JPH01126557A - Reagent for immunological measurement - Google Patents

Abstract

PURPOSE:To achieve a wider measuring range, a rise in sensitivity to a detection limit and a higher reproducibility, by using an alumina single crystal with a high vital affinity as solid phase to increase an antibody bondage and bonding force of the solid phase. CONSTITUTION:An polylysine layer, a glutaric aldehyde layer and an antibody or antigen layer are formed sequentially and the alumina single crystal with a high vital affinity is used to be a solid phase. With such an arrangement, a bonding force and 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 Field of Application The present invention is an immunoassay reagent using a solid phase, in which alumina single crystal (Al□03) is used as the solid phase, and a polylysine layer, a glutaraldehyde layer, and an antibody are used. Or, it relates to an immunoassay reagent that sequentially forms antigen layers to increase the amount of antibody binding to the solid phase and the antibody binding strength, thereby expanding the measurement range, increasing the sensitivity of the detection limit, and improving reproducibility. be.

B. Summary of the Invention The present invention relates to an immunoassay reagent comprising an alumina single crystal (A+*OS) as a solid phase, on which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed. It is.

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 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-lilue-book-diene-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 as an 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.

Means for Solving Problem E The immunoassay reagent according to the present invention uses alumina single crystal (A
), and its alumina single crystal (1 zoi) is used as the solid phase.

F action By using alumina single crystal (Human 1□03) 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 crosslinking glutaraldehyde increases, and the amount of antibody binding and antibody binding strength increase.

Example G (Evaluation of antibody binding amount using 11"'ic EA (carcinoembryonic antigen) Example 1) FIG. 1 is a diagram showing a method for preparing an immunoassay reagent according to an embodiment of the present invention.

The adjustment method is alumina single crystal (A+zO3) (under breath,
A1. s-ball) was mixed with Al (0.15 mol/l borate buffer (pH 8) containing 0.1 mg/- polylysine.
, 5) ) at room temperature overnight for polylysine treatment.

Wash with distilled water and add 5% glutaraldehyde aqueous solution.
Gluculaldehyde treatment was performed by immersing at 0°C for 2 hours. It was washed with distilled water and immersed in solution B (Q, 111 g/mi immunoglobulin G (abbreviated as IgG), 0.1 mol/l phosphate buffer (PH7, s)) at 4°C overnight. After further washing with liquid B, liquid C (0,01 mol/j
Phosphate buffer (p[(7,0), 0.1 mol/j N
After washing with a1l, 0.1*bovine serum albumin (abbreviated as BSA), 0.1X NaNs ffl, combined solution),
It was immersed in Solution C and stored at 4°C.

In this example, coated AI and s-ball 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 bound by competitive reaction, and shows the anti-CEA-IgG coated A1 prepared by the method shown in Figure 1.
．． s-ball in 1”■-〇 E A solution (approximately 2X10'C
pm [count bar/mini foot]) 100μ I,
CEA solution (0-11000n/mt') 100μl
The reaction was carried out overnight with a mixed solution of 100 μl of SC solution, and after washing, measurement was performed using a γ-well counter.

The amount of antibody bound to the anti-CEA-IgG-coated At and S balls prepared in FIG. 1 was measured by Ic EA 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.

FIG. 3 shows the results of Example 1 and Comparative Example 1, and A1. s
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, it was found that the amount of antibody bound to AI and s balls was relatively larger than that of polystyrene balls.

This is because AI,s 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-COD (abbreviated as EIA)
(glucose oxidase) labeled antibody preparation method diagram,
FIG. 5 is an operational diagram of the method for evaluating non-specific adsorption.

The IgG-COD labeled antibody preparation method first begins with a COD of approximately 3m.
Dissolve g70.3mN in 4 times the volume of O, 1mol/l phosphate buffer (PH7,0), GOD: GMB
S (succinimidyl-4-maleimidobutyrei-1-)
= 1:5G ratio and mixed it with O, 1 mol/l phosphoric acid l! using Sephadex G 25 (1x30 column) equilibrated with saline solution (pl(6,0)) at 12 nJ/
Desalt for 1 hr, separate 1 rnl aliquot, maleimide COD
I got it. Next, add 0.1mol/j' of 2m&' to IgG.
Phosphate buffer '75 (p[[6,o) 5mmol
/ l EDT, S-acetylmercaptosuccinic acid (S-acetylmercaptosuccinic acid: IgG
= 300: 1 ratio) in dimethylformite and added. After stirring at room temperature for 30 minutes, 0.1 mol/#
I.Lis-hydrochloric acid (p[H,O) 0.1-10.1m
ol/1 EDTA (pH 7,0) 0.02 ml 1.1 m
ol// Hydroxydiamine aqueous solution (pli7. O
) 0.1+nj were added to each and reacted at 30°C for 4 minutes. Sephadex G 25 (1x3
0 column) at a rate of 12 mN/hr,
1 me portion was collected and concentrated in a collodion bag while cooling with water to obtain SH-1gGJe. The obtained maleimide COD and SH-IgG were mixed in equimolar amounts, and after standing at 30°C for 1 hour, 4
The mixture was allowed to stand overnight at ℃. 0.1 mol/e phosphate buffer (p
HG, 5), 6 mt in Sephacryl 300 (1x90 column) equilibrated with 5 mmol/l EDT
Elute the above sample at a rate of
-COD labeled antibody was obtained. This is 0.1% Nap,,0
．． Add 1% BSA and store at 4°C.

As shown in FIG. 5, non-specific adsorption was observed in the anti-CEA-IgG coated A1. s ball was mixed with anti-CEA-IgG-GOD labeled antibody 0.1- diluted in C solution and C
Leave to stand overnight at room temperature in 0.2 ml of solution, wash with distilled water, and remove
．． Add 5 mol/I glucose, 0.3 mj of O, 1 mol/l acetate buffer (pH'5.1), and heat at 37°C.
Let it stand for a while.

0.1r+t' sampled, 2X1G-'+ol/
J'luminol, 0.2 mol/(l carbonate buffer (p
H9,8) 0.5mj, 6X10-'IIIol/
l Potassium ferricyanide water soluble w10.5+nl! was added, waited 15 seconds, and calculated the amount of luminescence for 16 to 45 seconds.

Comparative Example 2 Using anti-CE A'-IgG 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. In addition, the evaluation method is based on added IgG-GO
It was expressed as the ratio (%) of the amount of IgG-COD labeled antibody adsorbed on the solid phase to the D labeled antibody. The results are shown in Table 1.

Table IA1. The results showed that there was no difference between the S-ball and polystyrene ball sandwich assays in terms of non-specific adsorption of the enzyme-labeled antibody to the solid phase, which is one of the main factors influencing the sensitivity of the two methods. This is because after adsorbing the antibody to the solid phase, blocking with bovine serum albumin is A1. This is because even in the case of s, it is as effective as polystyrene.

(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 coating A1. s ball and anti-CEA-IgG
The coated polystyrene balls were placed in 0.1-ml of CEA standard (diluted with liquid C) and 0.2 ml of liquid C for 6 hours at room temperature.
Anti-CEA-IgG-G diluted with solution C after washing with distilled water
Add 0.1 mt' of OD-labeled antibody and 0.2 ml of C solution at room temperature.
After leaving to stand overnight and washing with distilled water, add 0.5 mo 171 glucose, 0.01 mol/l acetate buffer (p[5,1)
0.3 mj was added and left at 37° C. for 2 hours. 0.1-sampled, 2X10-'+ol/l luminol,
0.2 mol/l' carbonate buffer (pH 9,8) O, Sm
J', 8 x 10-''mol/J 7 Add 0.5 mj of potassium elycyanide aqueous solution, wait 15 seconds, 16-4
Calculate the amount of light emitted for 5 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 A1. It was found that s 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 s is large, and the immune 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 AI,s 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. S ball and polystyrene ball diurnal variation Number of measurements n = 8 Daily variation Number of measurements n = 6 In the table, () indicates the CEA concentration.

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

This is because the amount of antibody bound to the S 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 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. The g-ball was immersed 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. After further washing with solution B and three times with solution C, it is immersed in Cwl and stored at 4°C.

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

In the Fl preparation method, AI and S balls were immersed in a 5% glutaraldehyde aqueous solution at 30° C. for 2 hours to perform glutaraldehyde treatment. It was washed with distilled water and soaked overnight at BwR 4°C. Further, after washing with B solution and three times with C solution, it is immersed in C solution and stored 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, (
) shows the results of Comparative Example 5.

From the figure, A1. s is more CE
It was found that A was excellent in both the detection limit and measurement range.

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. The antibody bound to such a stable solid phase increases the amount of antibody bound and the antibody binding strength.

This time, we will use an enzyme (COD) 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-galactosigase, glucose 6-phosphate, dehydrogenase, alkaline phosphatase, etc.), luminescence [t (fluorescein isothiocyane-1)
・, tetramethylrhodamine isothiocyanate, etc.),
Chemiluminescent substances (aminoethylethylisoluminol, aminobutylethylisoluminol, aminopentylethylisoluminol, aminohexylethylisoluminol, etc.), radioisotope light? ('H.

C, 32P, 12'I, ""I, etc.) H Effects of the invention Highly biocompatible alumina single crystal (
Since the polylysine (Altos) was used as a solid phase, the amount of polylysine covered increased, and by the glutaraldehyde treatment, some amino groups of the polylysine were crosslinked with each other with glutaraldehyde, thereby stabilizing the solid phase. Antibody bound to such a stable solid phase increases the amount of antibody bound 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 s-ball and polystyrene ball. Figure 4 shows how to prepare gG-GOD-labeled antibody Fl. Figure 5 shows how to evaluate non-specific adsorption. Figure 6 shows the measurement range and detection. Comparison operation diagram of limits, Figure 7 is a CEA concentration-light emission change diagram, Figure 8 is an operation method diagram showing Comparative Example 3, Figure 9 is an operation method diagram showing Comparative Example 4, 1θ
The figure is an operating method diagram showing Comparative Example 5, and FIG. 11 is a comparison diagram of processing conditions showing the results.

Claims (2)

[Claims] (1) An immunoassay reagent characterized in that the solid phase is an alumina single crystal 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.