Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1992-02-07
1993-07-06
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 722, 435 732, 435 75, 435 79, 435 792, 435967, 435975, 436530, 436547, G01N 33569
Patent
active
052253228
DESCRIPTION:
BRIEF SUMMARY
large number of different monoclonals needed to test the virus adequately. Monoclonal antibodies are very time-consuming to produce and the specificity and cross-reactivities cannot be controlled except by lengthy selection procedures. Also, mutations can not be picked up unless the panel of monoclonal antibodies being used happens to have a specificity for the critical antigenic determinant.
Despite the efforts which have been made to develop accurate tests for detecting viruses, tests are required which can be performed accurately and with minimal need for specialized equipment. Tests which can be performed in the field are also needed. Poultry growers are often located far from research centers and agriculture department stations. Tests which can be performed in the field would allow poultry growers to avoid delay in treating birds and taking precautions for preventing the spread of the virus. Whether in the field or the laboratory, there is also a great need for tests which can accurately and rapidly distinguish between pathological and non-pathological subtypes of avian influenza virus.
The problems in providing fast accurate tests to detect avian influenza virus are also encountered in immunoassays for other substances, particularly if the substance contains multiple antigenic determinants or epitopes, such as would be present in living organisms and larger molecules. Although monoclonal antibodies are valuable for distinguishing between closely related organisms, immunoassays using monoclonal antibodies may give inaccurate results if the test sample contains an organism not having the epitope corresponding to the monoclonal antibody employed in the assay. This situation could easily arise if there has been a mutation in the organism deleting or modifying the antigenic determinant.
It is an object of this invention to provide novel methods for detection, identification and quantitation of antigens and hence the source or organism containing the antigen, such as viral subtypes and strains. It is also an object of the invention to provide novel methods for detecting, identifying and quantitating avian influenza virus subtypes and strains. It is a further object of the invention to provide kits for use in detecting, identifying and quantitating antigens and hence the organism containing the antigen, such as viruses, viral subtypes and strains.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1c show the dot blot assay for avian influenza virus strain H5N2/Mal/NY/82, FIGS. 1b and 1d show the dot blot assay for avian influenza strain H6N8/Ty/Mn/83 and FIG. 1e shows the dot blot system for H2N7/Ty/Mn/86.
FIGS. 2a and 2b show quantatitive representation of two viral proteins after computer-analyzed digitization.
FIG. 3A show a transblot of hyperimmune sera of two rabbits injected with avian influenza H5N2/Mal/NY/82 where the virus is adsorbed onto nitrocellulose and probed with biotinylated antibodies and FIG. 3B shows transblot of three different subtypes of avian influenza virus analyzed with a single virus to determine epitope differences among the subtypes.
FIGS. 4a and 4b show line and bar graphs, respectively, of the bands of lane 4 in FIG. 3a that have been quantitated by densitometry and computer-digitized. FIGS. 4c and 4d show line and bar graphs, respectively, of the bands of lane 5 in FIG. 3a that have been quantitated by densitometry and computer-digitized.
SUMMARY OF THE INVENTION
The invention provides methods of detecting an antigen in a test sample suspected of containing said antigen, and hence the source or organism containing the antigen. Polyclonal antibodies specific for the antigen are separated and then applied to a solid support. The separated antibodies are then contacted with a test sample suspected of containing the antigen under conditions selected to allow binding of the antigen to the antibodies. Antigen bound to the antibodies on the solid support is then contacted with antibodies specific for the antigen under conditions selected to allow binding of the antibodies to the
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Bidwell Carol E.
Saunders David
University of Pennsylvania
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