JPS629862B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to reagents and methods useful for performing enzyme immunoassays, and more particularly to methods for stabilizing the reaction product of peroxidase and its substrate. Stabilization of this reaction product allows for accurate measurements of immunoreagents such as antigens, antibodies, binding proteins, and haptens.

In a typical enzyme immunoassay, an enzyme such as peroxidase is introduced into the reaction medium as the enzyme that binds the immunoreagent (enzyme conjugate). Enzyme-labeled immunoreagents participate in test reactions as if they were unlabeled. However, its presence can be detected by adding a substrate and allowing it to react with the enzyme and observing the resulting reaction product. In order to accurately measure the reaction products, enzyme denaturants or irreversible inhibitors such as HCl, H ₂ SO ₄ ,
It was necessary to introduce _NaN3 or NaF to stop the enzyme-substrate reaction. These denaturants work by destroying enzymes and are unsatisfactory solely because of their potential danger in chemical means.

This risk can be reduced by introducing into _the reaction mixture a stabilizing amount of a soluble _salt of a reducing agent selected from the group consisting of S ₂ O ^2-5 and S ₂ O ^2-3 . By developing a method for stabilizing the product with peroxidase, they were able to significantly eliminate it.

Figure 1 shows horseradish peroxidase (horseradish peroxidase).
This figure shows the stabilization of the reaction product of peroxidase (HPO) and o-phenylenediamine (OPD) with sodium metabisulfite and sodium thiosulfate. As can be seen from this figure, uniform optical density can be obtained over a long period of time.

Figure 2 shows that the weak reducing agent sodium arsenite is unsuitable for stabilizing the reaction product of horseradish peroxidase (HPO) and o-phenylenediamine (OPD). As can be seen from this figure, an increase in optical density is observed over a long period of time.

Peroxidase, particularly horseradish peroxidase, is widely used in enzyme immunoassays to detect small amounts of immunoreagents.

Its presence in the reaction mixture can be detected and quantified by the following reaction equation.

Enzyme-substrate reaction (1) Peroxidase + H ₂ O ₂ enzyme complex (2) Enzyme complex + o-phenylenediamine (AH ₂ )
[A] Detectable product + enzyme + H ₂ O stabilization reaction formula (3) Enzyme complex + BH ₂ (reducing agent) →
B+Enzyme+H ₂ O (4) H ₂ O+BH ₂ →2H ₂ O+B The detectable product, Compound A, is a yellow compound that can be measured spectrophotometrically. The amount of compounds generated in step (2) is directly related to the amount of peroxidase present. When peroxidase binds to the immunoreagent,
The amount of the compound can be determined during any phase of the test by detecting the intensity of the enzyme-substrate reaction.

In order to accurately measure the amount of the detectable reaction product generated by the above reaction formula according to the present invention, it is necessary to use metabisulfite (S ₂ O ^2-5 ) or thiosulfate (S ₂ O ^2-3 _{) .} Process reducing agent like (1)
and substituting the substrate as electron donor in addition to the reaction medium after (2). The reduction potential of the reducing agent should be such as to maintain the concentration of reaction products at a constant level during the evaluation period. This reaction equation is shown in steps (1) and (2), and in Figures 1 and 2.
Illustrated in the figure.

Although the following examples illustrate the advantages of the present invention, it should be understood that the utility of the present invention is not limited to the particular enzymes and substrates used therein. be. For example, other peroxidases such as lactoperoxidase, verdoperoxidase and cytochrome peroxidase are also suitable, such as horseradish peroxidase, and other substrates such as mecidine, pyrogallol, benzidine. , p-phenylenediamine,
It is envisaged that 2,7-aminofluorene, p-toluidine and dimethylalanine are also suitable, as is o-phenylenediamine.

Example 1 Approximately 10 ng of horseradish peroxidase was added to 0.3 ml of 0.1 M citrate-phosphate buffer at pH 5.5 containing 0.02% H ₂ O ₂ and 3 mg/ml o-phenylenediamine (OPD). The reaction mixture was allowed to stand at room temperature for 30 minutes. Then 1ml of this mixture was diluted with 0.1M
It was added to 9 ml of Na ₂ S ₂ O ₃ solution. The maximum absorption of this reaction mixture was at 450 nm.

Example 2 In this example, 30 ng of horseradish peroxidase was added to 6 ml of the buffer of Example 1 ( _{H2O2 -OPD} -citrate-phosphate). The reaction mixture was allowed to stand at room temperature for 30 minutes. This reaction mixture was then
9 ml of 0.1 M Na ₂ S ₂ O ₅ solution, 9 ml of 0.5 M Na ₂ S ₂ O ₅ solution and 9 ml of 0.1 M citrate-phosphate buffer
Added to tube containing ml. The results obtained show that the metabisulfite solution stabilized the detectable reaction products for at least 5 hours. The absorbance of activated complexes in citrate buffer increased 2-fold within 1 hour.

Example 3 100 ng of horseradish peroxidase was added to three tubes each containing 1 ml of the buffer of Example 1 ( _{H2O2 -OPD} -citrate-phosphate). The reaction mixture was kept at room temperature for 30 minutes. To stop the reaction, 5 ml of 0.01M, _0.05M and 0.1M solutions of _Na2S2O5 were added to each of _the reaction tubes. The reaction mixture was examined at 450 nm for 24 hours. The results obtained showed that the 0.05M Na ₂ S ₂ O ₅ solution stabilized from the reactants during this period, but the 0.01M solution was not a strong enough reducing agent.

Example 4 4 ng of HPO was added to the _H2O2 - _OPD -citrate-phosphate buffer described above. The reaction mixture was kept at room temperature for 5 minutes. Upon stopping, add 0.3 ml of the reaction mixture to 0.1 M—Na ₃ AsO ₃ , 0.1 M—Na ₂ S ₂ O ₅ , 0.1 M
-KI and 0.1M-Na ₂ S ₂ O ₃ were added to 4 tubes each containing 1 ml each. The absorption spectrum of the mixture was examined at 450 nm for 22 hours.

The results obtained showed that both Na ₂ S ₂ O ₃ and Na ₂ S ₂ O ₅ stabilized the activated complex. Potassium iodide was unsuitable because the I ₂ generated interfered with measurements at 450 nm.
Na ₂ AsO ₃ was clearly not a sufficient reducing agent as the color intensity increased over time.

Example 5 HBcAg (Hepatiteis-B) in the reaction tray
core antigen) pre-coated polystyrene beads,
0.2 ml of a sterile dilution of an HBc (anti-hepatitis-B core) positive sample was added. 45% of this immunoreagent
The reaction was carried out at ℃ for 2 hours. The beads were removed from the reaction mixture and washed twice with 5 ml of water. Anti-HBc to washed bead:
0.2 ml of HRP conjugate was added. The beads and labeled conjugate were left at 45°C for 1 hour. Remove the beads from the reaction mixture again and wash with 5 ml of water 4 times.
H ₂ O ₂ - OPD - Citrate - Phosphate Buffer
Transferred to a reaction tube containing 0.3 ml. After standing at room temperature for 30 minutes, ₁ ml of 0.1M-Na ₂ S ₂ O was added to stop the reaction, and the absorption spectrum was measured at 45 nm using a dual wavelength analyzer. The obtained titration curve is
It has been shown that Na ₂ S ₂ O ₅ is suitable for use in a liquid immunity test environment.

[Brief explanation of the drawing]

Figure 1 shows horseradish peroxidase (horseradish peroxidase).
1 shows the stabilizing effect of sodium metabisulfite and sodium thiosulfate of the present invention on the reaction product of peroxidase (HPO) and o-phenylenediamine (OPD). Figure 2 shows the stabilizing effect of sodium metabisulfite of the present invention on the reaction product of HPO and OPD with citrate containing no stabilizer.
It is shown in comparison with a phosphate buffer and a buffer containing sodium arsenite as a stabilizer. In FIGS. 1 and 2, the horizontal axis represents elapsed time (unit time), and the vertical axis represents optical density at 450 nm. 〓〓〓...Curve of sodium metabisulfite, 〓〓〓...Curve of sodium thiosulfate, 〓〓〓...Curve of buffer without stabilizer, 〓〓〓...Sodium as stabilizer Curve of buffer solution containing arsenite,〓〓〓……
Curve for a buffer containing sodium metabisulfite as a stabilizer.

Claims (1)

[Claims] 1. In a reaction mixture of an electron donor and a peroxidase
Stabilization of the reaction product of a peroxidase with _a substrate as _an electron donor, characterized by adding a soluble salt of a reducing agent selected from the group consisting of S ₂ O ^2-5 and S ₂ O ^2-3 Law. 2. The method according to claim 1, wherein the enzyme is horseradish peroxidase. 3. The method according to claim 1, wherein the reducing salt is Na ₂ S ₂ O ₅ . 4. The method according to claim 1, wherein the reducing salt is Na ₂ S ₂ O ₃ . 5. The method according to claim 1, wherein the substrate is o-phenylenediamine. 6. The method according to claim 1, which is a method for stabilizing a reaction product of o-phenylenediamine and horseradish peroxidase. 7. The method according to claim 6, wherein the reducing salt is Na ₂ S ₂ O ₅ . 8. The method according to claim 6, wherein the reducing salt is Na ₂ S ₂ O ₃ .