 800,000 and use peroxidase-labeled complement C.
Obtain 1q40~.

(Example 2 of preparation of complement components bound to labeled substance) 2 ”?
/ ml! , complement C4 in saline 1 rnt per night,
0.2 ~ Fluoretscen isothiocyanate (FIT
C) Add carbonated buffer solution (pH 9,5) containing o, 1-,
Stir slowly for 4 hours at 10°C. Through this operation, the fluorescent substance F'ITC binds to complement C4. By passing this reaction product through a Sephadic G-25 column, unreacted FITC was separated and removed, and approximately 1.
Collect 8~.

(Execution 111) Complement fixation 11fj4 and below and 1
5 μt of the immobilized sample serum from No. 6 was added to each well, and 95 μt of rooxidase-labeled complement C1q (100-fold diluted with gelatin veronal solution) was added to each well, and the mixture was allowed to stand at room temperature for 1 hour. Next 0.051 Tween
Wash each well three times with v4tF2 buffer containing -20 and wash each well three times with v4tF2 buffer containing
, 2'-azinodi-(5-ethylbenzoceacrine sulfate) solution) was added thereto, and the mixture was reacted for 1 hour at room temperature. 1) After adding 100 μt of ¥1000 reaction stop solution, the absorbance at 414 nm was measured, and the absorbance was 0.029 for serum with a complement titer of 4 or less, and 0.579 for serum with a complement titer of 16. Ta.

(Example 2) HBs antigen of hepatitis virus was immobilized on polystyrene beads with a diameter of 6.35 mm, and transferred to a small sampling tube. This was then added with 0.1 ml of a 10-fold dilution of immobilized test serum in gelatin-veronal buffer, 0.1 ml of a 10-fold dilution of complement C4 labeled with tetraprosthetic FITC in the same buffer, and anti-C4.
0.1 d of fresh antibody-treated rabbit serum was added and allowed to stand at air temperature for 1 hour. Next, the beads were removed, veronal buffer i2.7d was added to the remaining solution, and measurement was performed using a fluorescence spectrophotometer at an excitation wavelength of 490 nm and a fluorescence wavelength of 520 nm. At the same time, a comparative test was conducted using normal serum and standard serum, and the antibody titer of the test l'c[L serum was determined by extrapolation.

(Example 6) Bovine serum albumin was dissolved in @acid buffered saline at 20 μt/ml at C4 degrees, dispensed into a 96-well microtiter plate, and kept at room temperature for 2 hours to be adsorbed to the wells. After removal of free bovine serum albumin, rabbit anti-bovine serum albumin antiserum (800% in gelatin-veronal buffer) was prepared.
50 μt of ˜6400-fold serial dilution) and 50 μL of alkaline phosphatase-labeled complement C1q (100-fold diluted with gelatin veronal buffer) were added, and the mixture was allowed to stand at room temperature for 30 minutes.

After washing each well, add 10 [3 μt of balanitrophenyl groove acid solution (Tris buffer, I) [-18,2], let it stand at room temperature for 60 minutes, stop the enzyme reaction, and then add A
Absorbance at 10 nm was measured, giving an absorbance of 0.568 to 0.054, depending on the pre-antivascular dose.

(Example 4) Purified herpes simplex virus antigen (6.55 mm17)
: Test serum (complement titer 4 or less and 16) that was adsorbed onto W listyrene beads, placed in a hand towel and a tube, and immobilized.
0.1 d of a 10-fold dilution of gelatin veronal solution and 0.1 d of a 10-fold dilution of peroxidase-labeled complement C1q in the same buffer.
1- was added at the same time and allowed to stand at room temperature for 1 hour. After washing, the beads were transferred to a towel tube on the side, and 0.3rrLt of orthophenylenediamine solution was added thereto, and the mixture was allowed to react at room temperature for 45 minutes. Stop the reaction by adding 1s12 ml of 1N salt,
The absorbance at nm was determined by dill. The absorbance of samples with complement titres below 4 and 16 is 0.018, Q, and 4, respectively.
It was 08.

(Effects of the Invention) According to the present invention, complement-fixing antibodies can be measured easily and with high accuracy. Furthermore, by measuring antibodies according to the present invention, clinical test results for infectious diseases, etc. can be quickly obtained, and appropriate therapeutic actions can be taken at an early stage.

As described above, the present invention provides a method for measuring complement-fixing antibodies that is extremely useful industrially.

Claims (1)

[Claims] 1. After immobilizing the antigen on a solid phase, the immobilized antigen is reacted with complement components to which an antibody and a label are bound, unbound substances are removed, and then the bound label is removed. 1. A method for measuring complement-fixing antibodies, characterized by quantifying the amount of a complement-fixing antibody. 2. The method for measuring complement-fixing antibodies according to claim 1, wherein the complement component is complement C1q. 3. The method for measuring complement-fixing antibodies according to claim 1, wherein the label is an enzyme.