JP2704760B2 - Agglutinating antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to an agglutinated antibody useful for an immune serum test, and more particularly, to a method for measuring an antigen by immunoturbidimetry (hereinafter referred to as TIA method). The present invention relates to an agglutinating antibody that can be measured with high sensitivity even at a concentration of antigen.

[Prior art] In clinical tests, immune serum tests are complicated and require a long time for measurement.In recent years, the development of the TIA method has enabled automation, and many clinical diagnostic agents based on the TIA method are currently provided. Have been.

The measurement principle of the TIA method is to optically measure the turbidity of an antigen-antibody complex generated by an antigen-antibody reaction. However, the TIA method has a disadvantage that the measurement sensitivity in a low concentration region is low. Therefore, a method of increasing the turbidity by increasing the particle size of the antigen-antibody complex to increase the measurement sensitivity has been performed, and such a method includes a latex nephelometry. However, the latex turbidimetric method has a problem in that the measurement range is short, and the prozone phenomenon appears earlier in the high concentration region than in the TIA method.

[Problems to be solved by the invention]

Therefore, it has been desired to develop a method capable of measuring an antigen in a low concentration region with high sensitivity without impairing the advantage of the TIA method that the measurement range is wide.

[Means for solving the problem]

Under such circumstances, the present inventors have conducted intensive studies and found that the above object can be achieved by using an agglutinated antibody containing an IgG polymer polymerized using a chemical crosslinking agent, and completed the present invention. did.

Thus, the present invention provides a specific
It is intended to provide an agglutinating antibody used for immunoturbidimetry, comprising a dimer or trimer of IgG obtained by polymerizing IgG using a chemical crosslinking agent.

The agglutinated antibody of the present invention is prepared by separating and purifying a γ-globulin fraction from an antiserum by a conventional method such as ammonium sulfate salting out or dialysis, and polymerizing the same using a chemical crosslinking agent.

As the chemical crosslinking agent, for example, glutaraldehyde,
Dithiobis (succinimidyl propionic acid), di-N
-(2-nitro-4-azidophenyl) cystamine-S,
S-dioxide, N, N'-phenylenedimaleimide, azophenylenedimaleimide, 2,2'-dicarboxy-4,4 '
Azophenyl diisocyanate and the like are used, and it is preferable to use 2 to 20 times the molar amount of IgG. Antibody polymerization can be performed, for example, in "Medicine Chemistry Experimental Method Course 4", immunochemistry,
According to the method described on pages 303-304, a glutaraldehyde solution is added to the buffer solution of the γ-globulin fraction with stirring, and the mixture is reacted at room temperature for 2 to 3 hours.

The γ-globulin fraction was analyzed by the FPLC (Fast
As shown in the gel filtration pattern by the Protein Liquid Chromatography system, IgG having a molecular weight of about 150,000 is used as the main active ingredient. However, the FPLC shown in FIG. As shown in the gel filtration pattern, an active ingredient having a molecular weight of about 300,000 to 500,000 appears. Then, the antibody having a molecular weight of about 300,000 to 500,000 is presumed to be a dimer or trimer of IgG based on the molecular weight.
The dimer has remarkably superior aggregation ability when reacted with the antigen in the TIA method (see FIGS. 2 and 3).

The aggregated antibody of the present invention is preferably as large as the content of IgG dimers or trimers. However, if the activity of IgG dimers is 20% or more of the total antibody activity, Is achieved.

The technique of the present invention can be applied to all antibodies to be used in the TIA method, such as anti-CRP antibody, anti-fibrinogen antibodies, anti-albumin antibody, anti-C ₃ antibody, anti-C ₄ antibody, anti
T _f, anti-C _p antibodies, anti-alpha ₂ M antibody, anti-alpha ₁ AT antibody, anti-H _p antibodies, anti-IAP antibody, anti-AT III antibody, anti-IgG antibody, anti-IgA antibodies, aggregation antibodies such anti-IgM antibodies Can be mentioned.

〔The invention's effect〕

The agglutinating antibody of the present invention has a strong agglutinating ability when corresponding to an antigen, and by using this, an antigen in a low concentration region can be measured with high sensitivity by the TIA method.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 (i) 100 ml of anti-CRP serum (goat) and 100 m of diethyl ether
were mixed under ice-cooling and stirred for 30 minutes to remove lipid fractions in serum. After defatting, the remaining diethyl ether in the antiserum was dialyzed against saline, and further 0.1 M Tris-HCl (pH 8.
0) It was dialyzed against a buffer. 80 ml of the anti-CRP defatted serum thus treated was mixed with the same amount of saline, and 131 ml of saturated ammonium sulfate was slowly added dropwise to the mixture. After the γ-globulin fraction was precipitated, the mixture was stirred for 30 minutes and left at 4 ° C. overnight. The precipitated γ-globulin fraction was collected by centrifugation,
The precipitate was dissolved by saline so that the liquid volume after dissolution was 40 ml.
After dissolution, dialyzed against 0.1 M phosphoric acid (pH 6.7)
γ-globulinated serum was used.

(Ii) Glutaraldehyde (25% aqueous solution) manufactured by Hanoi Chemical Co.
Is diluted 100 times with raw food. Glutaraldehyde aqueous solution diluted 100 times to 20 ml of anti-CRP γ-globulinated serum at room temperature
2.0 ml was slowly added dropwise, and after stirring for 30 minutes, the mixture was allowed to stand at 4 ° C. for 3 days. After the glutaraldehyde treatment, the reaction was stopped by adding 22 ml of a 0.1 M tricine-NaOH (pH 8.0) buffer (containing 0.1 M NaCl and 0.1% NaN ₃ ) to obtain an anti-CRP glutaraldehyde-treated serum (aggregated antibody). Was.

On the other hand, anti-CRP γ-
To 20 ml of the globulinized serum, add 2.0 ml of saline equivalent to the 100-fold diluted glutaraldehyde aqueous solution, and similarly add 22 ml
0.1M Tricine-NaOH buffer was added to obtain untreated anti-CRP serum.

(Iii) Gel filtration pattern of untreated anti-CRP serum and anti-CRP glutaraldehyde-treated serum by FPLC system was first.
This is as shown in FIGS. The gel filtration carrier was Superrose 6HR 10/30 (10 mm × 30 cm; molecular weight fractionation range 5000-5,000,000), and the eluent was 0.02 M phosphoric acid (pH 7.4).
Using a buffer solution (containing 0.15 M NaCl and 0.1% NaN ₃ ), the flow rate was set at 0.
It was performed at 6 ml / min, and fractionated at 0.6 ml / tube.

In addition, each fraction was examined for protein concentration, titer, and aggregation ability. For the protein concentration, each fraction was diluted 11-fold, and the absorbance was measured at a wavelength of 280 nm. The titer was measured by the Becker method. Aggregation ability was measured in the same manner as in Test Example 1 using each fraction as the second reagent and using 5 mg / dl of CRP antigen as a sample. The result is the second
As shown in the figure (untreated anti-CRP serum) and FIG. 3 (anti-CRP glutaraldehyde-treated serum).

Test Example In order to compare the agglutinating ability of the anti-CRP glutaraldehyde-treated serum obtained in Example 1 (ii) and the untreated CRP serum in TIA, the serum was further diluted 4-fold with 0.1 M Tricine-NaOH buffer, and the second dilution was performed. The reagent was used. In the first test, a commercially available auto TI
A-CRP S R1 CL “Nissui” was used. Hitachi 736 for measurement
Using an automatic analyzer of type -15, parameters were 2 point assay (photometry point 8-20), sample amount 15μ, R
1 amount 300μ, R2 amount 100μ, main wavelength 340nm, sub wavelength 700nm, K
Factor = 10000. The result is as shown in FIG.

Example 2 Example 1 in place of the anti-CRP serum (i) using an anti-C ₄ serum (goat) by the same procedure to obtain the anti-C ₄ .gamma.-globulin sera, Example 1 This (ii ) and was obtained treated with anti-C ₄ glutaraldehyde treated serum (aggregated antibody) as well.

Test Example 2 Anti-C obtained in Example 2 ₄ glutaraldehyde treated serum and untreated anti C ₄ serum Example 1 (ii) was prepared in the same manner as]
Was tested in the same manner as in Test Example 1 (provided that the sample amount was 20 μ (21-fold dilution), the R1 amount was 350 μ, and the R2 amount was 100 μ). The results are as shown in FIG.

[Brief description of the drawings]

FIG. 1 is a view showing a gel filtration pattern of an untreated anti-CRP serum and an anti-CRP glutaraldehyde-treated serum by an FPLC system. FIG. 2 is a diagram showing the protein concentration, titer, and aggregating ability of each fraction of untreated anti-CRP serum. FIG. 3 shows the protein concentration, titer, and the like of each fraction of the serum treated with anti-CRP glutaraldehyde.
It is a figure which shows an aggregation ability. Fig. 4 shows untreated anti-CRP serum and anti-C
FIG. 4 shows the reactivity of RP glutaraldehyde-treated serum in TIA. FIG. 5 is a figure that shows the reactivity in TIA untreated anti C ₄ serum and anti-C ₄ glutaraldehyde treated serum.

Claims (3)

(57) [Claims] 1. An agglutination method for use in immunoturbidimetry, comprising a dimer or trimer of an IgG specific to an antigen to be detected, which is polymerized using a chemical crosslinking agent. antibody. 2. The activity of an IgG 2-3 trimer is 20% of the total antibody activity.
% Of the agglutinated antibody according to claim 1. 3. The agglutinated antibody according to claim 1, wherein the chemical crosslinking agent is glutaraldehyde.