JPH01126552A - Reagent for immunological measurement - Google Patents

Abstract

PURPOSE:To enable a wider measuring range, a rise in sensitivity to a detection limit, by using alumina ceramics with a high vital affinity as solid phase to increase an antibody bondage and bonding force. CONSTITUTION:A polylysine layer, a glutaric aldehyde layer and an antibody or antigen layer are formed sequentially and alumina ceramics with a high vital affinity is used to be a solid phase. With such an arrangement, a bonding force and a bondage between the solid phase and polylysine are increased to enhance the amount of glutaric aldehyde in crosslinking, resulting in a greater antibody bondage and bonding force. This achieves a wider measuring range, a rise in sensitivity to a detection limit and the like.

Description

[Detailed Description of the Invention] A. Industrial Application Field The present invention is an immunoassay reagent using a solid phase, in which alumina ceramics is used as the solid phase, and a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed. The present invention relates to an immunoassay reagent that increases the amount of antibody bound to a solid phase and the antibody binding force, thereby expanding the measurement range, increasing the sensitivity of the detection limit, and improving reproducibility.

B. Summary of the Invention The present invention relates to an immunoassay reagent comprising an alumina ceramic solid phase, on which a polylysine layer, a gluculaldehyde layer, and an antibody or antigen layer are sequentially formed.

C Conventional techniques Immunoassay methods using solid phases can be roughly divided into 11
Classified into legal and non-competitive laws. A representative example of the former is the first antibody solid phase method, and the latter is the sandwich measurement method.

In this sandwich method, antibodies are adsorbed onto a solid phase and antigens are trapped there. Next, an enzyme-labeled antibody is bound to the antigen, and the amount of the antigen is measured from the amount of bound enzyme-labeled antibody.

Due to the above principle, in order to increase the detection sensitivity of the sandwich method, the amount of antibody bound to the solid phase and the antibody binding strength must be
It will have a big impact.

Conventionally, polystyrene, glass, acrylonitrile-butadiene-styrene copolymer resin (abbreviated as ABS), and the like have often been used as materials for the solid phase.

Most of the methods for binding antibodies to these solid phases involve physical adsorption. However, this method has problems such as a small amount of antibody bound to the solid phase, weak antibody binding strength of the solid phase, large variations in measured values, and large nonspecific adsorption.

Previously, in the patent application No. 60-45742 entitled "Method for preparing immunological assay reagents and reagents obtained thereby," the solid phase was treated with amino acids in order to solve the above problems. After that, it was treated with a bifunctional aldehyde and bound to an antibody, which has the effect of solving the above problems.

D. Problems to be Solved by the Invention However, it has been desired to further improve the detection limit, measurement range, and reproducibility of the immunoassay reagent.

The present invention has been made to solve these problems, and is an immunoassay reagent that has a large amount of antibody binding on the solid phase, strong antibody binding force on the solid phase, small variation in measured values, and low nonspecific adsorption. In addition to obtaining a P4 immunoassay reagent, the objective is to expand the measurement range, increase the sensitivity of the detection limit, and further improve reproducibility as an immunoassay reagent.

E Means for Solving Problems The immunoassay reagent according to the present invention uses highly biocompatible alumina ceramics in which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed, and the alumina ceramics are used as a solid phase. That is.

F action By using alumina ceramics with high biocompatibility as the solid phase, the binding strength and amount of binding between the solid phase and polylysine are increased. As a result, the amount of cross-linked gluturaldehyde increases, and the amount of antibody binding and antibody binding strength increase.

G Example (11'''Evaluation of antibody binding amount using I-CE A (carcinoembryonic antigen) Example 1゜Figure 1 is a diagram of a method for preparing an immunoassay reagent showing an example of the present invention. .

The adjustment method is to use an alumina ceramic ball (AtzOs).
) (hereinafter abbreviated as A1.C ball) to liquid A (0, II
ag/1111+0.15@ol/ containing polylysine
Polylysine treatment was performed by immersing the sample in a borate buffer (p[[8,5)] overnight at room temperature. Wash with distilled water,
% glutaraldehyde aqueous solution at 30° C. for 2 hours to perform glutaraldehyde treatment. Wash with distilled water,
Solution B (0.1 ng/mA' immunoglobulin G (abbreviated as IgG), 0.11 I+01/e phosphate buffer (p[(7
, 5)) Soaked overnight at 4°C. After further washing with solution B, solution C (0.01 mol/j phosphate buffer (pt)
7,0), 0.1a+ol/j Mail, 0.1X
Bovine serum albumin (abbreviated as BSA), 0.1XHa
After washing with N39 solution), it was immersed in solution C and stored at 4°C.

In this example, IgG was coated as anti-carcinoembryonic antigen (abbreviated as anti-CEA)-IgG. Adjusted the C ball.

Figure 2 is a diagram showing the procedure for measuring the amount of antibody binding by competitive reaction, and shows the anti-CEA-IgG coated A1 prepared by the method shown in Figure 1.
．． C ball "'■-〇 E A solution (approximately 2X10'C
pm [count par 1:)”) I) 100 p l
SCE A solution (0-11000n/m1) 10
The mixture was reacted overnight with a mixed solution of 0 μl and 100 μl of solution C, and after washing, measurement was performed using a γ-well counter.

Anti-CEA-IgG coated A1 prepared in FIG. The amount of antibody bound to the C ball was measured using 1"I-CE A as shown in FIG. 2. The results are shown in FIG. 3.

Comparative Example 1 An anti-CEA-IgG coated polystyrene ball was prepared in the same manner as in FIG. 1, and the amount of antibody bound was measured in the same manner as in FIG.

The results are shown in Figure 3. ' Figure 3 shows the results of Example 1 and Comparative Example 1, and A1. C
It is a diagram showing changes in the amount of antibody binding between balls and polystyrene balls. In the figure, (a) shows the results of Example 1, and (b) shows the results of Comparative Example 1.

From the figure, the amount of antibody bound is A1. It was found that the C ball was relatively larger than the polystyrene ball.

This is A1. This is because C has stronger biocompatibility than polystyrene.

(2) Evaluation example 2 of non-specific adsorption of enzyme-labeled antibodies to solid phase Figure 4 shows enzyme immunoassay (Enzyme I+mwaun).
o IKG when performing 5say (abbreviated as EIA)
FIG. 5 is a diagram of a method for preparing a GOD (glucose oxidase)-labeled antibody, and FIG. 5 is an operational diagram of a method for evaluating nonspecific adsorption.

IgG -G OD [mAntibody FI preparation method 1;! , :
! fG.

Approximately 3 mg of D70.3-4 times the amount of 0.1 mol//Phosphate a solution (pH 7,0> diluted oak, GOD 20 MB
S (succinimidyl-4-maleimidobutyrei 7-)
= 1:50 and was added to 12 ml/l' using Sephadex G 25 (1x30 column) equilibrated with 0.1 mol/l' phosphate buffer (p[(6,0)).
The mixture was desalted for 1 hr and separated into 1 ml portions to obtain maleimide GOD. Next, the IgG was treated with 2 mZ of 0.1 mol/l phosphate buffer (p[16,0) -51 +++ ol/l E
Add DTA and add S-acetylmercaptosuccinic acid (S
-Acetylmercaptosuccinic acid: IgG=300=
(in a ratio of 1:1) and added in dimethylformite. After stirring at room temperature for 30 minutes, 0.1 mol/jt')
Hydrochloric acid (pl(7,0) 0.1-10.1o+ol
/j EDTA (pH 7,0) 0.02ml, 1m
ol/j' Hydroxydiamine aqueous solution (pH 7.0)
0.1- was added to each and reacted at 30°C for 4 minutes. Desalting was carried out at a rate of 12 mj/hr using Sephadex G 25 (1x30 column) equilibrated with solution D, and 1 mj
Collect aliquots, concentrate in water-cooled collodion bags, and SH-
IgG was obtained. Obtained maleimide COD and SH-Ig
Equimolar amounts of G were mixed, left at 30°C for 1 hour, and then left at 4°C overnight. 0.1s+ol/e phosphate II buffer (p[(6
, 51,5a + 1/j Sephacryl 300 (2x90 Karagen) equilibrated with EDTA, elute the above sample at 6 mj/hr, collect 1 ml each, and collect IgG-COD.
A labeled antibody was obtained. This is 0.1% Nap, 0.1% B
Add to SA and store at 4°C.

As shown in FIG. 5, non-specific adsorption was performed by diluting the anti-CEA-IgG-coated AI, C balls obtained in FIG. Leave to stand overnight at room temperature in 0.2 mm of C solution, wash with distilled water,
．． Add 5a+ol/l glucose, 0.3mj of 0.01mol/l acetate buffer (pH 5,1), and heat at 37°C.
Let it stand for a while.

0.1d"t:/pull, 2X10-'mol/j
Le: / -le, 0.2 mol/j carbonate buffer (p
[(9,8) 0.5 ml, 6 x 10-' mol/l potassium ferricyanide aqueous solution 0.51 BI was added to each,
It was determined by waiting for seconds and calculating the amount of light emitted for 16 to 45 seconds.

Comparative Example 2 Using anti-CEA-IgG coated polystyrene balls prepared in the same manner as shown in Fig. 1, the amount of non-specific adsorption of the polystyrene balls was determined as shown in Fig. 5.

In Example 2 and Comparative Example 2, nonspecific adsorption of an enzyme-labeled antibody to a solid phase was compared. The evaluation method is based on added IgG-CO
It was expressed as the ratio (%) of the amount of IgG-GOD labeled antibody adsorbed on the solid phase to the D-labeled antibody. The results are shown in Table 1.

1IA1. Comparison of nonspecific adsorption between c-balls and polystyrene balls We found that there is no difference between the two in terms of nonspecific adsorption of enzyme-labeled antibodies to the solid phase, which is one of the main factors that determines the sensitivity of the sandwich assay method. Ta. This is true even when AI and C are blocked by bovine serum albumin after adsorbing the antibody to the solid phase.
This is because it is as effective as polystyrene.

(3) Evaluation of detection limits and measurement range FIG. 6 is a comparison diagram of measurement range and detection limit.

The operation is shown below. Anti-CE obtained by the procedure shown in Figure 1
A-IgG coated A1, choleoJ: human anti-CEA
- IgG coated polystyrene balls with CEAll semi-solution (C
Anti-CEA-I diluted with 0.1rnl of solution) and Solution C 0.2-, left standing at room temperature for 6 hours, washed with distilled water, and diluted with Solution C.
gG-GOD labeled antibody 0.1ml and C solution 0.2rnl+
ζLet stand overnight at room temperature, wash with distilled water, and then add 0.5 mol/
l glucose, 0.01 mol/j acetate buffer (pH 5
, 1) 0.3- was added and left at 37°C for 2 hours. 0.
1-Sampling, 2 x 10-'mol/1 Luminol, 0.2mol/j carbonate buffer (pH 9,8) 0
．． 5-16X10-”mol/j7x!Jii
7 Add 0.5 m# of potassium chloride aqueous solution to each sample, wait 15 seconds, and calculate the amount of light emitted for 16 to 45 seconds.

The detection limits and measurement ranges of Example 2 and Comparative Example 2 were compared. Figure 7 shows the results.
FIG. In the figure, (e) shows the results of Example 2, and (d) shows the results of Comparative Example 2.

From the figure A1. It was found that CEA was superior to polystyrene in both the detection limit and measurement range of CEA.

This is A1. Two points are mentioned: the amount of antibody bound by C is large, and the immunological activity of the antibody is not easily lost when the antibody is bound to the solid phase. Furthermore, the reason that the measurement upper limits of A1, C and polystyrene are the same is considered to be because the amount of enzyme-labeled antibody added is a limiting factor.

(4) Evaluation of reproducibility in detection limit Example 2 and Comparative Example 2 were compared for intraoral variation and daily variation in detection limit. The results are shown in Table 2.

Table 2A1. c Ball and polystyrene ball diurnal variation Number of measurements n = 8 Daily variation vh Number of measurements n = 6 In the table, () indicates the CEA concentration.

A1 at the detection limit. The reproducibility of C balls is better than that of polystyrene balls. This is AI.

C. This is because the amount of antibody bound to the balls is large and the individual differences in the solid phase to be compared are smaller than that of polystyrene balls.

(5) Evaluation of solid phase antibody binding adjustment method Comparative example 3゜ FIG. 8 is an operation method diagram showing Comparative Example 3.

The adjustment method is to soak in liquid B overnight at 4°C, wash with liquid B, wash three times with liquid C, then soak in liquid C at 4°C.
Save with .

Comparative example 4゜ FIG. 9 is an operation method diagram showing Comparative Example 4.

The adjustment method is A1. Ball C was immersed in Solution A at room temperature overnight to perform polylysine treatment. It was washed with distilled water and immersed in solution B at 4°C overnight. Further, after washing with B solution and three times with C solution, it is immersed in C solution and stored at 4°C.

Comparative example 5゜ FIG. 10 is an operation method diagram showing Comparative Example 5.

The adjustment method is A1. C-ball was immersed in a 5% aqueous glutaraldehyde solution at 30° C. for 2 hours to perform glutaraldehyde treatment. It was washed with distilled water and immersed in Solution B at 4°C overnight. After further washing with liquid B and three times with liquid C,
Immerse in Solution C and store at 4°C.

The measurement range and detection limit of Example 2 and Comparative Examples 3, 4, and 5 were compared using the method shown in FIG. FIG. 11 shows the results and is a comparison diagram of the processing conditions.

In the figure, (e) is Comparative Example 3, (f) is Comparative Example 4, (
g) shows the results of Comparative Example 5.

From the figure, A1. C is CE
It was found that both the detection limit and measurement range of A were violated.

In the preparation method shown in FIG. 1, the ball is coated with polylysine and then crosslinked with glutaraldehyde, so that the solid phase is stabilized. Antibody bound to such a stable solid phase increased the amount of antibody bound and the antibody binding strength.

This time, we will use an enzyme (GOD) as a label to detect the amount of antibodies.
Although only the case is described, similar results have been obtained not only with enzymes but also with fluorescent substances and radioactive isotopes. Examples of labeled objects are shown below. Enzymes (glucose oxidase, horseradish peroxidase, β-D-galactosidase, glucose 6-phosphate, dehydrogenase, alkaline phosphatase, etc.), luminescence
1., tetramethyl rogamine isothiocyane-1., etc.), chemiluminescent substances (aminoethylethylisoluminol, aminobutylethylisoluminol, aminopentylethylisoluminol, aminohexylethylisoluminol, etc.), radioactive isotopes ( sH114C, 2P,
125 ■, 1311, etc.) Effects of the H invention Since the solid phase is alumina ceramics with high biocompatibility, in which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed, the amount of polylysine covered increases.
The glutaraldehyde treatment stabilizes the solid phase because some amino groups of polylysine are crosslinked with each other with glutaraldehyde. Antibody bound to such a stable solid phase increases the amount of antibody binding and the antibody binding strength.

Therefore, the measurement range is expanded, the detection limit is increased, and the reproducibility is improved compared to the conventional method.

[Brief explanation of the drawing]

FIG. 1 is a diagram of a method for preparing an immunoassay reagent showing an embodiment of the present invention, FIG. 2 is a diagram of a procedure for measuring the amount of antibody bound by competitive reaction, and FIG. 3 is a diagram of A1. Figure 4 shows the change in antibody binding amount between C ball and polystyrene ball, Figure 4 shows how to prepare IgG-COD labeled antibody, Figure 5 shows how to evaluate non-specific adsorption, and Figure 6 shows how to evaluate non-specific adsorption.
The figure is a comparison operation diagram of the measurement range and detection limit, and Figure 7 is C.
EA concentration-emission amount change diagram, FIG. 8 is an operating method diagram showing Comparative Example 3, FIG. 9 is an operating method diagram showing Comparative Example 4, FIG. 10 is an operating method diagram showing Comparative Example 5, and FIG. 11 is the result,
It is a comparison diagram of processing conditions.

Claims (2)

[Claims] (1) An immunoassay reagent characterized in that the solid phase is alumina ceramics in which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed. (2) The immunoassay reagent according to claim 1, wherein the antibody is anti-carcinoembryonic antigen-immunoglobulin G.