JPH07270415A - Enzyme immunoassay - Google Patents

Abstract

PURPOSE:To prevent the sensitivity from fluctuating and carry out a stable and highly sensitive assay by obtaining the optimum hydrogen peroxide concentration based on a response curve between the hydrogen peroxide concentration and the sensitivity in a substrate solution and applying the concentration. CONSTITUTION:An aiming concentration of an object substance to be assayed is set based on the reaction conditions set for every kit and signal detection method and under the conditions, the hydrogen peroxide concentration in a substrate solution is changed, and a response curve between the obtained signal intensities and the hydrogen peroxide concentrations is drawn. Then, the hydrogen peroxide concentration at which the aiming optimum sensitivity value is obtained is calculated from the response curve. The concentration range is 0.002-0.5% (w/v), preferably 0.01-0.2% (w/v). The aiming sensitivity value is commonly set to be 0.8-2.5 as absorbance for the upper limit for the assay of the substance to be assayed in the case a where absorbance is employed. A labelled reference substance is mainly protein of such as antibodies, antigens, avidin, etc. A labelling method based on organic chemical bonding, an immunological labelling method, etc., are employed for the labelling method for a substance to be labelled by peroxidase. A sandwiching method using a peroxidase- labelled antibody is preferable for the measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive enzyme immunoassay (hereinafter abbreviated as EIA), and more specifically, it is characterized by optimizing the hydrogen peroxide concentration in a substrate solution. The present invention relates to the enzyme immunoassay method.

[0002]

2. Description of the Related Art EIA is a measurement method that combines the reaction specificity of an antibody with an antigen in an antigen-antibody reaction and the excellent performance as an enzyme labeling substance (very strong catalytic ability and substrate specificity in an enzyme reaction). A conventional radioimmunoassay (Radi
oimmunoassay, abbreviated as RIA below) 19
It was conceived in 1971 and has reached widespread use today.

Major enzymes used in EIA include peroxidase, β-galactosidase, alkaline phosphatase and the like. Peroxidase is generally often used because of its relatively low price and its good binding property to labeling substances.

In the kit whose measurement principle is EIA,
Sensitivity (defined as a dose-response curve) is one of the general measures to evaluate the quality, and represents the response variation [dR] per unit amount [dC] of the reaction substance, and dR / dC
And the same) [enzyme immunoassay (P.TIJSSEN, Tokyo Kagaku Dojin)]. In the present invention, this sensitivity is taken up, and the substance to be measured is used as the reaction substance, and the fluctuation of the finally detected signal (absorbance, fluorescence intensity, emission intensity, etc.) is used as the response fluctuation. When considering the quality of the kit, the reaction conditions (reaction time, reaction temperature, amount of chemicals used, etc.) and signal detection method (spectrophotometer, fluorescence detector, luminometer, etc.) are the measurement procedure and the equipment used.・ In order to obtain the required sensitivity, it is necessary to devise the composition of the parts that include the sensitivity adjustment factor among the kit components in order to obtain the required sensitivity due to the limited conditions and methods due to the restrictions of the equipment. Specific sensitivity optimization methods include a method of adjusting the amount of peroxidase bound to the labeling substance, a method of adjusting the concentration of the antibody to be used and the concentration of the enzyme labeling substance, and the stability of the component. The labeling substance, peroxidase, which is a raw material, is likely to vary depending on the composition.
Since the quality (reactivity, specific activity, etc.) of antibodies and the like varies depending on the lot, there are many problems such as the sensitivity of kit performance of these constituent reagents varies depending on the preparation lot of each constituent reagent.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a stable and highly sensitive enzyme-linked immunosorbent assay that prevents fluctuations in sensitivity.

[0006]

Based on such a technical background, the present inventors have earnestly studied a method for optimizing the sensitivity in EIA in view of the above-mentioned problems, and as a result, the substrate used in the enzyme reaction The inventors have found that the stable and high sensitivity of EIA using a peroxidase-labeled substance can be achieved simply and easily by optimizing the concentration of hydrogen peroxide in the liquid, and completed the present invention.

That is, in the present invention, in the enzyme immunoassay using a peroxidase-labeled substance, the optimum hydrogen peroxide concentration is obtained from the hydrogen peroxide concentration in the substrate solution and the response curve of sensitivity, and the concentration is used. It is a characteristic enzyme-linked immunosorbent assay.

Specifically, the concentration to be measured of the substance to be measured is set according to the reaction conditions and signal detection method set for each kit, and the hydrogen peroxide concentration in the substrate solution is changed under the conditions. , Drawing a response curve of the obtained signal intensity and hydrogen peroxide concentration. Next, the hydrogen peroxide concentration indicating the target sensitivity value (defined by the signal intensity at the desired concentration of the substance to be measured) to be optimized is determined from the response curve. The concentration range of hydrogen peroxide is 0.002-0.5% (w
/ V), preferably 0.01 to 0.2% (w / v). Although the sensitivity target value varies depending on the signal detection method, when the absorbance is used as the signal (response fluctuation), the absorbance is usually 0.8 to 2.5 per the upper limit of measurement of the substance to be measured.

As a method for forming a complex used in the enzyme immune system of EIA, a method of directly binding an enzyme to a substance to be labeled such as an antibody or an antibody-enzyme via a substance called avidin such as biotin-avidin system is used. There is a method of forming a complex.

The substance to be labeled used in the present invention is mainly an antibody (so-called immunoglobulin produced in vivo and various antibody fragments induced by enzyme treatment), an antigen, a protein such as avidin and the like. The method for producing these proteins also includes a recombinant method. As the antibody, a polyclonal antibody, a monoclonal antibody, or the like is used, but preferably, a monoclonal antibody having a uniform molecule and a certain constant is used.

As a method for labeling a substance to be labeled such as an antibody or avidin with a peroxidase, a labeling method by an organic chemical bond (maleimide method, periodate method, glutaraldehyde method, etc.), immunological labeling method (A method of crosslinking an enzyme-anti-enzyme antibody complex with an antibody by an anti-immunoglobulin antibody), a labeling method (avidin-biotin method, etc.) via a protein molecule other than an enzyme or an antibody, etc. [enzyme immunoassay (P.TIJSSEN (Author, Tokyo Kagaku Dojin)].

As a substrate for peroxidase, o-phenylenediamine (OPD), 3,3 ',
5,5'-tetramethylbenzidine (TMB), 2,
2'-azinobis (3-ethylbenzothiazoline) -6
-Sulfonic acid (ABTS), tyramine or p- by fluorescence method
Examples thereof include hydroxyphenylpropionic acid (HPPA) and luminol in the chemiluminescence method [enzyme-linked immunosorbent assay (protein nucleic acid enzyme, separate volume No. 31)].

As the enzyme immunoassay of the present invention, various measuring principles such as sandwich method and competitive method can be used, but the sandwich method using peroxidase-labeled antibody is preferably used. The sandwich method using a peroxidase-labeled antibody comprises a) a sample containing an antigen, an immobilized antibody in which an antibody capable of specifically reacting with the antigen to be measured is immobilized on a solid support, and an antigen specifically reacting with the antigen to be measured. The step of reacting with the peroxidase-labeled antibody obtained, b) the step of separating the immobilized antibody generated in step a) -the antigen to be measured-the peroxidase-labeled antibody from the unreacted peroxidase-labeled antibody, and c) being immobilized on a solid substance. An enzyme immunoassay method comprising a step of detecting a complex using a peroxidase contained in the complex. The carrier for the immobilized antibody is appropriately selected, and plastic test tubes, microtiter plates, glass beads, plastic beads, membranes, magnetic beads and the like are used.

The enzyme immunoassay method of the present invention can be used in both the so-called one-step sandwich method and the two-step sandwich method.

[0015]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 CA19-9 determination by a two-step sandwich EIA method
Optimization of hydrogen peroxide concentration when creating a kit Reagents The following reagents were used.

After immobilizing the anti-CA19-9 monoclonal antibody on a polystyrene immunomodule plate, it was stabilized by adding bovine serum albumin (hereinafter abbreviated as BSA) and sucrose, and then dried. CA19-9 solution (concentration 300 U / ml), peroxidase-labeled anti-CA19-9 antibody solution, more specifically, anti-CA19-9 antibody treated with mercaptoethylamine to introduce SH group, peroxidase Zease was treated with ε-maleimidocaproyloxysuccinimide to introduce a maleimide group, and a coupling reaction was performed, followed by purification with a hydroxyapatite column (peroxidase-labeled anti-CA19-9.
Preparation of antibody). After exchanging the buffer, it is stored in a 0.1 M acetic acid-citrate buffer solution containing the same labeled antibody and BSA, and is diluted 101-fold with the following reagent immediately before use and diluted.

0.1M acetic acid-citrate buffer containing 0.05 to 0.20% hydrogen peroxide 3,3 ′, 5,5′-tetramethylbenzidine was converted into N, N-dimethylformamide by a conventional method. Solution dissolved in 0.1M sodium citrate buffer, 1NH ₂ SO ₄ , 0.25% BSA, 0.05% polyoxyethylene
(20) 0.1 MTris containing sorbitan monolaurate
-HCl buffer and phosphate buffer containing 0.024% polyoxyethylene (20) sorbitan monolaurate

B. Operation method The sensitivity was adjusted by performing the measurement according to the following operation procedure.

(1) 400 wells per well
After dispensing μl, remove by suction (hereinafter, this unit operation is called a washing operation).

(2) Next, 100 μl of reagent is added per well.
Dispense, and further dispense 20 μl of reagent per well. After the plate is sealed with a plate seal, an antigen-antibody reaction is performed under shaking conditions at room temperature for 2 hours.

(3) After completion of the reaction, the liquid is removed by suction and the washing operation is repeated three times.

(4) 100 μl of the reagent was dispensed per well, the plate was sealed with a plate seal, and then an antigen-antibody reaction was carried out at room temperature for 30 minutes with shaking.

(5) Reagent and reagent 20:
The substrate solution mixed in 1 is prepared in advance.

(6) After completion of the reaction, the liquid is removed by suction, and the washing operation is repeated three times.

(7) 100 μl of the substrate solution was dispensed per well, the plate was sealed with a plate seal, and then a color reaction was carried out at room temperature for 30 minutes with shaking.

(8) After the reaction, add 1 reagent per well.
Discontinue the reaction by pipetting 00 μl, 450 nm and 650
Measure the absorbance at nm.

(9) After the measurement is completed, a correlation table and a correlation diagram of the hydrogen peroxide concentration and the absorbance difference (difference between the measured absorbance values at 450 nm and 650 nm) are prepared. Table 1 and FIG. 1 show the measurement results of the three lots from lots A1 to C1.

[Table 1] (10) The optimum concentration of hydrogen peroxide obtained from the response curve of FIG. 1 is shown in Table 2 when the target sensitivity is set to 1.8 for the difference in absorbance at a CA19-9 concentration of 300 U / ml.

[Table 2]

Example 2 Human IFN-β by 2-step sandwich EIA method
Optimization of hydrogen peroxide concentration when creating a quantitative kit Reagents The following reagents were used.

An anti-human IFN-β polyclonal antibody was immobilized on a polystyrene immunomodule plate, which was then stabilized by adding BSA or sucrose and dried, and a human IFN-β solution was added. (200 IU / ml), peroxidase-labeled anti-human IFN-β antibody solution, more specifically, anti-human IFN-β monoclonal antibody treated with pepsin to form F (ab ′) ₂ and treated with mercaptoethylamine. And Fab'-modified (SH group introduced) to which a peroxidase was treated with ε-maleimidocaproyloxysuccinimide to introduce a maleimide group, and a coupling reaction was carried out, followed by a hydroxyapatite column. Purify with (peroxida
Preparation of ze-labeled anti-human IFN-β antibody). After exchanging the buffer, the cells are stored in a 0.1 M phosphate buffer solution containing the same labeled antibody and BSA, and are diluted 6-fold with the following reagents just before use and used.

0.1M acetic acid-citrate buffer containing 0.014 to 0.020% hydrogen peroxide, and reagents as in Example 1, and 0.133% BSA, 0.067% poly. Oxyethylene (20) sorbitan monolaurate containing 0.1M phosphate buffer

B. Operation method The sensitivity was adjusted by performing the measurement according to the following operation procedure.

(1) 400 wells per well
After dispensing μl, remove by suction (hereinafter, this unit operation is called a washing operation).

(2) Next, 50 μl of the reagent is dispensed per well, and 100 μl of the reagent is further dispensed per well. After the plate is sealed with a plate seal, an antigen-antibody reaction is performed under shaking conditions at room temperature for 2 hours.

The operating procedures of (3) to (8) are the same as in Example 1.

(9) After the measurement is completed, a correlation table and a correlation diagram of the hydrogen peroxide concentration and the absorbance difference (difference between the measured absorbance values at 450 nm and 650 nm) are prepared. Table 3 and FIG. 2 show the measurement results for the three lots from lots A2 to C2.

[Table 3]

(10) Human IFN-β concentration of 2 was set as the target sensitivity.
The optimum concentration of hydrogen peroxide obtained from the response curve of FIG. 2 is shown in Table 4 when the absorbance difference at 00 IU / ml is 1.8.

[Table 4]

Example 3 Avidin-Biotin Amplification Two Step Sandwich EIA
Of human IL-6 assay kit
Optimization of hydrogen concentration A. Reagents The following reagents were used.

After immobilizing anti-human IL-6 monoclonal antibody on a polystyrene immunomodulator plate, BSA and sucrose were added to stabilize the plate, and the plate was dried to form a human IL-6 solution. (600 pg / ml), a biotin-labeled anti-human IL-6 antibody solution, more specifically, an anti-human IL-6 monoclonal antibody treated with pepsin to give F (ab ′) ₂ converted to NHS-L.
After being treated with C biotin to be biotinylated, it is stored in a phosphate buffer containing BSA. Immediately before use, dilute 11 times with the following reagent (10) and use.

Peroxidase-labeled avidin solution, more specifically, after peroxidase is treated with NHS-LC biotin to be biotinylated, streptavidin is added to introduce avidin, and then a phosphate buffer solution containing BSA is added. Store in the state. Immediately before use, use the following reagents (10) 11
Use after diluting twice.

0.1M Phosphate-citrate buffer containing 0.012-0.015% hydrogen peroxide, tablets containing 10 mg o-phenylenediamine dihydrochloride, 1NH ₂ SO ₄ , 1.0% BSA , 0.05% polyoxyethylene (2
0) Phosphate buffer containing sorbitan monolaurate, 0.25% BSA, 0.05% polyoxyethylene
A phosphate buffer containing (20) sorbitan monolaurate, and (10) a phosphate buffer containing 0.024% polyoxyethylene (20) sorbitan monolaurate.

B. Operation method The sensitivity was adjusted by performing the measurement according to the following operation procedure.

(1) For each reagent, add the reagent (10) to 40 wells.
After dispensing 0 μl, remove by suction (hereinafter, this unit operation is called a washing operation).

(2) Dispense 50 μl of the reagent per well, and further dispense 100 μl of the reagent, and then perform an antigen-antibody reaction under shaking conditions at room temperature for 2 hours.

(3) After completion of the reaction, the liquid is removed by suction, and the washing operation is repeated 3 times.

(4) 100 μl of the reagent was dispensed per well, the plate was sealed with a plate seal, and then an antigen-antibody reaction was carried out at room temperature under shaking for 1 hour.

(5) After completion of the reaction, the liquid is removed by suction and the washing operation is repeated 3 times.

(6) A 100 μl aliquot of the reagent was dispensed, the plate was sealed with a plate seal, and then a biotin-avidin reaction was performed under shaking conditions at room temperature for 30 minutes.

(7) Before completion of the reaction, a substrate solution prepared by dissolving 1 tablet of the reagent in 11 ml of the reagent is prepared in advance.

(8) After completion of the reaction, the liquid is removed by suction and the washing operation is carried out 4 times.

(9) A substrate solution was dispensed in an amount of 100 μl per well, the plate was sealed with a plate seal, and then a color reaction was carried out at room temperature for 30 minutes with shaking.

(10) After completion of the reaction, add 1 reagent per well.
The reaction was stopped by pipetting 00 μl, and 492 nm and 405
Measure the absorbance at nm.

(11) After the measurement is completed, a correlation table and a correlation diagram of the hydrogen peroxide concentration and the absorbance difference (difference between the measured absorbance values at 492 nm and 405 nm) are prepared. Table 5 and FIG. 3 show the measurement results for the three lots from lots A3 to C3.

[Table 5]

(12) The target sensitivity is 600 pg for IL-6 concentration.
Table 6 shows the optimum concentration of hydrogen peroxide obtained from the response curve of FIG. 3 when the difference in absorbance at 1.8 ml / ml is 1.8.

[0055]

[Table 6]

[0056]

INDUSTRIAL APPLICABILITY The present invention makes it possible to easily adjust the sensitivity to a target value in the EIA using a peroxidase-labeled substance by a simpler method than ever before. Therefore, a stable and highly sensitive enzyme immunoassay can be provided.

[Brief description of drawings]

FIG. 1 is a correlation diagram between the hydrogen peroxide concentration and the absorbance difference at a CA19-9 concentration of 300 U / ml.

FIG. 2 is a correlation diagram between the hydrogen peroxide concentration and the absorbance difference when the human IFN-β concentration is 200 IU / ml.

FIG. 3 is a correlation diagram between hydrogen peroxide concentration and absorbance difference at a human IL-6 concentration of 600 pg / ml.

Claims (1)

[Claims] 1. An enzyme immunoassay using a peroxidase-labeled substance, which is characterized in that the optimum hydrogen peroxide concentration is obtained from the response curve of the hydrogen peroxide concentration in the substrate solution and the sensitivity, and the concentration is used. Enzyme immunoassay.