JPH0989891A - Method and kit for measuring antigen or igm antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a target antigen by means of a uniform system of a liquid phase alone without using a solid phase, by making a complex of an IgM antibody and an antigen react with a non-active complement component C1, forming an active C1, and measuring a signal intensity. SOLUTION: An antigen, e.g. measles virus in a sample is reacted with, e.g. an IgM antibody such as measles IgM antibody or its analogue, thereby to form an antigen-antibody complex. A non-active complement component C1 is reacted with the complex, whereby an active C1 is formed. A signal- generating substrate (e.g. Z-GIy-Arg-SBu-i) is reacted with the formed active C1, and hydrolyzed through an enzymatic action. An intensity of a generated signal at this time is measured, and compared with a working curve formed by the antigen or antibody of the known amount. The amount of the antigen or antibody in the sample is consequently obtained. According to this method, such instable factors as labels, red corpuscles, etc., are not used, fixation of a solid phase and separation of BFs are eliminated, and a uniform system is served for manipulations, whereby measurements are achieved easily in a short time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for assaying an antigen or an IgM antibody using complement and a kit therefor.

[0002]

PRIOR ART AND PROBLEM TO BE SOLVED BY THE INVENTION Recently, a method for quantifying an antigen or antibody protein utilizing an antigen-antibody reaction has been remarkably developed. This includes those that use precipitation reactions, agglutination reactions, lysis reactions, complement fixation reactions, etc., and solid-phase methods that use beads or latex particles. Many of them use a label, which is called a radioimmunoassay, an enzyme immunoassay, a luminescence immunoassay, or the like depending on the type of the label. However, these measurements are unstable reagents, require a lot of human intervention,
In addition, depending on the label, an expensive dedicated analyzer may be required, leaving some problems.

As a measuring method utilizing the reaction of complement and antigen-antibody complex in the above-mentioned measuring method of antigen or antibody, a method in which an antibody bound to erythrocyte is reacted with antigen and complement to cause a hemolytic reaction Has already been proposed (Tokuhei 2-30
664 and Japanese Patent Publication No. 2-30665). But,
This method has the disadvantage that the red blood cells used are unstable. There is also a method (Japanese Patent Laid-Open No. 5-264551) in which a liposome encapsulating an enzyme is sensitized with an antigen, an antibody and a complement are reacted with the liposome to destroy the liposome, and the eluted enzyme is used as a marker substance. Further, there is also a method (JP-A-5-60758) in which a liposome encapsulating a fluorescent dye as a marker substance is used instead of the enzyme.

These conventional methods require that a marker substance be encapsulated in liposomes, and complement C1.
~ C9 active chain reaction is used, and as a complement, C1 ~
You have to prepare C9. Therefore, the present inventor, when developing a novel method for quantifying a protein, is caused not by the hemolytic reaction or lytic action of complement, but by the transfer of complement to an active form when the complement and the antigen-antibody complex react. We have continued to develop, considering the use of enzyme activity.

The principle of this measuring method is as follows. It is known that the mechanism of complement system activation includes the classical pathway and the alternative pathway. The classical pathway is activated primarily by immune complexes. The components C1 to C9 are known to be complement, and these are C1, C4, C2, C3 and C.
5, C6, C7, C8, C9 are activated in this order, and the finally formed C5b-C9 complex acts to cause a cytolytic reaction such as hemolysis or lysis. The above-mentioned prior arts ~ utilize this classical route.

In the measuring method of the present invention, first of all, the complement component C
Only C1, which is the first component of 1 to C9, is used, and it was considered to utilize quantitatively the reaction in which C1 directly binds to the antigen-antibody complex formed according to the amount of the antigen or antibody.

To explain this in more detail, the first component, C1, is Clq, C1r, C1 via Ca ^++.
Although each component of s is a high molecular weight polymer with a molecular weight of 700,000, Clq directly binds to the Fc part of the antibody of the antigen-antibody complex generated by the antigen-antibody reaction, and thereby activates C1r. It will be a factor that causes. At that time, Clq needs to directly bind to the Fc portion of the antibody of the two immune complexes.

C1r and C1s are zymogen-type protease precursors, which are activated C1r activated by Clq.
Acts as an activator of C1s, and activated C1s and activated C1r hydrolyze a synthetic substrate to develop a color. Active forms of C1r and C1s are enzymes belonging to serine proteases and are synthetic substrates with high specificity for C1-esterase (eg C
It can be measured using H ₃ OCO-Lys (ε-Cbo) -Gly-Arg-pNA.AcOH).

The present inventor has already bound I to the solid phase at a predetermined interval so that C1q can bind to two Fc simultaneously.
A method for quantifying a sensitive antigen or antibody by using a gG antibody has been disclosed (JP-A-4-241791).
issue). That is, this method uses IgM instead of IgM antibodies.
It utilizes the complement activating action of G antibody, and as described above, in order for Clq to become an activator, it is necessary to directly bind to the Fc part of two immune complexes. Therefore, a solid phase was essential to immobilize IgG at optimal intervals at positions where Clq could directly bind to IgG of two immune complexes. Therefore, the present inventors have conducted further studies for the purpose of avoiding the complexity of using a solid phase.

[0010]

Means for Solving the Problems In the present invention, it was found that it is possible to eliminate the need to bind IgG to a solid phase at a predetermined interval by using an IgM antibody or an IgM antibody-like substance instead of an IgG antibody. It has been completed. That is, in the pentamer IgM, Clq seems to have a convenient positional relationship for binding to two immune complexes. Therefore, the use of IgM or IgM antibody-analogous substance has the advantage that the measurement can be performed in a homogeneous system having only a liquid phase without using a solid phase.

That is, the present invention relates to (1) an IgM antibody or an IgM antibody or an IgM antibody in a sample, respectively.
A gM antibody analog or an antigen is reacted to form an antigen-antibody complex, which is reacted with an inactive complement component C1 to form an active form C1. A homogeneous system in a liquid phase in which a signal is generated by reacting with a signal-generating substrate which is hydrolyzed by an action to produce a measurable signal, and the intensity of the signal is measured and correlated with the amount of an antigen or an IgM antibody. The method for measuring an antigen or an IgM antibody, which is characterized in that

Further, (2) the inactive complement component C1 may be accompanied by a protease inhibitor, and
(3) An antigen containing (a) an IgM antibody or an IgM antibody analog, or an antigen, (b) a non-active complement component C1 or a composition containing the same, and (c) a substrate for measuring an active C1 enzyme activity. Alternatively, a kit for IgM antibody measurement is provided.

The above-mentioned "sample" includes any object suspected of containing the antigen or IgM antibody to be measured or intended by the measurer as the object of measurement.
The sample is usually a liquid, such as a solution, emulsion or suspension, which is either chemically prepared, is a wash obtained by washing a container, or is a living organism (e.g. vertebrate, preferably It may be a liquid derived from a mammal, especially a human. An example of a sample is a clinical sample, which includes blood (plasma, serum), lymph, spinal fluid, tissue fluid, saliva, tear fluid,
Including gastric juice, sweat, urine, etc.

The "antigen" to be measured includes all compounds and mixtures having an antigenic determinant or capable of inducing an IgM antibody. Examples of antigens are biological components (eg tumor-associated antigens, viruses, hormones, etc.),
Pharmaceuticals (eg, antiepileptic agents, cardiac glycosides, bronchodilators, antibiotics, etc.) and anaphylaxis inducers (eg, pollen, pests, etc.).

[Ig used for measurement capable of binding to antigen]
"M antibody" includes a polyclonal antibody and a monoclonal antibody. Polyclonal antibodies can be produced, for example, by injecting or contacting the antigen with an antibody-producing animal (eg, vertebrate, preferably mammal) or its antibody-producing system. The monoclonal antibody can be obtained, for example, by culturing antibody-producing cells obtained from a sensitized antibody-producing animal or fused cells of the antibody-producing cells and immortalized cells. The IgM antibody is obtained by separating it from the produced polyclonal antibody or monoclonal antibody. The IgM antibody-like substance refers to a substance having a structure or / and immunologically similar characteristics to an artificially synthesized IgM antibody, not an IgM antibody obtained by being biologically produced.

The "IgM antibody" to be measured is one that finds clinical significance in measuring IgM type antibodies, for example, Toxoplasma IgM antibody, measles I.
Examples thereof include gM antibody, rubella IgM antibody, Chlamydia trachomatis IgM antibody, and cold agglutinin IgM antibody.

As the "antigen" which can bind to the above IgM antibody and is used for the measurement, Toxoplasma, measles virus, rubella virus, Claudia trachomatis, cold agglutinin and the like are used.

An "antigen-antibody complex" is a conjugate of an antigen and an antibody, which is the result of a reaction between them. It should be noted that, at the same time as the IgM antibody, the IgG antibody also forms an antigen-antibody complex
The convenient presence of two IgG immune complexes at the position where non-active complement C1 may bind is possible, but negligible as it is considered very rare.

The "inactive complement component C1" is the component C1 or a mixture thereof which has not yet acquired enzyme activity. Here, the mixture includes intact complement itself and the fraction containing C1. The inactive Cl can be prepared by a conventional method such as human or animal serum, citrate-supplemented or EDTA-supplemented plasma (preferably fresh) is used as a starting material, and a conventional method for purifying complement C1. Purification is carried out by a certain globulin precipitation method or gel filtration method, or by affinity chromatography with IgG-Sepharose followed by gel filtration.

"Active C1" is a state in which enzymatic activity is acquired by, for example, a conformational change resulting from the binding of C1 to the Fc portion of the antibody of the two antigen-antibody complexes.

The "signal-generating substrate" is a molecule having an atomic group that can be detected by, for example, radiating or absorbing a wave having a specific wavelength and a portion that can be changed by an enzymatic reaction, and is an atom by an enzymatic action. It is a substance that can transform into a detectable form. Examples of such atomic groups are o- or p-nitro or 2,4-dinitrophenyl. Examples of substrates are Z-Gly-Arg-SBu-i, Z-Ala-
Arg-SBu-i, Z-Gln-Leu-Gly-Arg-SBzl, Z-M
et-Gln-Leu-Gly-Arg-SBzl, Z-Met-Gln-Leu-
Gly-Arg-Ala-DBHA, Z-Met-Lys-Gly-Arg-A
For example, la-DBHA. Suitable substrates for enzymatic activity are commercially available. Such substrates include, for example, CH
₃ OCO-Lys (ε-Cbo) -Gly-Arg-pNA.AcOH.

"Measuring the intensity of a signal and correlating it with the amount of an antigen or antibody" means, for example, making a calibration curve from the intensity of a signal obtained using a known amount of an antigen or antibody, and measuring the signal obtained from a sample. By the method of comparing with strength,
To calculate or plot the amount of antigen or antibody from the intensity of the signal.

The buffer system, stabilizer, activator, surfactant, preservative and proteolytic enzyme inhibitor (protease inhibitor) usually used can be added to the assay system of the present invention.

Examples of the buffer solution include common buffer solutions such as tris (hydroxymethyl) aminomethane, phosphoric acid / boric acid and salts thereof, veronal buffer solution and Good's buffer solution. Stabilizers include glycerol, lactose, sucrose, sorbitol, ethylene glycol, and other polyols, albumin, gelatin, and other proteins, glutathione, mercaptoethanol, dithiothreitol, and other SH group-protecting agents, and EDTA and other chelating agents. Be done. Examples of the activator include polyethylene glycol (P
EG), polyvinylpyrrolidone (PVP) and the like. Sodium dodecyl sulfate (SDS) is used as a surfactant.
Anionic surfactants such as, cationic surfactants such as dodecyltrimethylammonium chloride (DTAC), amphoteric surfactants such as N-dodecyl-betaine,
Non-ionic surfactants such as polyoxyethylene alkyl ethers, steroidal surfactants such as sodium cholate, etc., but are not limited thereto. Preservatives include sodium nitride, boric acid, phenol and various antibiotics.

Examples of the protease inhibitor include general protease inhibitors such as serine protease inhibitors, metalloprotease inhibitors and cysteine protease inhibitors, particularly soybean trypsin inhibitor, ε-aminocaproic acid, serpin and diisopropylflurophosphoric acid. 4- (2-aminoethyl) -benz
The serine protease inhibitor family such as enesulfonyl fluoride is preferred. The protease inhibitor is preferably mixed with the inactive complement component C1.

An example of a preferred method of implementation is as follows. A sample containing or suspected of containing the antigen or IgM antibody is added to the IgM antibody or IgM antibody analog or antigen to form an antigen-antibody complex. To this, C1 solution with protease inhibitor is added, and further a signal generating synthetic (artificial) substrate solution is added. The absorbance is measured at the absorption peak of nitrophenyl group at 405 nm. From the measured value, the amount of antigen or IgM antibody in the sample is known by a calculation formula or by comparison with a calibration curve.

More specifically, as an antigen measuring method,
An antigen that contains or is suspected of containing an antigen to be measured, and I showing specificity to the antigen to be measured
gM antibody or IgM antibody analogue is added in an amount of 0.01 μg / dl-
Buffer containing 100 mg / dl, preferably 1 μg / dl-5 mg / dl, more preferably 100 μg / dl-500 μg / dl (pH
4-10, preferably pH 6-9, more preferably pH
7-9, the reaction temperature is 4-40 ° C, preferably 25-37.
C., more preferably 37.degree. C.) for a certain period of time, and the complement C1 solution is added at 0.1 .mu.g / dl to 200 mg / dl, preferably 1
0 μg / dl to 20 mg / dl, more preferably 1 mg / dl to 5 mg / d
l is added and the reaction is continued for a certain period of time (pH 4 to 10, preferably pH 6 to 9, more preferably pH 7 to 9, reaction temperature is 4 to 40 ° C., preferably 25 to 37 ° C., more preferably 37 ° C.). Then, the synthetic substrate solution is added from 0.01 to
100 μmol, preferably 0.1 to 10 μmol, more preferably 0.5 to 5 μmol was added and immediately the change in absorbance was measured with time using a constant-heated kibbet. The concentration of the antigen analyte is calculated from the known calibration curve based on the change in absorbance per unit time.

As a method for measuring the IgM antibody, an IgM antibody sample containing or suspected of containing the IgM antibody to be measured, and an antibody-specific antigen of 0.01 μg
/ dl to 100 mg / dl, preferably 1 μg / dl to 5 mg / dl, more preferably 100 μg / dl to 500 μg / dl (pH 4 to 10, preferably pH 6 to 9, more preferably pH 7 to 9, reaction) The temperature is 4 to 40 ° C., preferably 25
To 37 ° C., more preferably 37 ° C.) for a certain period of time, and the complement C1 solution is added to 0.1 μg / dl to 200 mg / dl, preferably 10 μg / dl to 20 mg / dl, more preferably 1 mg / dl.
Add ~ 5mg / dl and react for a certain time (pH 4
-10, preferably pH 6-9, more preferably pH 7
~ 9, the reaction temperature is 4 ~ 40 ℃, preferably 25 ~ 37
C, more preferably 37 C). Then, the synthetic substrate solution is added in an amount of 0.01 to 100 μmol, preferably 0.1 to 10 μm.
ol, more preferably 0.5 to 5 μmol, is immediately added, and the change in absorbance is immediately measured with a kibbet which is heated at a constant temperature. The concentration of the IgM antibody test substance is calculated from the known calibration curve by the change in absorbance per unit time.

In order to carry out the above method as a clinical test, it is convenient to prepare a kit of the instruments and reagents to be used. Such a kit can include, for example, an antigen, an IgM antibody or an IgM antibody analog, an inactive C1, a signal-generating synthetic substrate, a buffer, and instructions describing the method of use.

[0030]

INDUSTRIAL APPLICABILITY The method of the present invention as described above can obtain various advantages in terms of operation, instruments used and reagents without using a label. Further, the operation is easy without using unstable things such as red blood cells. Furthermore, since solid phase immobilization and BF separation are not required at all and the procedure is carried out in a homogeneous system (homogeneous system), the procedure can be carried out in a short time, and there is an advantage that it can be used for various antigens or IgM antibodies.

[0031]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. Example 1 Human C-reactive protein (CRP) standard serum (Yatron) and anti-human CRP rabbit IgM antibody (homemade) were used as materials for the antigen-antibody complex. Inactive Cl was purified from human serum by a commonly used method. The signal-generating substrate is a synthetic substrate CH ₃ OCO-Lys- (ε-Cb for measuring Cl esterase.
o) -Gly-Arg-pNA.AcOH (Pentafarm:
Switzerland) was used. The C1 solution was added to the resulting antigen-antibody complex, and the substrate solution was further added. Color development absorption peak 405
The amount of antibody was determined by measuring the absorbance at nm.

Materials (a) 50 mM Tris-HCl buffer (250 mM NaCl, 5
(pH 8.4 containing mM CaCl ₂ ) (b) Anti-human CRP rabbit IgM antibody ((a) Protein amount 40 μg / ml in buffer) (c) Inactive Cl (200 μg / ml) (d) Synthetic substrate ( 3 μg / ml) (e) Test sample CRP standard serum

Method (1) Dispense 200 μl of the reagent (a) into a test tube. (2) Add 25 μl of antibody solution. (3) Add 25 μl of test sample. (4) Add 50 μl of Cl solution. (5) Incubate at 37 ° C for 5 minutes. (6) Add 100 μl of the synthetic substrate solution, and immediately measure the absorbance at 405 nm in a cuvette heated to 37 ° C. over time. (7) Calculate the amount of protein from the calibration curve based on the change in absorbance per unit time. (8) Absorbance at antibody concentration is calculated by the following formula. Measuring absorbance _{= (S T -S I) -} (RB T -RB I) S T: reaction time the absorbance of the subject in (T) S _I: Final reagent analyte absorbance after the addition (I _<T) RB T : Absorbance of reagent blank at reaction time (T) RB _I : absorbance of reagent blank after final reagent addition (I <
T)

Results The results obtained in Example 1 are shown in Figures 1 and 2. The reaction curve at each CRP concentration obtained by serially diluting CRP 9 mg / dl is shown in FIG. FIG. 2 shows the change in absorbance from 340 seconds to 400 seconds (1 minute) of the reaction curve of each CRP concentration as a concentration calibration curve. The specimen prepared here (10/10 = CRP 9 mg /
The graph (FIG. 2) showing the sigmoid curve in dl) is 8 /
It was shown that up to 10 measurements were possible. A sample of unknown concentration can be quantified using this sigmoid curve as a standard curve. Some recent analyzers measure several kinds of concentrations, create a multi-inspection curve, and automatically calculate the concentrations, so they can be applied to them.
The protein concentration and measurement time in this example of CRP are examples, and the optimum protein concentration and measurement time are set according to the sample to be measured.

[Brief description of drawings]

FIG. 1 is a graph showing a reaction curve of each concentration obtained by diluting CRP 9 mg / dl.

FIG. 2 is a graph showing a calibration curve of CRP.

Claims (3)

[Claims] 1. An IgM antibody or Ig as an antigen in a sample
IgM in sample reacted with M antibody analog
An antigen is reacted with an antibody to form an antigen-antibody complex, an inactive complement component C1 is reacted with this to generate active C1, and the active C1 is reacted with a signal-generating substrate to generate a signal. And measuring the signal intensity and correlating it with the amount of the antigen or IgM antibody in a homogeneous system only in the liquid phase. 2. The assay method according to claim 1, wherein the inactive complement component C1 is associated with a protease inhibitor. (A) The antigen or IgM antibody in the sample,
A homogeneous liquid phase containing a specific IgM antibody or IgM antibody-like substance or antigen, (b) non-active complement component C1 or a composition containing the same, and (c) a substrate for measuring active C1 enzyme activity Kit for measuring an antigen or IgM antibody in a system.