GB2067572A

GB2067572A - Partially oxidized enzyme conjugates

Info

Publication number: GB2067572A
Application number: GB8041533A
Authority: GB
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1980-01-21
Filing date: 1980-12-31
Publication date: 1981-07-30
Also published as: JPS56106155A; BE887128A

Abstract

A partially oxidized peroxidase is conjugated to an immunoreactant to provide a reagent useful in immunoreactions which demonstrates fewer erroneous results than enzyme conjugates prepared in the conventional manner.

Description

SPECIFICATION Partially oxidized enzyme conjugates This invention relates to a reagent and its method of preparation which is useful in conducting enzyme immunoassays. In particular, the reagent described in an immunoreactant conjugated to a peroxidase enzyme which has been partially oxidized.

It is well established that immunoreactants such as antigens, antibodies, haptens and binding proteins can be tagged or labeled with an enzyme which will not interfere with immunoreactive properties. After the reaction has been permitted to occur, a substrate susceptible to the enzyme can be added to a phase of the reaction mixture and the resulting activated complex can be monitored to indicate the quantity of enzyme labeled reactant present in that particular phase of the reaction.

Generally, the enzyme conjugate is prepared by oxidizing the enzyme and allowing it to couple with the immunoreactant to be labeled for a period of at least three hours.

The oxidizing agent, usually sodium meta periodate, is used to oxidize the carbohydrate moieties on the enzyme molecule. Normally, hydroxyl groups in the carbohydrate moiety are oxidized to aldehyde functional groups.

According to the prior art. This oxidation step employs meta periodate at about 0.08M which results in rather extensive or complete oxidation of the enzyme.

Once oxidized, the enzyme is allowed to react with the immunoreactant by incubating at room temperature for at least three hours and usually at long as over night.

This method of preparation results in what can best be described as a random polymerization of immunoreactant and oxidized enzyme which when employed in immunoassays results in an unacceptable number of erroneous results.

In particular, the incidence of false positive results has been dramatically reduced by preparing and employing an immunoreagent according to the instant disclosure. Essentially, what has been prepared and is a solution to the problem of false results is a reagent which comprises a partially oxidized peroxidase conjugated to an immunoreactant.

By controlling the oxidation of peroxidase by introducing meta periodate at a molar concentration of .02M, which is one-fourth that normally employed and by incubating the partially oxidized peroxidase for about one hour, which is one-third the time normally required, results in a more suitable immunoreagent. it is believed that a reagent prepared according to the disclosed method contains a more uniform number of monomers containing one immunoreactant and one peroxidase.

When prepared according to the conventional prior art method, there appears to be a greater incidence of high molecular weight polymer of immunoreactant and peroxidase.

The false results appear to be due to a steric hindrance effect caused by factors in blood samples (e.g., complement proteins, etc.). This effect blocks the binding of high molecular weight polymers decreasing the enzyme available for detection. The monomer prepared according to this invention lessens the effect.

Fig. 1 depicts the relative enzyme activity in two different enzyme conjugate reagents. One reagent employed peroxidase conjugated to IgG according to the conventional meta periodate method. The other reagent employed partially oxidized peroxidase conjugated to IgG. The two reagents were centrifuged in a sucrose gradient. As indicated by the graph, there was much more monomer demonstrated by the reagent prepared with the partially oxidized peroxidase.

As indicated above, reagents prepared according to the present invention may employ a variety of immunoreactants such as antibodies, antigens, haptens, and binding proteins.

Specific antigens contemplated within the scope of the present invention include hepatitis B surface antigen, hepatitis B core antigen, hepatitis A antigen, hepatitis B e antigen and their respective antibodies. Proteins such as insulin, growth hormone, AFP, TSH and thyroxine can also be conjugated according to the disclosed invention. Microorganisms such as bacteria viruses and Rickettsia are also candidates for enzyme conjugation according to the instant method.

The enzyme to be partially oxidized could be selected from a variety of peroxidases featuring carbohydrate moieties which are known to product predictable results.

The folowing elaboration will illustrate the preparation of a partially oxidized horseradish peroxidase (HRP) which is conjugated to an antibody to hepatitis B core antigen. This reagent can then be used in a variety of immunoassay procedures.

EXAMPLE 1 PREPARATION OF SEPHADEX G-25 COL UMN Five grams of Sephadex G-25 were added to 50 ml of distilled water and allowed to swell. Suitable swelling occurs between three and twenty-four hours. The swollen gel was packed into a column 1 cm x 50 cm. The column was washed with 0.01 M Na2CO3, pH 9.5. The flow rate was observed to be 1-2 ml/minutes by gravity.

EXAMPLE 2 PARTIALLY OXIDIZED HRP About 0.1 g of HRP was dissolved in distilled water to a final concentration of 10 mg/ml. Approximately 100-200 mgs NaHCO3 were dissolved in distilled water to a final concentration of 25 mg/ml. Sodium meta periodate, about 20-50 mgs, was dissolved in distilled water to a final concentration of 4.3 mg/ml. To 0.5 ml of the HRP solution was added 1 ml of the NaHC03 solution. The mixture was swirled and then 1 ml of sodium meta periodate was added and mixed gently. The resulting mixture was allowed to stand undisturbed for thirty minutes at room temperature. The partially reduced HRP was loaded into the Sephadex G-25 column and eluted with .01 M sodium carbonate pH 9.5 and adjusted to 1 mg/ml. The protein band was collected.

EXAMPLE 3 CONJUGATION OF ANTlHBc (HUMAN) IgG TO PARTIALLY OXIDIZED HRP Anti-H Bc (human) IgG (3 mg) in 0.5M sodium bicarbonate pH 9.5 was added to 1.5 ml of partially oxidized HRP. The mixture was gently stirred with a magnetic stirrer for one hour at room temperature. Conjugation was stopped by the addition of 0.1 2 ml freshly prepared sodium borohydride.

The conjugate prepared according to the foregoing may be further purified or may be employed without additional processing.

Claims

1. A reagent useful in enzyme immunoassays which comprises a partially oxidized peroxidase conjugated to an immunoreactant.

2. A reagent according to Claim 1 wherein the peroxidase is horseradish peroxidase.

3. A reagent according to Claim 1 wherein the immunoreactant is an antigen.

4. A reagent according to Claim 1 wherein the immunoreactant is an antibody.

5. A reagent according to Claim 4 wherein the immunoreactant is antibody to hepatitis B core antigen.

6. An enzyme immunoassay reagent comprising essentially of a peroxidase which has been partially oxidized by meta periodate and conjugated to an immunoreactant.

7. A reagent according to Claim 6 wherein the oxidizing agent is sodium meta periodate.

8. A reagent according to Claim 6 wherein the peroxidase is horseradish peroxidase.

9. A reagent according to Claim 6 wherein the immunoreactant is an antibody.

1 0. A reagent for conducting a competitive enzyme immunoassay to detect antibodies to hepatitis B core antigen which comprises partially oxidized horseradish peroxidase conjugated to antibody to hepatitis B core antigen.

11. A method for preparing a peroxidase conjugated immunoreactant which comprises partially oxidizing the peroxidase prior to conjugation with the immunoreactant.

1 2. A method according to Claim 11 wherein the peroxidase is horseradish peroxidase.

1 3. A method according to Claim 11 wherein the immunoreactant is an antibody to hepatitis B core antigen.

14. A method according to Claim 11 wherein the immunoreactant is an antibody to hepatitis B surface antigen.

1 5. A method according to Claim 11 wherein the immunoreactant is an antibody to hepatitis B e antigen.

1 6. A method according to Claim 11 wherein the immunoreactant is an antibody to soluble rubella virus antigen.

1 7. A method according to claim 11, and substantially as described in any one of the Examples herein.

1 8. A reagent prepared according to any one of claims 11 to 17.