JP2670498B2 - Immune complex assay - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel immunological assay method for an immune complex (Immune Complex, hereinafter abbreviated as “IC”).

[Conventional technology]

Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA)
In collagen diseases such as, IC is detected in the bloodstream at a high rate, and its clinical significance has been examined in detail, but in recent years, in many other diseases other than collagen diseases such as malignant tumors and blood diseases. In addition, IC is recognized at a high rate, and its clinical significance and role in the pathological condition are being analyzed.

An antigen invading or appearing in a living body binds to an antibody to form an IC, but is generally rapidly removed by reticuloendothelial cells. However, a large number of ICs are generated,
When untreated by reticuloendothelial cells, IC deposits in tissues and causes its damage.

Therefore, the measured blood concentration of IC is information that gives a clue to the diagnosis of these diseases, and the transition of the concentration is valuable information for grasping changes in the pathological condition and as an index of treatment. .

In recent years, many methods have been developed to quantitatively measure this IC, which is present in a trace amount in blood, and the number of ICs exceeds 30 types. When various measuring methods are roughly classified according to their measuring principles, methods that utilize the property that IC activates complement (Clq solid-phase method, etc.).

A method of measuring IC using the physicochemical properties of IC (eg, polyethylene glycol method).

Cellular receptors (complement receptor and Fc receptor)
Method that utilizes the property that IC binds to (Raji cell method, etc.).

And so on.

[Problems to be solved by the invention]

Although there are many methods for measuring IC as described above, good correlation is not always obtained among the measurement values obtained by each measuring method. The reason for this is that, in addition to technical problems such as sensitivity and reproducibility, there is the interference effect of the test serum specific to each measurement method, and the IC to be detected.
It is said that there is a problem with the diversity of.

However, among them, the method using Clq is considered to be clinically useful, considering that many of the tissue disorders observed in autoimmune diseases are caused by complement fixing IC. In principle, the classical path (clas
Clq is the first complement component that binds to the IC that activates complement via the sical pathway).
lq solid-phase assay (CSP method), Clq deviation test
(CDT method) and Clq binding assay (CBA method) are typical methods. Among them, the CSP-EIA (CSP-enzyme immunoassay) method is currently widely used because of its specificity and ease of operation.

This method begins with the solid phase (plate or tube)
Immobilize Clq. Then, a sample (serum) from which the endogenous complement of IC binding has been removed by EDTA treatment is added to capture the IC. Subsequently, after washing, it is reacted with an enzyme-labeled anti-human IgG antibody to bind to the human IgG component in IC. After further washing, IC is measured by colorimetrically quantifying the enzyme amount of the bound enzyme-labeled IgG antibody by a color reaction due to a reaction with a substrate. Although this method is relatively superior to the other method, it has the following problems.

When immobilizing Clq, Ig was added as impurities to the Clq solution.
When G is mixed, the IgG is also immobilized. Finally, the enzyme-labeled IgG antibody used as an indicator binds to the IgG component in the IC and is immobilized I
It also binds to gG. As a result, the chromogenic reaction is raised by the amount of immobilized IgG, which narrows the dynamic range of this measurement system and thus narrows the IC detection width. This also leads to a decrease in the sensitivity of this measurement system.

By the way, even if assayed as a single standard by a protein chemical assay method (SDS-polyacrylamide electrophoresis method, disc electrophoresis method, immunoelectrophoresis method, etc.),
In the CSP method, the effect of contaminant IgG at a level that cannot be detected by the protein chemical assay is often not negligible. In addition, it is highly possible that the above-mentioned degree of IgG is mixed in by the Clq purification method which is generally used at present.

[Means to solve the problem]

In view of such circumstances, the present inventor has made diligent studies in order to utilize the advantages of the CSP-EIA method as it is and to eliminate the drawbacks of the method. As a result, the IC has a plurality of Clq binding sites (at least two places).
Focusing on the characteristics of ICs that exist above, we have devised a completely new Clq sandwich (CSP-EIA) method that does not use enzyme-labeled IgG antibody, that is, sandwiching with Clq and enzyme-labeled Clq (indicator). Therefore, it was presumed that the problems of the current CSP-EIA method using the enzyme-labeled IgG antibody could be solved by switching to the use of the enzyme-labeled Clq. Then, as a result of earnest research, it was confirmed that the present measurement method was possible, and the present invention was achieved.

That is, in the present invention, Clq is first immobilized on a solid phase (plate or tube). Then, a sample (serum) from which the endogenous complement of IC binding has been removed is added by EDTA treatment, and the IC is captured. Then, after washing, react with substance-labeled Clq,
It binds to the human IgG component in C. After further washing, IC is measured by quantifying the bound substance-labeled Clq substance.

Next, the present invention will be further described with reference to preparation examples and examples, but the present invention is not limited thereto as long as the gist thereof is not exceeded.

Preparation Example 1 Purification of Clq Animal serum such as human is used as a raw material for the solid-phased Clq and the substance-labeled Clq of the present invention. Several methods are known for this production method, among which the method of increasing rice (K.Yonemasu and RMstroud: Journal of Immun
ology, 106, 304-, 1971) has the highest yield and can be purified in a short time. In this method, Clq was obtained by further improving this purification method.

Preparation Example 2 Preparation of peroxidase-labeled Clq IJ Simpson's method (Journal of Immunological Me
thods, 67, 167-172, 1984), Clq was labeled with peroxidase. The cross-linking method may be the glutaraldehyde method or the maleimide method instead of the periodoic acid method of this method.

Example 1 Peroxidase labeling as an indicator
CSP (EIA) method using Clq 96-well microplate (made of polystyrene) Preparation example 1
The Clq purified by the method described above was adjusted to a concentration of 10 μg / ml with PBS, 200 μm each was added, and the mixture was left at 4 ° C. overnight for coating.

Next, the Clq solution was aspirated and discarded, and then each well was washed 3 times with PBS. Then, PBS containing 2% BSA was dispensed in an amount of 250 μm and left at room temperature for 2 hours for blocking.

After discarding the liquid and washing 3 times with PBS, a simulated I
150 dilution series of AHG (Aggregated human IgG) which is C
Each μ was dispensed into each well and left at room temperature for 3 hours.

After aspirating and discarding the reaction solution and washing 3 times with PBS, 150 μl of peroxidase-labeled Clq was dispensed into each well,
Leave at 4 ° C. overnight.

The reaction solution was aspirated and discarded, and after washing three times with PBS, a substrate solution containing 2,2'-azinodi- [3-ethylbenzthiazolinesulfonic acid (6)]; ABTS (Boehringer Mannheim) and hydrogen peroxide was added. In addition, the mixture was reacted at room temperature for 90 minutes, and the absorbance at OD ₄₁₅ was automatically measured by EIA-READER (Fig. 1).

As a result, this measurement system showed good reactivity to AHG.

〔The invention's effect〕

As described in detail above, this method is a completely new IC measurement method, and it has been shown that IC measurement by this measurement is possible.

[Brief description of the drawings]

 FIG. 1 is a diagram for explaining the present invention.

Claims (3)

(57) [Claims] 1. A method for measuring an immune complex in a human body fluid by an immunological assay method, in which the solid complex complement Clq is reacted with the body fluid to capture the immune complex in the body fluid. A method for measuring an immune complex, which comprises binding a substance-labeled Clq to the captured immune complex and measuring the amount of the labeled substance bound to the bound substance-labeled Clq to detect and quantify the immune complex. 2. A patent characterized in that Clq is derived from various animals such as humans, rabbits, cows, horses, rats, mice, guinea pigs, frogs and goats, and immobilized Clq and substance-labeled Clq. The method according to claim 1. 3. A substance-labeled Clq, which is a compound or composition containing an enzyme, a radioactive substance, a coenzyme, an enzyme substrate, a cell function regulator, a dye, an electron-accepting substance or a metal, is used as the substance. The method according to claim 1.