JPH0318758A - Agglutinating antibody - Google Patents

Abstract

PURPOSE:To measure the antigens in a low concn. region by a TIA method with a high sensitivity by using the agglutination antibody contg. the polymer of IgG polymerized by using a chemical crosslinking agent. CONSTITUTION:The agglutination antibody is prepd. by separating and refining a gamma-globulin fraction by the conventional method, such as ammonium sulfate salting-out or dialysis, from antiserum and polymerizing this fraction by using the chemical crosslinking agent. For example, glutaraldehyde or the like is used at 2 to 20 times the mol of the IgG as the chemical crosslinking agent. The higher content of the dimer and trimer of the IgG in the agglutination antibody is more preferable and the activity of the dimer and trimer of the IgG is specified to >=20% of the total antibody activity.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to the measurement of N-collection antibodies useful in immune serum tests, more specifically, in the measurement of antigens by immunoturbidimetry (hereinafter referred to as TIA method). This invention relates to an agglutinated antibody that can be measured with high sensitivity even at low concentrations of antigen.

[Conventional technology]

Among clinical tests, immune serum tests are complicated to operate and require a long time for measurement, but in recent years, automation has become possible with the development of the TIA method, and many clinical diagnostic reagents based on the TEA method are currently provided.

The measurement principle of the TIA method is to optically measure the turbidity of an antigen-antibody complex produced by an antigen-antibody reaction. However, the TIA method has a drawback of low measurement sensitivity in a low concentration region. Therefore, a method has been used to increase the turbidity and measurement sensitivity by increasing the size of the particles of the antigen-antibody complex, and one such method is the latex nephelometric method. However, the latex nephelometric method has a problem in that the measurement range is narrow and the prozone phenomenon appears earlier in the high concentration region than in the TIA method.

[Problem to be solved by the invention]

Therefore, it has been desired to develop a method that can measure antigens in a low concentration range with high sensitivity without sacrificing the advantage of the TIA method of having a wide measurement range.

[Means to solve the problem]

Under such circumstances, the present inventor has conducted extensive research and found that the above object can be achieved by using an aggregating antibody containing an IgG polymer polymerized using a chemical crosslinking agent, and has devised the present invention. Completed.

Therefore, the present invention provides polymerized Ig using a chemical cross-linking agent.
The present invention provides an aggregated antibody containing G dimers or trimers.

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

Examples of chemical crosslinking agents include glutaraldehyde, dithiobis(succinimidylbropionic acid), di-N-(2-nitro-4-azidophenyl)cystamine-S,
S-dioxide, N,N'-phenylene dimaleimide,
Azophenyl dimaleimide, 2,2'-dicarboxy-4,4' azophenyl diisocyanate, etc. are used, and it is preferable to use these in an amount of 2 to 20 times the mole of IgG. For the polymerization of antibodies, for example, "Medical Chemistry Experimental Methods Course 4"
, Immunochemistry, pp. 303-304, a glutaraldehyde solution was added to the buffer solution of the T-globulin fraction under stirring, and the mixture was reacted at room temperature for 2 to 3 hours. It will be done.

The T-globulin fraction was analyzed by FPLC (Fa
st Protein Liquid Chroma
As shown in the gel filtration pattern obtained by the tography system, the main activity is IgG with a molecular weight of approximately 150,000, but by treatment with the chemical cross-linking agent described above, the FPLC shown in Figure (B) As shown in the gel filtration pattern, an active fraction with a molecular weight of approximately 300,000 to 500,000 appears. Based on the molecular weight, this antibody with a molecular weight of about 300,000 to 500,000 is estimated to be an IgG dimer or trimer, and this IgG dimer has a high agglutination ability when reacting with an antigen in the TI8 method. It is significantly superior (see Figures 2 and 3).

The aggregated antibody of the present invention preferably has a higher content of IgG dimers and trimers, but if the activity of IgG dimers is 20% or more of the total antibody activity, the object of the present invention can be achieved. is achieved.

The above technology of the present invention can be applied to all antibodies used in the TIA method, such as anti-CRP antibody, anti-fibrinogen antibody, anti-albumin antibody, anti-C3 antibody, anti-C, anti-Tr antibody, anti-C, Antibody, anti-α, M antibody, anti-α. A
Examples include agglutinating antibodies such as T antibody, anti-1{2 antibody, anti-IAP antibody, anti-ATI[I antibody, anti-1gG antibody, anti-1gA antibody, and anti-IgM antibody.

〔Effect of the invention〕

The agglutinating antibody of the present invention has a strong agglutinating ability when reacting with an antigen,
If this is used, antigens in the low concentration range can be measured with high sensitivity by the TIA method.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 (i) Anti-CRP serum (goat) 100- and diethyl ether 100- were mixed under water cooling and stirred for 30 minutes to remove the lipid fraction in the serum. After delipidation, residual diethyl ether in the antiserum was dialyzed against normal saline, and further O. lM}
Dialysis was performed using Lis-HCl (pH 8.0) buffer. Anti-CRP defatted blood treated in this way i'tif80ml2
and the same amount of raw food, and add 13% of saturated ammonium sulfate to the mixture.
1 ml was slowly added dropwise. After precipitating the T-globulin fraction, it was stirred for 30 minutes and left at 4°C overnight. The precipitated γ-globulin fraction was collected by centrifugation, and the precipitate was dissolved with saline so that the volume of the solution after dissolution was 40. After dissolution, it was dialyzed against a 0.1M phosphoric acid (pl+6.7) buffer to obtain anti-CRP r-globulinated serum.

(ii) Glutaraldehyde manufactured by Hani Chemical Co., Ltd. (25%
Aqueous solution) is diluted 100 times with normal saline. Anti-CR at room temperature
Slowly drop 2.0-fold of a 100-fold diluted glutaraldehyde aqueous solution into m20 ml of Pγ-globulinated blood,
After stirring for 30 minutes, the mixture was allowed to stand at 4°C for 3 days.

After glutaraldehyde treatment, 22rnl of 0.1M toI
J Shin-Na011 (pH 8.0) buffer (0.1
M NaCj! The reaction was stopped by adding anti-CRP glutaraldehyde-treated serum (containing 0.1% NaN3).
Agglutinated antibody) was obtained.

On the other hand, anti-CRP r without glutaraldehyde treatment
- To 20 ml of globulinated serum, add 100-fold diluted glutaraldehyde aqueous solution and the same amount of 2.0H saline,
Similarly 22ml! O. 1Ml thin NaOH buffer was added to give untreated anti-CIIP serum.

(iii) The gel filtration patterns of untreated anti-CRP serum and anti-CRP glutaraldehyde-treated serum using the FPLC system are shown in (8) and (B) of FIG.

In addition, the gel filtration carrier contains 3upperrose 6}IR
10/30 (10 mm x 30 cm; molecular weight fractionation range 50005000000), 0 for the eluent
．． 02M phosphate (ptl7.4) buffer (0.15M
NaCA', containing 0.1% NaN3) at a flow rate of 0. 6ml! / min, 0. 6 m
f! / fractions were collected in a tube.

In addition, each fraction was examined for protein concentration, titer, and agglutination ability. Protein concentration was determined by diluting each fraction 11 times and using wavelength 280
Absorbance was measured at nm. The titer was measured by Betka's method. For the agglutination ability, each fraction was used as the second reagent, and CRP antigen 5
mg/dj! The sample was measured in the same manner as in Test Example 1. The results are shown in Figure 2 (untreated anti-CRP serum) and Figure 3 (anti-CRP glutaraldehyde-treated serum).

Test Example 1 In order to compare the agglutination ability in TIA of the anti-CRP glutaraldehyde-treated serum obtained in Example 1 (11) and the untreated CRP serum, each was further diluted 4 times with 0.1 MIJ Thin-NaOH buffer, and Two reagents were used. Further, as the first reagent, commercially available Cl8-CRP SRI CL "Nissui" was used. A Hitachi 736-15 automatic analyzer was used for the measurement, and the parameters were a 2-point assay.
<Photometry point 8-2 0>, sample amount t5μA, R
L amount 300uj! , R2 amount 100μl, dominant wavelength 3 4
0 nm, subwavelength 70D nm. K Facto
It was carried out at r=10000. The results are shown in Figure 4.

Example 2 Anti-C. r-globulinated serum was obtained and treated in the same manner as in Example 1(ii) to obtain anti-C, glutaraldehyde-treated blood r? fCM collection antibody)
I got it.

Test Example 2 In TIA of the anti-C4 glutaraldehyde-treated serum obtained in Example 2 and the untreated anti-C serum [prepared in the same manner as in Example 1 (ii)]! II concentration was the same as Test Example 1 (however, the sample amount was 20 μf (21-fold dilution), and the Rl amount was 350 μf.
The test was carried out using a volume of 100 μl (μl, n2). The results are shown in Figure 5.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing gel filtration patterns of untreated anti-CIIP serum and anti-CRP glutaraldehyde-treated serum by an FPLC system. FIG. 2 is a diagram showing the protein concentration, titer, and agglutination ability of each fraction of untreated anti-CRP serum. FIG. 3 is a diagram showing the protein concentration, titer, and agglutination ability of each fraction of anti-CRP glutaraldehyde-treated serum. FIG. 4 is a diagram showing the reactivity of untreated anti-CRP serum and anti-CRP glutaraldehyde-treated serum in TEA. Figure 5 shows the T of untreated anti-C4 serum and anti-C, glutaraldehyde-treated serum.
It is a figure showing the reactivity in EA. that's all? ．． sl- 1 I ■ Figure 4 CRP antigen Jt Imq/di) (△) Untreated anti-CRP serum 10 υ1 N (B) Anti-CRρ glutaraldehyde-treated serum Sanoble TP 6.0'/o

Claims (1)

[Scope of Claims] 1. An aggregated antibody containing IgG dimers or trimers polymerized using a chemical crosslinking agent. 2. The aggregated antibody according to claim 1, wherein the activity of dimer to trimer of IgG is 20% or more of the total antibody activity. 3.Claim 1, wherein the chemical crosslinking agent is glutaraldehyde.
or the agglutinating antibody described in 2.