JPH01126555A - Reagent for immunological measurement - Google Patents

Abstract

PURPOSE:To achieve a wider measuring range, a rise in a detection limit and a higher reproducibility, by using a crystallized glass with a high vital affinity as solid phase to increase an antibody bonding force and bondage of the solid phase. CONSTITUTION:A polylysine layer, a glutaric aldehyde layer and an antibody or antigen layer are formed sequentially and a crystallized glass with a high vital affinity is used to be a solid phase. 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 a higher reproducibility.

Description

[Detailed Description of the Invention] A. Industrial Application Field The present invention is an immunoassay reagent using a solid phase, in which crystallized glass 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 binding to a solid phase and the antibody binding strength, thereby expanding the measurement range, increasing the sensitivity of the detection limit, and improving reproducibility.

B Summary of the Invention The present invention uses crystallized glass as a solid phase, and on the solid phase,
The present invention relates to an immunoassay reagent comprising a polylysine layer, a glucuraldehyde layer, and an antibody or antigen layer formed in this order.

C. Conventional Technology Immunoassay methods using solid phases are broadly classified into competitive methods and non-competitive methods. A representative example of the former is the first antibody solid phase method, and the latter is the sandwich liquid 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. The object of the present invention is, of course, to obtain an immunoassay reagent that can expand the measurement range, increase the sensitivity of the detection limit, and further improve reproducibility.

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

F action By using crystallized glass 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 (Evaluation of antibody binding amount using 11” r-c E A (carcinoembryonic antigen) Example 1゜ Fig. 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 a crystallized glass pole (S 1o2-Na2
0-CaO-P2O3- to 20-MgO glass, Si
O2-MgO-CaO-Pad.

A liquid (hereinafter abbreviated as C, G ball) (one of the materials of Ca0-P2O, system coordination crystallized glass) (abbreviated as C and G balls)
0.15mol/containing 0.1+++g/-polylysine
Polylysine treatment was performed by immersing the sample in a boric acid buffer (pH 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,
BM (0,1+ng/m# Immunoglobulin G (IgG
), 0.1 mol/l phosphate buffer (pH 7.5
)) Soaked overnight at 4°C. After further washing with solution B, solution C (0°01 mol/c' phosphate buffer (plt?
, o), 0.1 mol/e NaCl, 0.1 z bovine serum 7 to 100 ml (abbreviated as BSA), 0. After washing with lXNaN3 mixed solution), it was immersed in solution C and stored at 4°C.

In this example, coated C0G balls were prepared using anti-carcinoembryonic antigen (abbreviated as anti-CEA)-IgG as IgG.

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 C0 prepared by the method shown in Figure 1.
Add the G ball to the ``I-CE A solution (approximately 2 x 10'Cpm
I count bar 1) 100μ t', CE
A solution (0-11000n/ml) 100pl,
React overnight with a mixed solution of Solution C 1007zp, wash, and then measure using a γ-well counter.

The amount of antibody bound to the anti-CEA-IgG-coated C0G balls prepared in FIG. 1 was measured by I-CEA 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 FIG.

FIG. 3 shows the results of Example 1 and Comparative Example 1, and is a diagram of changes in the amount of antibody binding between C and G balls and polystyrene balls.

In the figure, hl shows the results of Example 1, and (b) shows the results of Comparative Example 1.

From the figure, it was found that the amount of antibody bound to the C0G balls was relatively larger than that of the polystyrene balls.

This is because C0G 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 Immunoassay).
IgG-CO when performing As5ay (abbreviated as ET person)
FIG. 5 is a diagram of a method for preparing a D (glucose oxidase) labeled antibody, and FIG. 5 is an operational diagram of a method for evaluating non-specific adsorption.

In the IgG-COD labeled antibody preparation method, first, GOD of about 3n
tg10.3rrvl is 4 times the amount, 0.1mol/l!
Dissolved in phosphate buffer (p[17,0), GOD: G
MBS (Succinimidyl-4-maleimidobutyreI)
・) = 1:50 ratio, and it was added at a ratio of 0.1 mol/
l phosphate buffer (ptte.

Sephadex G 25 (1x30
Column) was used to desalt at a rate of 12+nj/hr, and 1- portions were collected to obtain maleimide COD. Next, add 2 to IgG.
0.1 mol/I of rne! Phosphate CI bomb (1(
pl[6,O) 5 m-ol/l ED
Add T and add S-acetyl mercapto (S-
Acetyl mercap 1-succinic acid: IgG =
300:1) in dimethylformite and added. After stirring at room temperature for 30 minutes, 0.1 mol/l
l.Lis-hydrochloric acid (p[17,0) O.

In(0.1mol/j EDT person(pH7.0)0
．． 02mj! , 1 mol/J Hydroxydiamine water soluble 'a (pH 7,0) 0. iml was added to each and reacted for 4 minutes at 30°C. Desalting was performed at a rate of 12 ml/hr using Sephadex G 25 (IX30 column) equilibrated with Di&, fractionated into 1 me portions, and concentrated in a collodion bag while cooling with water to obtain SH-IgG. The obtained maleimide COD and SH-IgG were mixed in equimolar amounts, left at 30°C for 1 hour, and then left at 4°C overnight.

0.1 mol/4 phosphate buffer (pH 6,5), 5 m
Iol/l! Cephacryl 30 equilibrated with EDT humans
The above sample was eluted at 6 ml'/hr on 0 (1x90 column) and fractionated into 1-unit fractions to obtain IgG-COD-labeled antibodies. This was added to give 0.1% NaN3.0.1% BSA and stored at 4°C.

As shown in Fig. 5, non-specific adsorption was performed using anti-CEA-IgG-coated C, G balls obtained in Fig. 1 with anti-CEA-IgG-GOD-labeled antibodies 0 and 1 rnl diluted in solution C and C.
Liquid Q, 2mj! After leaving it at room temperature overnight and washing with distilled water,
0.5a+ol/l glucose, 0.01mol/j
Add 0.3 ml of acetate buffer (pH 5,1) and heat at 37°C.
It was left to stand for 2 hours.

0, 1ml sampling, 2X10-'11o1/
l luminol, 0.2@ol/l carbonate buffer (pH 9,
8) 0.5 ml of 0.5 mj, 8×10 −3 mol/l potassium ferricyanide aqueous solution 71i was added to each, waited 15 seconds, and determined by calculating the luminescence amount for 16 to 45 seconds.

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

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-COD labeled antibody adsorbed onto a solid relative to the D labeled antibody.

The results are shown in Table 1.

Table IC, Comparison of nonspecific adsorption between G balls and polystyrene balls 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. Got the results. This is because, after adsorbing the antibody to the solid phase, blocking by bovine serum albumin is as effective as polystyrene even in the case of C and G.

(3) Evaluation of detection limit 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 C, G balls and anti-CEA-IgG coated polystyrene balls were mixed with CEA standard solution (diluted with C solution) 0
．． bt! ! Cw! Anti-CEA-IgG diluted in iL
-GOD-labeled antibody 0.1mj and C solution 0.2-1 at room temperature
After leaving to stand overnight and washing with distilled water, add 0.5 mol/j glucose, 0.01 mol/j acetate buffer (pH 5,1) 0
．． 3- was added, and the mixture was left standing at 37°C for 2 hours. 0.1-sampled, 2X10-'+ol/l Luminol, 0
．． 2mol/j carbonate buffer (pH 9,8) 0.5m#,
Add 6 x 10-' mol/j 7 x 0.5- of potassium ricyanide aqueous solution, wait for 15 seconds, and calculate the amount of luminescence for 16 to 45 seconds.

The detection limits and measurement ranges of Example 2 and Comparative Example 2 were compared. FIG. 7 shows the results and is a CEA concentration-emission amount change diagram. In the figure, (e) is Example 2, (
d) shows the results of Comparative Example 2.

From the figure, it was found that C and G were superior to polystyrene in both the detection limit and measurement range of CEA.

This is due to two reasons: the amount of antibody binding to C0G is large, and the immunoactivity of the antibody is less likely to be lost when the antibody is bound to the solid phase. Furthermore, the reason that the measurement upper limits for C0G 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 2 Comparison of reproducibility in detection of C and G balls and polystyrene balls Intraoral variation Number of measurements n = 8 Daily variation Number of measurements n = 6 The parentheses in the table indicate the CEA concentration.

The reproducibility of C and G balls at the detection limit is better than that of polystyrene balls. This is because the amount of antibody bound to the C0G ball is large, and the individual differences in the solid phase to be compared are smaller than that of the polystyrene ball.

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

The preparation method was to soak in liquid B overnight at 4°C, wash with liquid B, wash three times with liquid C, and then soak in Ct&.
Store at °C.

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

The preparation method was to immerse the C0G ball in liquid 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 preparation method was to perform glutaraldehyde treatment by immersing C and G balls in a 5% glutaraldehyde aqueous solution at 30° C. for 2 hours. It was washed with distilled water and immersed in Solution B at 4°C overnight. After further washing with B solution and 3 times with C solution, C
Immerse in liquid and store at 4°C.

The measurement range and detection limit of Example 2 and Comparative Examples 3 and 4.5 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, C and G adjusted using the method shown in Figure 1 have a higher CEA.
It was found that the detection limit and measurement range were excellent.

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 have only described the case of COD as an enzyme, but similar results have been obtained using peroxyguse, alkaline phosphacuse, β-D-galactonase, and the like.

Furthermore, similar results have been obtained not only with enzymes but also with fluorescent substances and radioactive isotopes.

Labeled substances as labeled antibodies include enzymes (glucose oxyguse, horseradish peroxidase, β-D-galactosigase, glucose 6-phosphate, dehydrogenase, alkaline phosphatase, etc.), luminescent substances (fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, etc.). Ne-
1, etc.), chemiluminescent substances (aminoethylethylisoluminol, aminobutylethylisoluminol, aminopentylethylisoluminol, aminohexylethylisoluminol, etc.), radioisotopes ('H, "C,"p
, 128I, 1llI, etc.) H Effects of the Invention Since the solid phase is crystallized glass with high biocompatibility, in which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed, the coverage of polylysine increases and By treatment with chloraldehyde, some amino groups of polylysine are crosslinked with each other with glutaraldehyde, thereby stabilizing the solid phase. 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 other adjustment methods.

[Brief explanation of the drawing]

Figure 1 is a diagram of a method for preparing an immunoassay reagent showing an embodiment of the present invention, Figure 2 is a diagram of a procedure for measuring the amount of antibody bound by competitive reaction, and Figure 3 is a diagram of the amount of antibody bound to C and G balls and polystyrene balls. Figure 4 is a diagram of how to prepare the IgG-COD standard antibody, Figure 5 is a diagram of how to evaluate non-specific adsorption, Figure 6 is a diagram of comparison of measurement range and detection limit, and Figure 7 is a diagram of how to prepare the standard antibody for IgG-COD. C.E.
A: Concentration vs. luminescence amount change diagram; Figure 8 is an operating method diagram showing Comparative Example 3; Figure 9 is an operating method diagram showing Comparative Example 4; Figure 2O is an operating method diagram showing Comparative Example 5; shows the results and is a comparison diagram of processing conditions.

Claims (3)

[Claims] (1) An immunoassay reagent characterized in that the solid phase is crystallized glass 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. (3) The crystallized glass is SiO_2-Na_2O-
CaO-P_2O_5-K_2O-MgO glass, S
iO_2-MgO-CaO-P_2O_5 glass, C
The reagent for immunoassay according to claim 1, characterized in that it is made of one of aO-P_2O_5-based coordination glass ceramics.