WO1981001414A1

WO1981001414A1 - An improved method of non homogenous enzyme immunoassay

Info

Publication number: WO1981001414A1
Application number: PCT/AU1980/000090
Authority: WO
Inventors: P Duffy
Original assignee: Charles Hospital Dev; P Duffy
Priority date: 1979-11-19
Filing date: 1980-11-19
Publication date: 1981-05-28
Also published as: EP0040224A4; EP0040224A1; JPS56501583A

Abstract

Method of non-homogenous enzyme immunoassay including the initial step of binding an analogue of ligand to an enzyme to form an enzyme-ligand analogue complex. This complex is then contacted with antibody for the ligand so as to bind the complex thereto. The enzyme analogue may then be displaced from the binding sites of the antibody by addition of the original ligand to which the antibody has been derived or of a different analogue having a higher specificity for the antibody than the enzyme labelled analogue. Subsequently the liberated enzyme analogue is separated from the enzyme analogue bound to the antibody and a quantity of liberated enzyme analogue is measured thereby determining the amount of ligand in a sample by comparison of the amount of liberated enzyme analogue with known concentrations of ligand.

Description

"AN IMPROVED METHOD OF NON HOMOGENOUS ENZYME IMMUNOASSAY" This invention relates to an improved method of the technique known as non homogenous enzyme immunoassa , described, for example, in "Quantitative Enzyme Immunoassay", Ed. E. Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7, 1978) .

The technique of non homogenous enzyme immunoassay is based on the competition for the active site of an anti¬ body between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme ligand phases, enzyme assay of either fraction can then be made. To aid in the separation of the phases, it is often convenient to bind the antibody to an insoluble particle such as the wall of the reaction vessel itself, or a polysaccharide particle. Plotting of the percent bound against the ligand concentration, results in a typical immunoassay curve. The enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-anti- body derivatives and using a solid phase ligand to assist in the separation step.

The antibody characteristics required for the development of a successful non homogenous enzyme immunoassay for a particular ligand are those of high specificity for the ligand, as well as a high apparent affinity constant (as 1/mol) for the ligand.

It has been generally observed in accordance with the invention that an antibody demonstrating a high specificity and a high affinity constant towards a particular ligand will also bind substances which are closely similar in chemical structure to the ligand itself. Such substances are herein referred to as "analogues of the ligand".

The affinity constant of the antibody observed with respect to the analogue may differ markedly from that observed for the antibody with the ligand itself. It is the object of

OMPI WIPO ^ the present invention to describe a generalised system of non homogenous enzyme immunoassay, which utilises an analog with a lower affinity constant for the antibody than the ligand itself. Accordingly, the invention provides a method of non homogenous enzyme immunoassay including the steps of: (i) binding an analogue of a ligand to an enzyme to form an enzyme-ligand analogue complex; (ii) contacting the enzyme-ligand analogue complex with antibody for the ligand so as to bind the complex to the antibody;

(iii) displacing the enzyme-analogue from the binding sites of the antibody by addition o the original ligand to which the antibody has been derived or of a different analogue having a higher specificity for the antibod than the enzyme labelled analogue; (iv) separating the liberated enzyme analogue fr the enzyme analogue bound to the antibody; and (v) measuring the quantity of liberated enzyme analogue and thereby determining the amount of ligand in a sample by comparison of the amount of liberated enzyme analogue with known concentrations of a ligand. Thus, to prepare a suitable assay for a ligand, f which an antibody has been prepared by appropriate means, a suitable analogue of the ligand is selected such that it is bound specifically by the antibody, though to a much lower affinity than is the case for the original ligand itself. Such an analogue is then covalently joined to an appropriat enzyme by known methods of chemistry.

As a further procedure, preparations of the antib are linked by appropriate means to an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead. Such a "formed solid phase" antibody is required to retain its function as an antibody. By appropriate means, the enzyme-labelled analogue is then brought into contact with the solid phase antibody, such that all the binding sites of the solid phase antibody are occupied by the enzyme-labelled analogue. Such an. enzyme-labelled analogue may then be displaced from the binding site of the solid phase antibody by addition under appropriate conditions of the original ligand to which the antibody has been derived, or a different analogue having a higher affinity for the antibody than the enzyme-labelled analogue. By separation of the liberated enzyme analogue from the enzyme analogue remaining bound to the solid phases , it is found that the quantity of liberated enzyme analogue, as measured by whatever means, bears an analytical relation¬ ship to the quantity of antigen or other ligand which causes the displacement. By comparison of the amount of enzyme analogue liberated by known concentration of ligand in appropriate standards, the measurement of an unknown concen¬ tration of the ligand in a sample can be effected.

To allow performance of the assay on future occasions, known amounts of the enzyme-labelled analogue bound to the antibody can be dispensed into vials and subjected to the process known as freeze-drying. By adding a required volume of water to the vial to reconstitute the active enzyme-analogue-antibody-complex, the assay can then be performed directly. With appropriate precautions to maintain stability of the reagent, it would be possible to utilise a previously defined calibration curve from which the amount of ligand in a sample can be calculated without the necessity of so performing a calibration curve at that time. This method thus has application in times of emergency when speed

and simplicity of an assay is required.

By way of an example, an assay for the anti- epilepsy drug dilantin, in the patients taking this drug is described. Antibodies were raised to dilantin in goats by appropriate means and the antibodies demonstrated an affinity for dilantin of 10 1/mole, while having an affini for meth l-dilantin of 10 1/mole. Methyl dilantin was the covalently joined to bacterial beta-galactosidase using water soluble carbo-dii ide, and the antibody covalently bound to sepharose beads. Aliquots of the sepharose antibo methyl dilantin-beta-galactosidase complexes were formed an all available antibody binding sites saturated. Following this these beads were exhaustively washed with phosphate buffered saline by appropriate means such that only enzyme methyl dilantin bound to the antibody remained on the beads Tubes were set up containing 250 uL of Buffer, 20 uL of sepharose antibody-methyl dilantin-beta-galactosidase and 20 uL of dilantin serum calibrators containing dilantin in the concentrations of 0,7,14 and 35 mg/L respectively. The tubes were incubated with gentle shaking at 37 degrees for 30 minutes at which time they were centrifuged and 100 uL removed and assayed for beta galactosidase activity by colorimetric means. The results were as follows:

Tube No. Dilantin Cont b-Galactosidase activity (Mg/L) (A405/min/ml)

1 0 0.0128

2 7 0.0190

3 14 0.0232

4 35 0.0314 Thus it can be seen that there is a proportional increase in enzyme activity released with increasing amount of dilantin in the sample. By freeze-drying 20 uL aliquots of the antibody-methyl dilantin beta galactosidase sepharose solution in appropriate vials a curve similar to that above could be obtained when this experiment was repeated. Thus i

was possible to estimate the amount of dilantin in a test serum obtained from a patient taking dilantin in this manner.

It will be appreciated that the method of the invention as described above will be applicable generally to any suitable ligand or ligand analogue such as the systems phenobarbitone (meta-nitro phenobarbitone) theophylline (l-methyl-3-carboxy butyl xanthine) thyroxine (3,3' 5 - triiodo-5 '-bromo thyronine) and triiodothyronine (3,5 - diiodo-3'-bromo thyronine). In the abovementioned systems the ligand analogue is shown in parentheses.

SUBSTITUTE SHEET⁴

Claims

1. A method of non homogenous enzyme immunoassay including the steps of:

(i) binding an analogue of a ligand to an enzyme to form an enzyme-ligand analogue complex; (ii) contacting the enzyme-ligand analogue complex with antibody for the ligand so as to bind the complex to the antibody; (iii) displacing the enzyme-analogue from the bin ing sites of the antibody by addition of th original ligand to which the antibody has been derived or of a different analogue having a higher specificity for the antibod than the enzyme labelled analogue;

(iv) separating the liberated enzyme analogue from the enzyme analogue bound to the anti¬ body; and (v) measuring the quantity of liberated enzyme analogue and thereby determining the amount of ligand in a sample by comparison of the amount of liberated enzyme analogue with kn concentrations of ligand.

2. A method as claimed in Claim wherein the ligand i dilantin, the ligand analogue is methyl-dilantin and the enzyme utilised is bacterial beta-galactosidase.

3. A method as claimed in Claim 1 or 2 wherein the antibody is bound to a solid substrate.

4. A method as claimed in Claim 3 wherein the solid substrate is the inner wall of a glass tube or a small insoluble particle.

5. A method as claimed in Claim 4 wherein the small insoluble particle is a polysaccharide or a glass bead.

SUBSTϊTUTE SH^^cT Λ T™⁵