Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1983-03-31
1986-05-13
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 7, 435188, 436531, C12Q 170, G01N 5300, G01N 33545, C12N 996
Patent
active
045886806
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
TECHNICAL FIELD
No simple effective method is currently available for rapidly detecting viruses such as influenza virus in biological fluids. Isolating and identifying viruses from biological specimens typically requires several days to weeks and the usual procedures cannot be readily performed in most clinical laboratories. By the time an infecting viral agent has been identified, its identity tends to be of little more than academic interest to the physician faced with the immediate problem of therapy. The availability of a rapid diagnostic method for influenza viruses (and structurally related viruses) would prevent unnecessary antibiotic therapy in the cases of many infections. Moreover where the patient has been affected with certain viruses, such as influenza type A virus, knowledge of such a fact may indicate treatment with other therapeutic agents such as amantadine.
BACKGROUND ART
Many others are currently interested in developing assay systems which will permit the direct detection of viruses in biological specimens. Most approaches seem to be modeled on the double antibody sandwich similar to that found to be effective for rotavirus detection. For that purpose antirotavirus antibody is adsorbed on a bead of size convenient for handling. The antirotavirus antibody bead is then exposed to a stool specimen suspected of containing rotavirus. Following exhaustive washing of the bead any rotavirus present is "sandwiched" with a second antirotavirus antibody to which enzyme has been conjugated. An enzyme assay is then performed to detect the presence of viral particles.
Nearly all of the reports described assay systems which exploit the double-antibody sandwich (DAS) approach with conjugation of .sup.125 I or enzyme (alkaline phosphatase, or horseradish peroxidase) to the second antibody, or indirect analysis, which involves the use of a third antibody to which an enzyme or radiolabel has been conjugated. In a limited number of instances competitive inhibition with labelled or quantified antigen has been suggested along with DAS.
The inventors are also aware of one report of direct adsorption of viral antigen to a substrate used to detect intact picornavirus. This approach can be sucessful with picornaviruses if there is no competing protein antigen in the system.
A severe limitation in detecting virus particles through assay for surface antigens is the widespread existence of proteases as in biological specimens. If samples are not immediately frozen and maintained in a frozen state, surface antigens may be degraded and rendered non-reactive with the antibodies intended to detect them.
Two reports of influenza virus detection systems appear in the literature. The first method (Berg et al., 1980) involves a minimum of 24 hours to carry out and uses fluorescent or radioactive substrates to indicate the presence of influenza virus. The procedure is quite complicated and requires special equipment to detect the fluorescence or radioactivity of the indicator. A second approach (Yolken, et al., 1980) employs "capture" antibody on a Microtiter plate to adsorb neuraminidase in intact viral particles. A neuraminidase assay with a fluorescent substrate (methyl umbelliferyl neuraminic acid) indicates the presence of virus. However, the neuraminidase antigen (particularly Nl) varies greatly in its stability as an enzyme among different subtypes. Inactivation of this enzyme prevents analysis. Further limitations associated with this assay are those arising from its dependence on fluorometry (which precludes the use of visual analysis), and the occurrence of non-specific reactions which yield spurious results.
DISCLOSURE OF INVENTION
In accordance with the present invention we have developed a new method for detecting viruses which does not depend on isolating or detecting the viral particle itself, but rather on detecting a major structural protein component of the virion, the M-protein. Use of the M-protein as the detected antigen in a solid phase system for viral assay has a number of advan
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Bucher Doris J.
Khan Mohamed W.
Kharitonenkov Igor G.
Huleatt Jayme A.
Mount Sinai School of Medicine of the City University of New Yor
Wiseman Thomas G.
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