Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-03-16
1998-12-15
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 79, 435 792, 435 795, 435961, 435969, 435 793, 435 794, 427 213, C12Q 168, A61L 5103
Patent
active
058494804
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The subject of the present invention is a hapten or anti-hapten antibody assay device and use thereof.
The haptens are small non-immunogenic molecules, that is to say incapable by themselves of promoting an immune reaction by forming antibodies, but which are capable of being recognized by antibodies obtained by immunizing animals under known conditions, in particular by immunization with the hapten-protein conjugate.
BACKGROUND
Various techniques have been proposed for assaying haptens in a sample, among which are the so-called competitive techniques. For example, some authors have described an antigen phase competitive technique in which a hapten, which is identical to the hapten to be assayed, and which is attached to a solid phase, competes with the hapten in the sample for its binding with a labelled specific antibody introduced in a defined quantity. This technique has been described for example for the assay of thyroxine, cortisone and testosterone (see especially Patent Applications EP 089806 and WO 83/03306).
However, because of their small size, it is impossible to adsorb haptens directly onto a solid phase consisting of a polymeric material while retaining the availability towards the binding site of a specific antibody. Thus, in conventional techniques (see especially Clinica Chemica Acta, 162 (1987) 199-206 Elsevier), the hapten is grafted onto a protein or polypeptide ligand for its immobilization onto a solid phase. This technique has, nevertheless, a major disadvantage inherent to the non-immunogenicity of haptens. Indeed, the production of anti-hapten antibodies necessitates the immunization of an animal against haptens coupled to proteins or polypeptides, so as to render them immunogenic, and the anti-hapten antibodies produced in response can interfere, by cross-reactions, with proteins or polypeptides to which the hapten is coupled for its binding onto a solid phase. Moreover, the access of the antibody to the hapten attached onto the support can be obstructed by phenomena of intramolecular reorganization of the proteins or polypeptides under certain physicochemical conditions, which can introduce significant errors in the accuracy of a test.
SUMMARY OF THE INVENTION
Consequently, the subject of the present invention is a support appropriate for the assay of haptens which overcomes the abovementioned disadvantages and which makes it possible, in addition, to control the number of haptens immobilized onto the solid phase and therefore to carry out a real quantitative assay. In addition, the invention provides a support permitting improved orientation of the immobilized haptens and therefore increased accessibility of the latter for their binding with antibodies during the assays.
It has in fact been discovered, surprisingly, that it is possible to overcome the abovementioned disadvantages by using, as attachment intermediate, a nucleic acid fragment.
The subject of the invention is therefore a hapten assay device comprising a solid support and a reagent covalently linked to a ligand to form a conjugate which makes it possible to immobilize the said reagent onto the said solid support, the said reagent being recognized by antibodies capable of recognizing the hapten, characterized by the fact that the said ligand is a nucleic acid fragment.
It is understood that the reagent may be either the hapten to be assayed or a compound having a sufficient immunological analogy with the hapten so that antibodies recognizing one also recognize the other.
The term hapten designates the molecule to be assayed, or an analogue capable of reacting with an antibody specific for the molecule to be assayed.
For example, the haptens may be peptides, glycosylated peptides, metabolytes, vitamins, hormones, prostaglandins, toxins or various medicinal products.
The heptan may be chosen for example from:
a glycosylated peptide such as the N-terminal sequence of the beta subunit of human haemoglobin;
thyroidal hormones especially thyroxine, triiodothyronine; steroidal hormones especial
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Battail Nicole
Cros Philippe
Kurfurst Robin
Piga Nadia
Ashton Rosemary
Bio Merieux
Housel James C.
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