GB2070767A

GB2070767A - Stabilizing the reaction product formed in enzyme immunoassays

Info

Publication number: GB2070767A
Application number: GB8103120A
Authority: GB
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1980-03-03
Filing date: 1981-02-02
Publication date: 1981-09-09
Also published as: FR2477176A1; JPS56137155A; FR2477176B1; BE887771A; DE3107904A1; JPS629862B2; GB2070767B

Abstract

The addition of a soluble salt of a reducing agent selected from the group consisting of metabisulphites or thiosulphates (S2O5= or S2O3=) to a reaction mixture of a substrate and a peroxidase stabilizes the reaction product to permit accurate quantification of that product. Use of the sodium salts is exemplified.

Description

SPECIFICATION Method of stabilizing the detectable reaction product formed in enzyme immunoassays This invention reveals and describes reagents and methods useful in conducting enzyme immunoassays. In particular, a method for the stabilization of the reaction product of a peroxidase and its substrate is disclosed. Stabilizing this product permits the accurate determination of immunoreactants such as antigens, antibodies, binding proteins and haptens.

In the typical enzyme immunoassay, an enzyme such as peroxidase is introduced to the reaction medium as a enzyme bound or attached to an immunoreactant (enzyme conjugate). The enzyme labeled immunoreactant will participate in the assay reaction as if it were unlabeled; however, its presence can be detected by adding a substrate to react with the enzyme and observing the resulting reaction product. In order to accurately measure the reaction product it has been necessary to stop the enzyme-substrate reaction at the optimum point by introducing an enzyme denaturant or irreversible inhibitor such as HCI, H2SO4, NaN3 or NaF. These denaturants work by destroying the enzyme and are unsatisfactory simply because of their potential hazard in commercial channels.

This hazard has been largely eliminated by developing a method for the stabilization of the reaction product of the substrate and peroxidase which comprises introducing to the reaction mixture a stabilizing quantity of a soluble salt of a reducing agent selected from the group consisting of S205= and S2O3.

Techniques according to this invention will now be described in more detail by way of example and with reference to the accompanying drawings, in which: Figure 1 depicts the stabilization of the reaction product of horseradish peroxidase (HPO) and o-phenylene diamine (OPD) by sodium metabisulfite and sodium thiosulfate as evidenced by the uniform optical density over a period of time.

Figure 2 depicts the inadequacy of sodium arsenite, a weak reducing agent, for stabilizing the reaction product of horseradish peroxidase (HPO) and o-phenylene diamine (OPD) as evidenced by the increase in optical density over a period of time.

Peroxidase, particularly, horseradish peroxidase is widely used in enzyme immunoassay to aid in detecting minute quantities of immunoreactants. Its presence in the reaction mixture can be detected and quantified according to the following reaction scheme: Enzyme-Substrate Reaction (1.) Peroxidase + H202 = Enzyme complex (2.) Enzyme complex + o-phenylene diamine (AH2) + [A] detectable product + enzyme + H2O Stabilization Scheme (3.) Enzyme complex + BH2 (reducing agent) B B + enzyme + H2O (4.) H2O + BH2 + 2H2O + B Compound A, the detectable product, is a yellowish compound that can be measured spectrophotometrically. The quantity of compound A generated in step (2) is directly related to the amount of peroxidase present.When peroxidase is conjugated to an immunoreactant, the quantity of that component can be determined in any phase of the assay by detecting the intensity of the enzyme-substrate reaction.

In order to accurately determine the quantity of detectable reaction product generated by the reaction sequence according to claimed invention, a reducing agent such as metabisulfite (S205=) orthiosulfate (S203=) is added to the reaction medium after steps (1) and (2) to replace the substrate as an electron donor.

The reducing potential of the reducing agent shouid be such that it will maintain the concentration of the reaction product at a constant level during the period of evaluation. This reaction scheme is set forth in steps (1) and (2) and depicted in Figures 1 and 2.

The subsequent examples will demonstrate with particularity the advantages of the claimed invention; however, it should be noted that the utility of the invention is not confined to the particular enzyme and substrate employed. For example, it is envisioned that other peroxidase such as lactoperoxidase, verdoperoxidase and cytochromeperoxidase would be as suitable as horseradish peroxidase and other substrates such as mesidine, pyrogallol, benzidine, p-phenylene diamine, 2,7-diaminofluorene, p-toluidine and dimethyl analine would be as suitable as o-phenylene diamine.

Example 1 About 10 ng of horseradish peroxidase (HPO) was added to 0.3 ml of solution containing 0.1 M of citratephosphate buffer at pH 5.5 containing 0.02% H202 and 3 mg/ml of o-phenylene diamine (OPD). The reaction mixture was left undisturbed for 30 minutes at room temperature. One ml of this mixture was then added to 9 ml of 0.1 M Na2S205. The adsorption maximum of this reaction mixture was 450 nm.

Example 2 This time 30 ng of horseradish peroxidase was added to 6 ml of the peroxidase, OPD and citrate-phosphate buffered solution prepared in Example 1. Again, the reaction mixture was allowed to sit for 30 minutes at room temperature. Then, 1 ml of the reaction mixture was added to tubes containing 9 ml of 0.1 M Na2S205 and 9 ml of 0.5M of the same salt and 9 ml of 0.1 M citrate phosphate buffer at 450 nm for 5 hours. The results demonstrate that the metabisulfite solutions stabilized the detectable reaction product for at least five hours.

-The adsorbance of the activated complex in citrate buffer increased two times within one hour.

Example 3 One hundred ng of horseradish peroxidase were added to three tubes containing 1 ml each of peroxidase, OPD and citrate-phosphate buffer solution prepared in Example 1. The reaction mixture was allowed to remain at room temperature for 30 minutes. To stop the reactions, 5 ml of 0.01 M, 0.05M and 0.1 M solutions of Na2S205 were added to each of the reaction tubes. The reaction mixtures were monitored at 450 nm for 24 hours. The results demonstrated that 0.05M and 0.1 M Na2S20f; solutions stabilized the color from the product for that period of time, but the 0.01 M solution was not sufficiently strong reducing agent.

Example 4 Four ng of HPO were added to the H2O2, OPD and citrate-phosphate buffered solution. The reaction mixture was allowed to remain at room temperature for 5 minutes. At stop time, 0.3 ml of the reaction mixture was added to four tubes containing 1 ml of 0.1 M Na3AsO3, 0.1 M Na2S205, 0.1 M Kl and 0.1 M Na2S203 respectively. The adsorbance spectra of the mixture was monitored at 450 nm for 22 hours.

The results indicated that both Na2S203 and Na2S205 stabilized the activated complex. Potassium iodide was not suitable because the 12 generated interferred with the determination at 450 nm. Na2AsO3 was evidently not a strong enough reducing agent since it allowed the color intensity to increase as time increased.

Example 5 To polystyrene beads precoated with HBcAg (hepatitis B core antigen) in 3 reaction tray, was added .2 ml of serial dilutions of anti-HBc positive sample. The immunoreactants were allowed to react for two hours at 45 . The beads were rernoved from the reaction wells and washed twice with 5 ml of water. To the washed beads was added .2 ml of anti HBc:HRP conjugate. The beads and labeled conjugate were allowed to stand for one hour at 45" C. The beads were again removed from the reaction wells, washed four times with 5 ml of water and transferred to reaction tubes containing 0.3 ml of the peroxide, OPD and citrate-phosphate buffered solution. After 30 minutes at room temperature, 1 ml of 0.1 M Na2S205 was added to stop the reaction and the adsorbance spectrum was determined at 450 nm with a dual wavelength analyzer. The resulting titration curve showed that Na2S205 was suitable for use in an immunoassay environment.

Claims

1. A method for the stabilization of the reaction product of a substrate and peroxidase which comprises introducing to the reaction mixture a stabilizing quantity of a soluble salt of a reducing agent selected from the group consisting of S2O5= and S2O3.

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 Na2S205.

4. The method according to Claim 1 wherein the reducing salt is Na2S203.

5. The method according to Claim 1 wherein the substrate is o-phenylene diamine.

6. A method for the stabilization of the reaction product of o-phenylene diamine and horseradish peroxidase which comprises introducing to the reaction mixture a stabilizing quantity of a soluble salt of a reducing agent selected from the group consisting of S205= and S203=.

7. The method according to Claim 6 wherein the reducing salt is Na2S205.

8. The method according to Claim 6 wherein the reducing salt is Na2S203.

9. A method of stabilisation of the reaction product of a substrate and peroxidase, substantially according to any one of the Examples herein.