JPS61102558A - Measuring method of antigen - Google Patents

Abstract

PURPOSE:To make possible the easy and quick quantitative determination and measurement of a labeling material by immobilizing a material having affinity to an antigen to a solid phase then adding a complement constituting component conjugated with the antigen and labeling material, etc. thereto to effect reaction and removing the non-conjugated matter. CONSTITUTION:The material having affinity to the antigen, for example, the substrate of enzyme or inhibitor, etc. are immobilized to the solid phase. Cleaning, etc. of the solid phase are thus made easy unlike the liquid phase. An antigen to be examined is then added to the material having affinity to the immobilized antigen to conjugate the antigen with the material having affinity to the antigen. The complement constituting component conjugated with the labeling material is further added to the conjugate. The labeling material such as a dye which permits easy measurement is selected. An antibody is further added thereto according to need to bring the conjugate into reaction. The conjugate consisting of the material having affinity to the antigen, the antigen, the complement constituting component conjugated with the labeling material, etc. is immnobilized to the solid phase and therefore the unconjugated complement constituting component and reaction inhibiting material are easily removed by cleaning. The labeling material of the conjugate adsorbed to the solid phase is then quantiatively determined. The efficiency of the measurement is thus improved and the measurement of the antigen quantity with high accuracy is made possible.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is applicable to microorganisms such as bacteria, gramydia, and viruses, various physiologically active substances such as interferon and lymphokines, specific antigens in cancer, and specific antigens in immune disorders. It is a measurement method that can be used to detect, identify, quantify, or test sexual substances, allergens in allergic diseases, or drugs such as hormones, and it can also be used for rapid definitive diagnosis of various diseases, a guideline for determining the effectiveness of therapeutic actions, doping tests,
It can be widely applied to the detection of foreign substances in products and the inspection of health and hygiene issues.

(Problems to be Solved by the Invention) Various antigen measurement methods using antigen-antibody reactions have been attempted for the detection, identification, quantification, and testing of microorganisms, immunoglobulins, physiologically active substances, drugs, etc. . These measurement methods are widely used in various fields due to the specificity of their reactions and the usefulness of being able to measure trace amounts. However, traditional measurement methods (yes, they are complicated to operate, sometimes use expensive equipment, sometimes require specialized laboratories, and also require sufficient knowledge and training for the person performing the measurement). Since this measurement method is highly useful, there is a strong demand from industry for measurement automation.However, because the operation is complicated and requires expensive equipment, measurement automation is not generally used. Currently, there are 5.

Making this measurement method easier and faster, and achieving a measurement of 48 degrees, will also expand the scope for automation using inexpensive equipment, which will become a priority in the chestnut producing world. Big fi(
(“It is something that should be put into one pile.

2. Antibiotics against microorganisms, physiologically active substances or drugs, etc. 1! J
As one of the methods to measure using the antigen-antibody reaction, the 7111 standard method, which applies the complement fixation reaction, is used.
There is.

T+1ifV, the binding reaction utilizes a reaction in which each component from C1 to C9-4 of the rtIi body sequentially binds to the specific antibody bound to the antigen in a fixed I'l+'i order C1. , Ralo. In other words, an excess of complement is added to the antigen-antibody complex that has been harvested, and the residual rfiiH body (,t) that is not bound to it is measured by a hemolysis method. 1 liter of antigen is calculated based on the result.The dark a method is that complement acts on sensitized red blood cells made from sheep red blood cell antibodies and anti-sheep red blood cell immune antibodies, and as a result, The hemolysis of sheep red blood cells is used as an indicator, but the measurement procedure is extremely laborious and requires skilled technique and knowledge.Furthermore, the sensitivity is relatively low, and the time required for measurement is 2 days. The present inventors have conducted extensive research in search of a method to more easily measure the amount of antigen. At that time, the complement C1 component binds first, and then the complement C9 component binds for 711!
We focused on the phenomenon of continuous binding and activation. Therefore, we discovered that it is possible to measure the amount of antigen very easily by directly labeling the complement component and measuring the labeled product. and.

Furthermore, it has been discovered that by labeling complement C1q in the complement component, the efficiency of measuring the complement fixation reaction can be improved and the amount of antigen can be measured with a high degree of accuracy.

In addition, using Enzyme 1 Indicator CIq, autoimmune disease
A technique for measuring antigen-antibody complexes present in the serum of patients with disease and patients with QC has been disclosed by Simpson et al.
logical methods67.167-172
．． 1984). However, the purpose of this technique is not to test for autoimmune diseases, but rather to measure antigen-antibody combinations that have already formed within the patient's body. Therefore, it is clearly different from the present invention, which uses known antibodies to measure antigens that specifically react with the antibodies, and is used for investigations such as identification of infectious disease pathogens and drug identification.

(Means for Solving the Problems) The present invention involves immobilizing a substance with affinity for an antigen on a solid phase, adding the antigen, and then binding a labeled substance to the complement component and, if necessary, the antibody. This is a method for measuring antigens, which is characterized by adding one reaction, removing unbound substances, and then subjecting the fabric to one plate.

Next, the present invention will be explained in detail.

In the present invention, the substance having affinity for the antigen refers to a substance that is easily absorbed by the antigen, and 11 substances can be used. For example, antibodies, sites that harbor antigen-binding groups of antibodies (e.g. Fab, Ftab', F(ab')2%)
, ``substrates or inhibitors of 1 yen, protein A held by staphylococci, divine drugs possessed by certain living organisms, receptors for viruses, etc.In addition, biological tissues containing substances that have affinity for antigens. It can be used even if it is not made from 4L1.These substances with antigen KQ affinity are immobilized on a solid phase.
Operations such as cleaning become easier. The solid phase may be any material as long as the substance that has affinity for the adsorbed antigen does not easily detach.
For example, synthetic polymers made of plastic materials such as polyvinyl chloride or polystyrene, natural polymers such as paper,
Microtiter wells, polystyrene beads, and the like are typical examples where cells or structured tissues can be used. In addition, physical adsorption,
Solid-phase surface #IK due to biological covalent bonding or fcFi infection etc.
If you fix it, you'll get seven. In addition, when cells or tissues are used as the solid phase, if a substance having affinity for the antigen is already bound, it is not necessary to artificially fix the substance to the tissue twice.

Next, a wave-breaking antigen is added to the immobilized substance that has an affinity for the antigen, and the antigen is bound to the substance that has an affinity for the antigen. The antigen and antigen used herein are the targets of the measurement of the present invention, and are not limited in any way as long as they have the property of binding to a substance that has an affinity for antigens, rather than antibodies. Typical examples of antigens include viruses and +1' (TI bacteria and other microorganisms and their products, biological components in animals and proboscis, physiological substances in plants, drugs, etc.). Examples include facial fluids, urine, spinal fluid, saliva and other body fluids, animal meat,
Aqueous solutions obtained from processed products such as plants, rivers, sewage, wastewater, etc. are used.

Note that when using a sample in which an antigen is already bound to a solid phase made of cells or tissue, it is of course not necessary to add the antigen.

A complement component bound to a labeled substance is added to the bound product of the antigen and a substance that has affinity for the antigen. Complement is animal @
Tl is composed of more than ten types of proteins contained in fresh blood, and is a group of proteins involved in defense against infection, immune response, inflammation, etc. of living organisms. Complement is mainly activated by antigen-antibody complexes, and exhibits amazing biological activities such as immune conjugation reactions, anti-phagocytosis reactions, agglutination reactions, sterilization and malignancy reactions, and solubilization of immune complexes. The complement component may be any component of the sleeve system, such as complement Cd, C2, C5゜(lq
etc. can be selected as appropriate. In particular, complement C1q is the part of the complement system that first binds to antigen-antibody complexes.

Complement C1q can bind to antigen-antibody complexes independently without regard to other complement components; therefore, complement C1q
By labeling complement C1q, it is possible to measure the antigen titer without using any other complement components, so it is particularly preferable to use complement C1q because it simplifies the measurement.

As a FA preparation method for the sleeve component, a general separated phase production method for proteins with physiological activity can be used. For example, the adjustment method described in "Complement Science", Ishiyaku Publishing, 1981, pp. 141-158 can be used, but the method is not limited to this method.

As the label to be bound to the complement component, any burr can be used as long as it has a V quality that can be easily measured by common analytical methods. For example, enzymes, coenzymes, d? round substance, pigment,
The nature of the enzyme can be selected as appropriate.

Since the r17 powder acts as a catalyst at 7 o'clock, the reaction temperature,
By changing the reaction time, the measurement sensitivity can be freely adjusted. Otherwise, i1% element amplifies a trace amount of antigen (;1
It is a more preferable label because it can be determined by As the enzyme, for example, gluoxidase, alkaline phosphatase, β-galactosigase, alcohol hydrogenase, etc. can be used.

/rli fT rit component component The binding of the labeled substance is between the complement's proboscis l paleo e+ and the
Any bonding method may be used as long as it does not cause any harm. When a binder is used, periodic acid, glu, taraldehyde or maleimide derivatives can be used.

Furthermore, add the antibody by adding L and S. If there is an antibody or a part of the antibody that has a parent binding group for the antigen, or a part of the antibody that has a sleeve-binding group, It is not necessary to add the antibody again.However, when using a substance having the requirements for anti-p(1) and without a cuff binding group, it is necessary to add the antibody again. It is necessary to add an antibody to bind the body components.When adding the antibody, there are no restrictions as long as it is after the addition of the antigen.
It may be applied at the same time as the K component or before or after. The antibody used here must, of course, specifically bind to the antigen to be analysed, and must also be capable of binding to complement components. Generally, antibodies are immunoglobulins contained in the serum of animals, and have complement-fixing properties.
gM, IgG, etc.

Antibodies can be produced by using natural antibodies present in serum, or by administering or infecting a human with a bacterial antigen. Antibodies are immunoglobulin purified and separated from blood a, or non-1-clined blood (1?
But it can be used.

There are no particular restrictions on the conditions for the reaction between the antigen, the antigen and the label, the sleeve components, and the antibody.Just by mixing these, a reaction will occur.
(The reaction time and temperature will vary depending on the angle of the antigen, etc., but those skilled in the art can easily set the reaction conditions.The point is that the biological activity of complement Conditions that do not cause deactivation can be selected as appropriate.

Since the conjugate, which consists of substances that have practical application to antigens, antigens, and complement components bound to labeled substances, is immobilized in the same phase, unbound complement components and reaction inhibitors can be removed by washing. It's easy to do.

The label of the conjugate adsorbed to the same phase is then quantified. As a quantitative method, in addition to visual measurement, various types of autopsy using 11 to 20 years of age, measurement of absorbance of visible and ultraviolet IfJl, pulse count measurement of fluorescence intensity 1, etc. can be used as appropriate. For the quantitative determination of the labeled substance, we not only directly measured the amount of the labeled substance in the conjugate, but also used a certain amount of the labeled substance and then not subjected it to binding;
Therefore, it can be quantified.

By quantifying the labeled substance, the specific amount of antigen can be determined.

(JAI adjustment f+ll of oil body Ij' blue composition) IflLr'ft 100 me, O, Cl
The resulting precipitate was dialyzed against 5g of a 26M+-ethylene glycol tetraacetic acid (EGTA) aqueous solution (pH 7,5) for 15 to 24 hours, collected by centrifugation (20,000G, 20 minutes), and 0.01M Ethylenediaminetetraacetic acid (E
Dissolve in 20 ml of 0.75 M sodium chloride aqueous solution (pH 5,0) containing "D'rA) ) T5.0) 5
The precipitate was centrifuged (2
0.0OOG, 20 minutes). This procedure yields about 3i proteins, of which more than 95i are complement C1q. Next, the above protein was added to 0, +15 M
) An aqueous solution (p) containing lis(hydroxymethyl)aminomethane, 1M sodium chloride, 0.005M EDTA and 10 liters of sucrose (p) [4) It can be dissolved in 1 tnl and provided to the present invention as complement C'lq. Furthermore, the above operation may be repeated to further purify complement 1CIq.

(Example 1 of g1'4 complement component bound to labeled substance) Rabbit purified C1q3 [1~ was mixed with 0.05 M tris(hydroxymethyl)aminomethane, 1 M sodium chloride,
Dissolve in an aqueous solution (pH 7,4) containing 0.005 mM F, DTA and 10 tisucrose.

Add 0.1 ml of 0.1M dithiothreitol solution to this and leave to react at room temperature for 1 hour. Next, the reaction solution is passed through a Sephadic G-25 column and the protein fraction is collected. This protein content is filtered through ultra filtration to approximately 10m/I! % reduction to obtain reduced complement C'1q30~.

Next, wasabi-derived wasabi oxidase 20 ~ was dissolved in 6 ml of phosphate buffer (pl(7,li)), and dimethylformamide A ml was added.Additionally, 2% dC maleimidomethyl]duclohexane 1-carboxylic acid Add 0.2 ml of succinimide ester (CHMJ dimethylformamide solution) and allow to react at room temperature for 1 hour. Pass the reaction solution through a Sephadex G-25 column to recover CHM-bound, 'oxyguse 20+q.

For reduced complement C1q30~ and C) IM binding, mix Roxyguse 20~ and incubate for 15 hours at ~10°C.
With a Sepharose 6B column, molecular weight of 400,000 ~
A total of 800,000 ryo samples were collected, and oxidase-labeled complement C14 was collected.
Get 40~.

(Column 2 of complement components bound to labeled substance) 2~/
Add at complement C4 physiological saline solution 1-K, 0.2 cape fluorescein isothiocyanei) (FIT (1-containing carbonate buffer (f) + 19.5) 0.1-, and stir slowly at 10°C for 4 hours. FIT which is a fluorescent substance in operation
C binds complement C4. By passing this reaction product through a Sephadex G-25 column, unreacted FTTC was separated and removed, and approximately 1.81% of FITC-bound complement C4 was obtained. Collect Ii.

Example 1 A patient's cervical swab solution (0.1 iK) was placed in a 96-hole microtiter well to which a virus antibody (Fab) had been adsorbed, and the guinea pig anti-simple virus antibody (Fab) was placed at room temperature for 60 minutes. PBS (phosphate buffer βIZ4 containing 0.85% sodium chloride)
The non-mobilized guinea pig anti-simple virus was washed 3 times and diluted at the appropriate time in each well. ? ? (Complement-fixing antibody titer 16 to 52) 0.05- and boruoxidase-labeled complement C1q0,05me were added, and the mixture was allowed to stand for 60 minutes under electrofusion. Next, PBS containing 0.05% Tween 20
Wash each well six times with t,o,-ABTS (
Hydrogen peroxide-containing 2゜2'-azinodi-(5-ethyl-
0.1 rnt of benzothiazoline sulfate) solution
was added and reacted for 1 hour at room temperature. Next, after adding 0.05 g of 0.054 sodium nitride aqueous solution, which is an enzyme reaction stop solution, the absorbance at 414 nm was measured, and the absorbance value was 0.030 for cervical swabs from herpes simplex virus-negative patients, and 0.030 for positive patients. So 0.11'5.0.50
Values such as 0.0.550 were obtained.

Example 2 Goat anti-hepatitis virus antibodies were immobilized on polystyrene beads with a diameter of 6.55 and transferred to a small test tube.

Next, 0.1- and N
Dilution of complement C4 labeled with AD (cotinamide adenine dinucleotide) in the same buffer i0.1rn1.
Furthermore, 0.1 rnt of fresh rabbit serum treated with anti-C4 antibody was added, and the mixture was shaken for 1 hour in a constant temperature water bath at 37°C. Next, the pieces were washed with PBS.

Separately prepared NAD quantification reagent 2-1 + 50 units alcohol dehydrokenase, 0.25 μM ethanol, 1μ
The beads were placed in 50mM Tris.HCLfl containing Mnitroprutetrazolium and 2 units of diaphorase, pH 9.01, and reacted at 67°C for 15 minutes. The absorbance of the reaction solution at 550 nm was measured. The 550 nm absorbance value for negative serum is 0.092, and for positive serum it is 0.092.
It showed a value of 100 or more.

Example 6゜ Lymphocyte fraction was prepared from mouse tile cells according to a conventional method,
After washing with PBS, incubate the cells with PBS 4 degrees I x 107/
-I adjusted my schedule. Anti-mouse 'rhy- with its 0.1rnt
i, 2 0.05nIt of allo serum and FITC [complementant C1qO,05-] were mixed and left at room temperature for 1 hour. The cells were then thoroughly washed with PBS and observed under a fluorescence microscope. As a result of observation, 37% of the cells showed fluorescence.

Example 4. Phosphorus in the serum of a patient with leukemia W. 108 o-balls were suspended in 1 mL of phosphoric acid slowing solution and treated with an ultrasonic cell disrupter for 2 minutes. Add DNA Sepharose 0.5 to the centrifuged supernatant.
rn1. was added and reacted at 37°C for 60 minutes. D.N.
A Sepharose contains DNA, +? Limerase, terminal deoxynucleotide transferase (TdT)
DNA-related enzymes such as Next, 0.1 mt each of peroxidase-labeled complement C1q and immobilized rabbit anti-TdT serum were added, and a reaction was carried out at 37°C for 30 minutes.

Next, the DNA Sepharose was washed thoroughly with PBS, and 1 l of KH,O,-ABTS solution was added thereto at 7°C.
After reacting for 60 minutes, 1 ml of a 0.05% enriched sodium aqueous solution as a reaction stop solution was added, and the absorbance of the supernatant at 414 nm was measured. TdT at absorbance value below 0.075
The test result was negative, and the absorbance value was 0.100 or higher, indicating TdT positive, and it was determined that the patient had sexually transmitted leukemia.

(Effects of the Invention) According to the present invention, antigen sensitivity can be measured easily and with high accuracy. Furthermore, by measuring the antigen according to the present invention,
The results of clinical tests for infectious diseases, etc. can be obtained quickly, and appropriate treatment can be carried out at an early stage. Also, so-called doping tests and determining the type of meat can be easily carried out. .

As described above, the present invention is an industrially extremely useful method for measuring antigens.

Claims (1)

[Claims] 1. After immobilizing a substance that has affinity for the antigen on a solid phase, the antigen is added, and then a complement component to which a labeled substance is bound and, if necessary, an antibody are added to carry out a reaction. A method for measuring an antigen, which comprises quantitating a labeled substance after removing unbound substances. 2. The method for measuring an antigen according to claim 1, wherein the complement component is complement C1q. 3. The method for measuring an antigen according to claim 1, wherein the label is an enzyme.