WO1987003691A1

WO1987003691A1 - Determination of test substances

Info

Publication number: WO1987003691A1
Application number: PCT/US1986/002681
Authority: WO
Inventors: Francis Cole
Original assignee: Dennison Manufacturing Company
Priority date: 1985-12-12
Filing date: 1986-12-12
Publication date: 1987-06-18
Also published as: EP0250557A1; JPS63501983A; EP0250557A4

Abstract

Determination of whether or not a test substance, such as a pregnancy antigen (AG), is present by combining (1) the test substance (AG) with a first labeled material, e.g. a first enzyme labeled antibody (ABA-E1) in a vial (30). The resulting product is added (2) to a second labeled material, e.g. a second enzyme labeled antibody (ABA-E2), bound to a membrane (22) which overlays a porous member (21) both within a housing (10) to which a substrate is applied (3) from vial (40) to react with one of the labels and produce a reagent that in turn reacts with an indicator and the other label to provide a test indication such as a color change.

Description

DETERMINATION OF TEST SUBSTANCES

BACKGROUND OF THE INVENTION

This invention relates to testing for substances, particularly by "assays" and more particularly by "immunoassays.

An assay is a procedure for determining whether or not a test substance is present or absent, and, where necessary, in what amount.

In the case of an "immunoassay" the testing involves the "immunological" agents, which are produced by or cause a response in, the immune system of a living body. The agents produced by the immune system are "antibodies". They result from the reaction of the body against foreign substances, like viruses, or may be generated in response to a physical change in body chemistry, like pregnancy. "Antibodies" are the response to the presence of "antigens". For example, during pregnancy, the body produces elevated levels of a hormone known as human chorionic gonadotrophin (HCG) , so called because it originates in the reproductive organs. A non-human body reacts to HCG exposure by producing anti¬ bodies known as anti-HCG. Another example is the gonococcus (GC) bacteria that produces the venereal desease known as gonorrhea. The body reacts by producing antibodies known as anti-GC.

Since the reaction to an antigen is an antibody, it has been recognized that the determination of either can be made using the labelled complement. Thus, where it is desired to detect the pregnancy antigen (HCG hormone) , the correspon¬ ding antibody can be labelled with an indicator. The labelled antibody is then applied to a test sample which may contain the complementary antigen. If the antigen is present, the labelled antibody binds to the antigen and the label or indicator now bound to the antigen can be detected by a variety of methods. Since these techniques employ antibodies and antigens associated with the immune system, they are known as immuno- assays. Originally, the labels indicators were radioactive and/or fluorescent materials. Thus, when the antigen, and an antibody labelled with a radioactive or fluorescent material, were brought together, the presence of the label permitted detection of a binding reaction between the antibody and the antigen.

Radioimmunoassays are objectionable in that they involve the potential hazards of radioactive materials. Fluoroimmunoassays, in turn, have the objection of requiring fluorescent dyes (fluorochromes such as fluorscein and rhoda ine) . These can be detected only by fluorescent microscopes, which are expensive and not widely available.

Radio and Fluoroimmunoassays thus were primarily for laboratories and not suitable for home diagnosis. To overcome this difficulty, enzymes have been used as the labelling material. The anibody or antigen that is to be detected is used with its labelled counterpart. Where detection is to be made of an antigen the labelled counterpart is an enzyme labelled antibody. All that is necessary is to use an indicator which can react with an enzyme and thus indicate its presence.

In the typical enzyme immunoassay, antibodies that are specific to a test antigen are first absorbed in excess on a solid surface such as a plastic tube. A test solution containing the antigen is then added, and the antigen binds to the antibody. The "solid phase", composed of all material bound to the antibody, is then thoroughly washed to remove unbound components. The test then proceeds by identifying the bound antigen.

In the sandwich antibody ELISA method, for example, an enzyme labelled second antibody, preferably having binding sites different from those of the first antibody, is added and reacts with specific determinant sites on the bound antigen. The enzyme labelled second antibody is added in excess to assure that all antigen in the solid phase, and bound to the. first antibody, will also be bound to enzyme labelled second antibody. The enzyme labelled second antibody molecules will bind to each antigen molecule in a fixed ratio depending on the specific available binding sites at the antigen. The solid phase is again washed to remove excess second antibody and any other unbound constituents. An enzyme "substrate", i.e. a substance which reacts with the enzyme, is then added in solution in excess amounts to make contact with the bound solid phase. For the peroxidase enzyme, the substrate may be a solution of hydrogen peroxide and a chromogenic material.

It is apparent that the typical test procedure for enzyme immunoassays, as described above, requires separate washing steps in order to remove excess material.

In addition, the constituents of the indicator material, which are commonly hydrogen peroxide and a chromogenic material, must be kept apart until the time for the assay. If the indicator materials are mixed prematurely there is a decay and the resultant solution is not satisfactory for use after a delay interval. Indeed, the chromogenic reagent and hydrogen peroxide must be mixed in a fresh batch just prior to each use because they tend to oxidize and become spontaneously colored when left in storage for as little as one hour.

Accordingly, it is an object of the invention to facili¬ tate the determination of test substances and the assaying for test materials. A related object is to facilitate immunoassay, particularly enzyme immunoassay, procedures.

Another object of the invention is to simplify the rinsing requirements of the prior art. A related object is to completely eliminate rinsing after formation of a test "sandwich".

Still another object of the invention is to reduce the amount of time required in enzymatic assays.

Yet another object of the invention is to eliminate the need for combining a chromogen with an activator such as hydrogen peroxide. A related object is to eliminate any adverse consequences from the premature combination of a chromogen with an activator. SUMMARY OF THE INVENTION

In accomplishing the foregoing and related objects the invention provides for determining whether or not a test substance is present by combining a sample, which may contain the test substance, with a first-labelled material. The result of this combination is then combined with a second labelled material and a substance is applied to react with one of the labelled materials to produce a reagent that is able, in turn, to react with the other of the labelled materials.

In accordance with one aspect of the invention, an indicator reacts with the reagent and one of the labels to produce a color change. The indicator is a leuco dye, desirably tetramethylbenzidine, or a derivative, and the labels are different enzymes. One of the enzymes can be an oxidase which reacts with a substance, such as glucose or cholesterol to produce a peroxide. The other of the enzymes can be peroxidase which reacts with a peroxide and tetramethylbenzidine to produce a color change.

In accordance with yet another aspect of the invention, the second labelled material is bound to a porous support. When the test substance is present, it binds jointly to the first labelled material and the second labelled material. Peroxide produced by one of the labels reacts with the other label and an indicator, such as tetramethylbenzidine, to produce a color change indicative of the presence of the test substance. When the first labelled material is unbound, i.e., when the test substance is absent, the amounts of reagent, indicator and second label having been preadjusted such that the unbound first reagent is carried away before a color change occurs.

In accordance with a further aspect of the invention, a test kit is provided including a first labelled material, a second labelled material, means for permitting a first reaction of the first material test sample, means for permitting a reaction of the second material with the result of the first reaction, and means for applying an indicator to the further reaction. This provides a color change when a test substance is present. The first material is desirably labelled with a first enzyme, and the second material is desirably labelled with a second enzyme. The indicator is desirably a benzidine or a derivative of benzidine. The first material is supported by a first vessel and the second material is supported by a second vessel. In practice, the first vessel is a test tube on which the first material is liquid or lyophilized preparation and the second vessel is a container for a porous support for the second material. Consequently, when the complement of the second material is present in a test sample, it is able to bind to the second material on the support. Since it is bound to the support along with the first labelled complement, a reaction such as a color change that takes place on the support can provide an indication of whether or not the test substance is present. For this purpose it is important that the support be visible from the exterior of the second vessel or housing.

In accordance with yet another aspect of the invetion, a structure for indicating the presence or absence of a test substance is produced by providing a housing, including a porous membrane within the housing, using the porous membrane to support a labelled material, and overlying the porous member with the porous membrane. The labelled material desirably has an enzyme label which is an oxidase, such as glucose oxidase. The porous membrane is fitted in the mouth of the housing and is visible when the mouth is uncovered in order to provide an indication of color change when a test substance is present. The porous membrane that supports the labelled material desirably has a lesser degree of porosity than the porous member that supports the membrane. The reason for this is that when a test substance is absent and the first label is washed from the membrane into the more porous underlying member, and no color change is observed in the porous layer. DESCRIPTION OF THE DRAWINGS Other aspects of the invention become apparent after considering several illustrative embodiments taken in conjunction with the drawings in which:

Fig. 1 is a test housing in accordance with the invention: and

Fig. 2 is an illustration of a test assay in accordance with the invention making use of the test housing of Fig. 1 and auxiliary test components

DETAILED DESCRIPTION

With respect to the drawings. Fig. 1 shows a test kit 10 which includes three basic constituents: a test housing 20, a preliminary mixing vial 30, and an indicator vial 40. The test housing is shown with a part of its side wall broken away to reveal the realtionship of interior constituents 21 and 22 which are respectively a porous support and an overlying porous membrane.

The specific relationship of the underlying porous support 21 and the overlying membrane 22 are set forth in greater detail in the cross sectional view of Fig. 2. The housing 20 includes a mouth 23 which exposes a substantial portion of the surface of the membrane 22 and permits the application of fluids to the housing 21. For that purpose, the upper portion of the housing has retaining walls 21-4 to 24-4 which are fitted above the base 25.

The mixing vial 30 has an interior wall 31 that is coated with a first labelled material, such as an enzyme labelled antibody. For convenience, the enzyme labelled antibody is designated as AB-E,.

The remaining constituent of the kit 10 is the indicator vial 40. This contains materials including a chromogen which will bring about a color change when the fluid of the indicator is applied to the test housing as discussed in detail below in connection with Fig. 2. The indicator vial 40 contains a chromogen such as tetramethylbenzidine or one of its derivatives, and a "substrate" for an enzyme. The "substrate" is so designated - 7 -

because it reacts with an enzyme label. In the case of Fig. 1 the enzyme in question is linked to an antibody that is on the porous membrane 22. For convenience the enzyme labelled antibody is designated as AB-E₂.

The test procedure, in accordance with the invention, is illustrated in Fig. 2. A test sample which possibly contains a test substance such as an antigen (AG) is applied to the vial 31. If antigen is present in the test sample, it binds to the enzyme labelled antibody in vial 30 in accordance with equation 1 (1) :

AG-AB^E^ (1)

The result of this first step is to produce in the test sample, when the test substance is present, an enzyme-linked antibody that is in turn bound to the complementary antigen. The contents of vial 30 are then deposited as indicated by the arrow for step No. 2 on the membrane 12 that contains the second enzyme linked antibody. If the test antigen is linked to the first antibody as in accordance with equation 1, the presence of this material with the enzyme linked antibody on the membrane 22 produces the reaction shown in equation (2) below:

E₂-AB_A-AG-AG_A-E₁ (2)

The next step is to apply the contents of the indicator vial 40 as indicated by the arrow No. 3. Since the indicator vial contains a "substrate" that reacts with the enzyme E₂, this produces a reagent, for example, hydrogen peroxide as shown in equation (3) :

S+E₂ —>H₂o₂. (3)

The result of equation (3) in turn produces a reaction with the first enzyme E-. as shown in equation (4) :

^H2°2 ^{+ I + E}ι —> color change ⁽4)

Other aspects of the invention are described below. Within membrane 22 glucose oxidase in various concentrations is immobilized while keeping the level of co-immobilized antibody constant. This procedure is commonly referred to as a titration. There are several dilutions that range from a concentration level of 30 mg. per illiliter in a stock solution of 1.4 mg. per ml. of monoclonal antibody (LH262G9H10) to 1.8 micrograms per ml. The stock solution of glucose oxidase (GO) and AB and in a PBS buffer of pH 7.2. Exact levels of glucose oxidase are 30, 15, 7.5, 3.7 and 1.8 micrograms per ml.

Simultaneously, dextrose is diluted in citrate phosphate buffer having a pH of 5.0. Concentrations of this substance are 50, 12.5, 3.12, 0.78 and 0.195 grams per liter. Additionally, 0.9 milligrams of Igepal CA 720 are added to each of the solutions to form a volume of 14.9 milliliters.

The foregoing immobilization procedure is begun by applying 3.0 milliliters of AB-GO mixture per membrane and allowing a 10 second drying time. A blocking phase is completed by putting through a carnation miller PBS buffer under vacuum and drying the membranes in a desiccator.

The assay steps are as follows:

0.25 milliliters of lyophilized conjugate is reconstituted with 0.25 milliliters of sample that can contain LH hormone. Upon dissolution of the lyophilized materials, the liquid is poured over the membrane, containing the immobilized AB and GO of the assembled test device. This device consists of from top to bottom, a membrane, a semi-rigid porous disc and an absorbent matrix.

Other aspects of the invention will be apparent to those of ordinary skill in the art.

Claims

1. The method of determining whether or not a test substance is present, which comprises the steps of:

(1) Combining a sample, which may contain the test substance, with a first labelled material;

(2) Combining the result of step (1) with a second labelled material;

(3) Applying, to the result of step (2), a substance which reacts with one of the labelled materials to produce a reagent that is able to react with the other of the labelled materials.

2. The method of claim 1 wherein an indicator reacts with said reagent and the label of one of the labelled materials to produce a color change.

3. The method of claim 2 wherein the indicator is leuco dye, including tetramethylbenzidine, or a derivative thereof, and the labels are different enzymes, including peroxidase.

4. The method of claim 3 wherein one of the enzymes is an oxidase which reacts with a substance to produce a peroxide.

5. The method of claim 4 wherein the other of the enzymes is a peroxidase which reacts with a peroxide and tetramethylbenzidine to produce a color change.

6. The method of claim 5 wherein said second labelled material is bound to a porous support.

7. The method of claim 6 wherein said test substance, when present, binds jointly to said first labelled material and second labelled material, and peroxide produced by one of the labels reacts with the other label and said indicator to produce a color change indicative of the presence of said test substance.

8. The method of claim 6 wherein said first labelled material is unbound when said test substance is absent and said reagent is not produced by the first label and no reaction of said second label and said indicator can occur.

9. The method of claim 6 including the step of washing away unbound first color change from said support by said indicator before a color change can take place.

10. A test kit comprising: a first labelled material; a second labelled material; means for permitting a first reaction of the first material with a test sample; means for permitting a reaction of the said second material with the result of said first reaction; and means for applying an indicator to the further reaction to provide a color change when a test substance is present.

11. A test kit as defined in claim 10 wherein said first material is labelled with a first enzyme; said second material is labelled with a second enzyme; and said indicator is a benzidine.

12. A test kit as defined in claim 10 wherein said first material is supported by a first vessel, and said second material is supported by a second vessel.

13. A test kit as defined in claim 12 wherein said second vessel includes a porous membrane for said second material, whereby a sandwich can be formed on said membrane which is a complex of the first material, the test sample and the second material.

14. A test kit as defined in claim 13 wherein said porous membrane is visible from the exterior of said second vessel.

15. The method of producing a structure for indicating the presence or absence of a test substance which comprises the steps of:

(1) providing a housing;

(2) including a porous member within said housing;

(3) using a porous membrane to support a labelled material; and

(4) overlying said porous member with said porous membrane supporting said labelled material.

16. The method of claim 15 wherein said material has an enzyme label.

17. The method of claim 16 wherein said enzyme is an oxidase.

18. The method of claim 17 wherein said oxidase is glucose oxidase.

19. The method of claim 15 wherein said porous member is fitted in the mouth of said housing and is accessible when said mouth is uncovered.

20. The method of claim 15 wherein the porosity of said member is greater than that of said membrane.