JPH01126549A - 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 carbon with a high vital affinity as solid phase to increase an antibody bondage and bonding force of the solid phase. CONSTITUTION:A polylysine layer, a glutaric aldehyde layer and an antibody or antigen layer are formed sequentially and carbon 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 Application Field The present invention is an immunoassay reagent using a solid phase, in which carbon 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 strength, 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 carbon as a solid phase, on which a polylysine layer, a glutaraldehyde layer, and an antibody or antigen layer are sequentially formed.

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 then impregnated onto the solid phase. 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, acrylny-1-lyl-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 improve the detection limit, measurement range, and reproducibility of t+- and W 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.

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

F action By using carbon with high biocompatibility for 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 1 Evaluation of antibody binding amount using I-CE A (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 carbon (hereinafter abbreviated as CRB, ball).
A) Night (0.15a containing 0.1mg/- polylysine
Polylysine treatment was carried out by immersing the sample in an acid buffer (pH 8,5) at room temperature overnight. Wash with distilled water and soak in 5% glutaraldehyde aqueous solution at 30°C for 2 hours.
Glucuraldehyde treatment was performed by immersion for a period of time.

The cells were washed with distilled water and immersed in solution B (0.1 mg/- immunoglobulin G (abbreviated as IgG), 0.1 mol/l phosphate buffer (pl(7,5)) at 4°C overnight. After washing with solution C (0,01 mol/l phosphate buffer (pH 7,0), 0.1 mol/J NaC1,0,1
x Bovine serum 7 Rubumin (abbreviated as BSA), 0.1XNa
) Js mixed solution), immersed in C solution and stored at 4°C.

In this example, a ball coated with Crb was prepared using anti-carcinoembryonic antigen (abbreviated as anti-CEA)-IgG.

Figure 2 is a diagram showing the procedure for measuring the amount of antibody binding by competitive reaction.
ml count bar e: fu 1) 100μl,
CE A solution (O~11000n/rnI) 1
The reaction was carried out overnight with a mixed solution of 100 µl of 00 pl and C solution, and after washing, measurement was performed using a γ-well counter.

Anti-CEA-IgG coated Cr b prepared in Figure 1.

The amount of antibody bound to the ball was measured by T-CE A as shown in Figure 2.The results are shown in Figure 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, showing Crb,
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) show the results of Comparative Example 1.

From the figure, it was found that the amount of antibody sprayed in the Cr b ball was relatively larger than that in the polystyrene ball.

This is because Crb has stronger biocompatibility than polystyrene.

(2) Evaluation of nonspecific adsorption of enzyme-labeled antibodies to solid phase Example 2 Figure 4 shows the IgG-COD (glucose oxidase)-labeled antibody WJ during enzyme immunoassay (abbreviated as Enzyme Immuno 5say ETA). It is a 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 g70.3- in 4 times the amount of 0.1 mol/l phosphate buffer (pH 7, o), GOD: GMBS (succinimidyl-4-maleimidbutyrei 1-) = 1:
O, 1 mol/l phosphoric acid t
Sephadex G equilibrated with S buffer (pH 6,0)
25 (1x30 column) at a rate of 12 rnl/hr and fractionated into 1-unit fractions to obtain maleimide GOD. Next, add 2 ml of 0.1 mol/l phosphate buffer (
pH 6, O) Add 5 mmol/l EDTA,
S-acetylmercaptosuccinic acid (in the ratio S-acetylmercaptosuccinic acid: IgG = 300:1) is dissolved in dimethylformite and added. After stirring at room temperature for 30 minutes, 0.1nol/J'l Lis-HCl (pF[7,0) 0.1-1O, 1mol/l E
DTA (pl[7,0) 0.02mj, 1mol/J
k Droxydiamine aqueous solution (pH 7, O) 0. Im
j was added to each and reacted at 30°C for 4 minutes. Desalting was carried out at a rate of 12 ml/hr using Sephadex G 25 (1 x 30 color L) equilibrated with Solution D, aliquoted in 1-by-1 portions, concentrated in a collodion bag while cooling with water, and S H-IgG.
I got it. The obtained maleimide COD and S H-IgG
The mixture was mixed in equimolar amounts and allowed to stand at 30°C for 1 hour, and then at 4°C overnight. 0.1@ol/l phosphate buffer (pH 6,5),
5 m1o1/l' 6nd! /hr, the above sample was eluted, separated into 1-hour fractions, and IgG-GODIL
Ka antibody G['Jta. This is 0.1% Nap3.0.1
%BSA and store at 4°C.

For non-specific adsorption, as shown in FIG. 5, the anti-CEA-IgG-coated Cr water obtained in FIG. 1rnl
and C solution 0.2- left at room temperature overnight, and after washing with distilled water,
0.5mol/J glucose, O,0IIIol/
Add 0.3-l acetate buffer (pH 5,1) and heat at 37°C.
It was left to stand for 2 hours.

0.1 d sampling, 2×10-'mol/j'luminol, 0.2 mol/l carbonate a solution (pH 9,8
) Add 0.5 ml of 0.5 mt', 6 x 10-' mol/4 potassium ferricyanide aqueous solution, wait 15 seconds,
It was determined by calculating the amount of light emitted for 6 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-COD labeled antibody adsorbed on the solid phase to the D labeled antibody. The results are shown in Table 1.

Table ICrb, Comparison of nonspecific adsorption between balls and polystyrene balls The results show that there is no difference in nonspecific adsorption of enzyme-labeled antibodies to the solid phase between the two, which is one of the main factors that determines the sensitivity of the sandwich assay method. I got it. This is true even if the antibody is blocked by bovine serum albumin after adsorption to the solid phase.
This is because 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-IgGi-covered Crb, ball and anti-CEA-TgG
Covered polystyrene ball with CEA standard solution (diluted with C solution)
Anti-CEA-IgG-GOD was left standing at room temperature for 6 hours on 0.1- and 0.2- nights, washed with distilled water, and diluted with Solution C.
Leave to stand overnight at room temperature in 0.1 ml of labeled antibody and 0.2 rnl of solution C, wash with distilled water, and add 0.5 mol/l glucose, O, O, 1 mol/l acetate buffer (pH 5,110,
3- was added, and the mixture was left standing at 37°C for 2 hours. 0.1- sampled, 2x10-ma mo1/l Luminol, 0.2 m
ol/l carbonate buffer (p[(9,8) 0.5d, 6X
1G-"mol/j 7 Potassium Elycyanide aqueous solution 0.
Add 5d each time, wait 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 CRB is superior to polystyrene in both the detection limit and measurement range of CEA.

This is because the amount of antibody binding to Cr b is large;
When the antibody binds to the solid phase, the immune activity of the antibody is lost (there are two points to mention).Also, the upper limit of measurement for Crb and polystyrene is the same, because the amount of enzyme-labeled antibody added is the limiting factor. I think this is because it is.

(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 Cr b, ball and polystyrene pole diurnal variation raJ Number of measurements n = 8 Daily variation Number of measurements n = 6 In the table () indicates CT! , indicates 19 degrees.

The reproducibility of the Cr b ball at the detection limit is better than that of the polystyrene ball. This is because the amount of antibodies bound to Crb and balls is large, and the individual differences in the solid phase to be compared are smaller than those 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.

In the yJ preparation method, Crb and balls were immersed in liquid A at room temperature overnight to perform polylysine treatment. Wash with distilled water, B
It was soaked overnight at 4°C in liquid. After further washing with B solution, C
After washing with solution three times, immerse in solution C and store 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 Crb and 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 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, (fl is Comparative Example 4, (
g) shows the results of Comparative Example 5.

From the figure, Crb adjusted by the method shown in Figure 1 has a higher CE
It was found that both the detection limit and measurement range of A were 1 tatami smaller than B.

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.), luminescent substances (fluorescein isothiocyane-1,
, tetramethylrhodamine isothiocyanate, etc.), chemiluminescent substances (aminoethylethylisoluminol, aminobutylethylisoluminol, aminopentylethylisoluminol, aminohexylethylisoluminol, etc.), radioisotopes ('H.

14C, sap, 12@■, 1411, etc.) H Effects of the invention Since the solid phase is carbon 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. However, by the glutaraldehyde treatment, 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 the conventional method.

[Brief explanation of the drawing]

Fig. 1 shows a method for preparing an immunoassay reagent showing an embodiment of the present invention, Fig. 2 shows a procedure for measuring the amount of antibody bound by competitive reaction, and Fig. 3 shows antibodies of C, r b , pole, and polystyrene balls. Figure 4 shows the change in binding amount of gG-GOD-labeled antibody 1! Figure 5 is a diagram of the method for evaluating non-specific adsorption, Figure 6 is a comparison diagram of the measurement range and detection limit, Figure 7 is a diagram of changes in CEA concentration vs. luminescence amount, and Figure 8 is a comparison diagram. An operating method diagram showing 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 a comparison diagram of processing conditions, showing the results.

Claims (2)

[Claims] (1) An immunoassay reagent characterized in that the solid phase is carbon 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.