Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1996-11-20
1998-06-23
Scheiner, Toni R.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
435 5, 435 71, 435 792, G01N 3353
Patent
active
057704570
DESCRIPTION:
BRIEF SUMMARY
This application was filed under 35 USC 371 as the national stage of PCT/EP95/01829, which was filed May 15, 1995.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an immunoassay for the determination of antigens, especially high molecular weight antigens.
2. Description of the Related Art
Immunological assay systems in which monoclonal antibodies are used as the specific and selective indicator have been used for more than a decade. A large number of different methods of performing these assays have been developed and subsequently used. One essential element of immunological assay methods is the separation of the bound antigen or antibody from molecules in which no antigen-antibody reaction takes place ("bound to free separation"). In the majority of the assay systems employed this separation is carried out by immobilizing one of the assay components on a solid phase ("solid phase technology"). The immobilized molecules can consist of an antigen, an antibody or specific anchoring molecules (e.g. avidin, biotin conjugate, etc.) for the indirect fixation of immunoassay components.
The solid phase generally consists of plastics (PVC, polystyrene), latex, glass, metal or porous (filter) membranes of a wide variety of compositions. The immunoassay components (the antigen or the antibody) are then immobilized on this solid phase by adsorption or covalent binding. In the course of the incubation the bound portion of the incubation mixture (antigen-antibody complex) which is fixed to the solid phase by the immobilized assay component is removed by decanting, centrifugatiion or filtration etc.
This conventional immunoassay technique is limited by the possible variations in the biochemical or immunological properties of antigens or antibodies. Thus not all antigens can be immobilized on solid phases without undergoing changes in structure or behaviour which result in immunological (and consequently analytical) deviations from their corresponding native forms. Also it is not always possible to fix antibodies to all types of solid phase. While polyclonal antibodies can be immobilized with sufficient efficacy in almost any solid phase design, monoclonal antibodies cannot always be immobilized in satisfactory yields, or cannot be immobilized at all. The possible method of binding assay components to a solid phase by covalent bonds is also highly complicated, if at all usable for routine applications.
The by far the most widely used solid phase for immunoassay techniques, the mictrotitre plate (MTP) consisting of polystyrene, cannot always be coated with a sufficient quantity of monoclonal antibodies for effective use in assays. At present a number of methods are used in immunoassay technology for carrying out solid phase immunoassays.
In the sandwich technique at least two specific antibodies are used, for which the recovery, optimization, production and characterization of two separate antibodies is required. In addition, a comparatively complicated setup of incubation and washing stages is required. Also it is not always possible to use polystyrene MTP's for monoclonal antibodies (insufficient immobilization), but instead PVC material has to be used, or the monoclonal antibody has to be fixed indirectly by another technique (biotin/avidin etc.).
When antigens are immobilized ("half-sandwich ELISA") their structure can possibly change as a result of adsorption, thus resulting in deviations from the native molecule. Considerable optimization efforts -are still required in this area.
The problem addressed by the present invention was that of developing an immunoassay which avoids the above disadvantages.
SUMMARY OF THE INVENTION
In the following an immunoassay design for specific antibodies in particular for monoclonal antibodies is described which is distinguished by a considerably simplified assay procedure (reduction in time), a considerable shortening of the development phase (cost reduction) and an increase ion versatility (use with all types of solid phase) compared with
REFERENCES:
patent: 4828985 (1989-05-01), Self
Harlow & Lane Antibodies, A Laboratory Manual. Cold Spring Harbor Press, pp. 555-557, 1988.
Brown, J.C. & Koshland, M.E., Proc. Natl Acad Sci USA 72:5111-15, 1975.
Brown, J.C. & Koshland, M.E., Proc. Natl. Acad. Sci. USA 74:5682-86, 1977.
Barkas, T., and Watson, M.J. Immunol. 36:557-61, 1979.
Doth Margit
Stocker Ronald Helmut
Bayer Aktiengesellschaft
Scheiner Toni R.
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