Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1996-11-22
2000-02-22
Stucker, Jeffrey
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 71, 435 79, 435 792, 435968, 435974, 436820, 5303871, 5303883, 530826, C12Q 170
Patent
active
060278749
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an immunoenzymatic conjugate consisting of substance(s) having immunological activity and glycosylated labelling enzyme(s) in copolymer form. The invention also relates to the method for the production of the conjugate according to the invention and to the use of the said conjugate for diagnosis.
Various conjugates and methods for the production of immunoenzymatic conjugates are known from the prior art. These immunoenzymatic conjugates generally consist of a substance having immunological activity and a labelling enzyme which may be glycosylated. The methods for the production of these conjugates are varied, and there are, in particular, methods such as those employing periodate coupling techniques, which have given rise to more sophisticated methods employing homo- or heterobifunctional agents. The object of these methods is to improve the coupling of the enzyme to the substance having immunological activity in order to obtain conjugates which enable the specificity and/or sensitivity of the diagnostic results to be increased.
The coupling method of Nakane (The Journal of Histochemistry and Cytochemistry, vol.22,No.12, pp.1084-1091, 1974) describes, after oxidation of the carbohydrates of the enzyme with periodate, the direct coupling of the protein via its free amine groups. Patent EP 209,155 describes a method enabling the immunological reagent to be coupled to the protein portion of the enzyme. According to this method, the enzyme is subjected to an oxidation using periodic acid before or after its coupling with the immunological reagent. The oxidation product thereby obtained is then reduced with sodium borohydride. As regards the coupling, this is carried out using traditional reagents, such as glutaraldehyde, or succinimide derivatives. This method does not employ any copolymerization step. The conjugates originating from this method enable false positive results arising from so-called "problem" sera to be eliminated.
Similarly, Patent Application EP 601,318 describes a method of coupling the immunological reagent to the enzyme via a diamine followed by a heterobifunctional reagent (succinimide derivative), the diamine having reacted beforehand with the oxidized portion of the carbohydrate of the enzyme. A non-copolymerized conjugate, in which the enzyme-immunological reagent link occurs on the carbohydrate portion of the enzyme. In effect, according to this coupling method, the carbohydrate groups borne by the protein are used to graft the protein to the antibody directly.
More recently, Patent Application EP 560,912 and U.S. Pat. No. 5,191,066 describes a method for the production of immunoconjugates (antibody-alkaline phosphatase) using the carbohydrate portion of the proteins, thereby enabling the immunological recognition site of the antibody to be preserved.
Some methods enabling immunoenzymatic conjugates to be obtained in the form of copolymers are also known.
U.S. Pat. No. 4,693,969 describes a reagent for immunoassays of the sandwich type and a method for its production. The reagent for immunoassays, containing an immunoenzymatic conjugate in polymerized form, enables the sensitivity of the assays of substances present at low concentrations in biological samples, such as hormones, to be increased. This reagent is obtained by synthesizing, in a first stage, antibody-enzyme immunoconjugates in monomer form using a coupling agent (m-maleimidobenzoic acid N-hydroxysuccinimide ester). In a second stage, the monomers are subjected to a polymerization reaction with glutaraldehyde or carbodiimide.
Application EP 430,510 describes a polymer based on "detectable" units joined to one another with a coupling agent. These "detectable" units either have antigenic activity, or form part of a fluorescent, chemoluminescent, chromogenic or enzymatic system. The "detectable" units are joined to one another with a hydrophilic coupling agent which is a 1,4-di(aminoalkyl)piperazine derivative. To obtain these polymers, the "detectable" units are chemically modified so as to con
REFERENCES:
patent: 4256833 (1981-03-01), Ali et al.
patent: 4693969 (1987-09-01), Saxena et al.
patent: 5191066 (1993-03-01), Bieniarz et al.
Cognet Gilles
Cucurou Christophe
Gadelle Stephane
Le Sager Carine
Pasteur Sanofi Diagnostics
Stucker Jeffrey
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