Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-08-07
1995-05-16
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435967, 435972, 435975, 436537, 436548, G01N 33535, G01N 33542, G01N 33577
Patent
active
054159989
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an homogeneous immunoassay for determining the amount of a ligand in a liquid test sample and to kits for carrying out such assay.
In this specification the term ligand is to be taken to include any substance (e.g. antigen, antibodies), against which anti-ligands can be produced (e.g. antibodies and anti-antibodies) and accordingly includes within its scope haptens, which may have been rendered immunogenic for the purpose of producing antibodies.
Immunoassay techniques rely upon the formation of a complex between the antigen being assayed and antibodies which are added as part of the immunoassay procedure. Means are provided whereby the amount of antigen: antibody complex formation is detectable. There are several known methods of immunoassay employing antibodies which are labelled so as to be analytically identifiable. "Sandwich" or "two-site" techniques involve the formation of a complex between the antigen and two antibody reagents. A convenient method of detecting complex formation between an antigen in a liquid sample and two antibody reagents is to provide one labelled antibody reagent and an unlabelled reagent bound to a solid phase support so that the complex can readily be isolated. Where a radio-active label is employed this technique is known as immunoradiometric assay (IRMA).
One difficulty with this type of assay is the production of sufficiently pure labelled antibody. Whilst this can be done, it is a laborious procedure and hence relatively expensive. Recently, this problem has been reduced by the availability of monoclonal antibodies. Sandwich-type immunoassays involving the use of two monoclonal antibody reagents, a labelled soluble monoclonal antibody, and a monoclonal antibody which is bound to a water-insoluble solid phase, are described, for example in published European patent application N.0048357.
It is a feature of the technique described above that a significant incubation period is normally required to ensure that the reaction goes (so far as possible) to completion. This is due, at least in part, to the fact that the antigen in solution is required to react with antibody bound to a solid phase.
To avoid these problems, homogeneous immunoassays have been developed. Their use is however limited to the determination of drugs, hormones and plasma proteins. The homogeneous immunoassay known as EMIT (Biochem. Biophys. Res. Comun. 47:846,1972) has been applied successfully for the determination of small molecules like steroid hormones. In a modified EMIT the activity of the enzyme used as tracer is reduced when the conjugate enzyme-hapten (E-L) is bound to the antibody (AB). This seems to be due either to a reduced affinity of the substrate (S) towards the active site of the enzyme in presence of AB, or to a steric hindrance, or to a conformational modification of the enzyme. A further type of EMIT rests upon inhibition of the enzymatic tracer by the conjugated hapten. The tracer re-acquires its activity when the antibody (AB) against the hapten (L) binds the conjugate hapten-enzyme (E-L). A version of this method was developed for large antigen molecules (Anal. Biochem. 102: 167,1980) like IgG but its reported sensitivity seemed to be low. Fluorescence excitation transfer immunoassay (J. Biol. Chem. 251:4172, 1976) is based on the energy transfer between two fluorescent molecules, one on the antibody and the other on the antigen. Again, free analyte inhibits the complex formation between the antibody and the labelled antigen.
The enzyme channelling method (ECIA) (Anal. Biochem. 1056: 223, 1979) (Appl. Biochem. Biotechnol. 6,53-64 1981) makes use of an antibody and an antigen labelled with two different enzymes so that the product of the first enzyme reaction is the substrate of the second one. The overall reaction rate is greatly enhanced if the two enzymes are co-immobilised instead of being present separately in solution. Other techniques like the antigen-labelled fluorescence protection assay (Clin. Chem 25:1077, 1979) have also received attention.
REFERENCES:
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patent: 5130234 (1992-07-01), Hoshino et al.
patent: 5168057 (1992-12-01), Oh et al.
G. Gorog et al, "Use of bispecific hybrid antibodies for . . . a homogeneous enzyme immunoassay", J of Immunol. Methods v 123 (1989) pp. 131-140.
N. Holmes et al, "Enhancement of Monoclonal Antibodies . . . " J. of Biol. Methods v 258 No. 3 (1983) pp. 1580-1586.
W. Moyle et al, "A Circular Antibody Complex is Responsible for Increased Affinity . . . " J. of Immunol. v. 131 No. 4 (1983) pp. 1900-1905.
K. Rubenstein et al, "`Homogeneous` Enzyme Immunoassay . . . " Biochem Biophys Res Comm. v 47 No. 4 (1972) pp. 846-851.
I. Gibbons et al, "Homogeneous Enzyme Immunoassay . . . Employing .beta.-Galactosidase", Anal. Biochem. v 102 (1980) pp. 167-170.
Celada Franco
Gorog Gyorgy
Applied Research Systems ARS Holding N.V.
Saunders David
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