Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Patent
1996-12-20
1998-08-04
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
4352351, C12N 700
Patent
active
057892326
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for preparing antigens of viral origin from infected animal cells and to the use thereof in enzyme-linked immunosorbent assays (ELISAs).
In modern immunodiagnosis on patients' sera it is a proven and widely used technique to determine the antibody status against a wide variety of antigens, for example with the aid of an ELISA. This entails microtiter plates being coated with the antigen against which antibodies are to be detected, and subsequently being incubated with serum. If antigen-specific antibodies are present, they bind to the offered antigen and can then be detected by a detection system with, for example, peroxidase-conjugated anti-antibodies via an enzyme-mediated substrate conversion with development of a color which can be measured by photometry.
Preferably employed for producing antigens for coating the plates are animal cell cultures which form the required product or can be stimulated thereto. To establish the humoral immunity status against certain virus specificities using a suitable ELISA, for example cell cultures are infected with the appropriate viruses and harvested some time later, when the infection has affected the entire cell lawn. The infected cells are then starting material for preparing the viral antigen required for coating the assay plates.
N. E. Cremer et al., J. Clin. Microbiol. 21, pages 869-874 (1985) describe preparing antigens from cultures infected with varicella zoster virus (VZV)--a representative of the human herpesviruses--in such a way that cell homogenates are prepared from animal cells by disruption with ultrasound or multiple freeze/thaw treatment, which then contain nuclei and cytosolic constituents in suspension. The medium chosen for taking up the cells before the disruption treatment is isotonic phosphate-buffered saline (PBS) or alkaline glycine buffer. The resulting cell homogenates are subjected to a low-speed clarifying centrifugation in order to sediment cell nuclei and other larger constituents. The removed supernatants from the centrifugation--referred to as (cell) homogenate antigens hereinafter--can then be employed without further purification for coating microtiter plates for the ELISA. The preparation of antigens as described, for example, by M. Lehtinen et al., Intervirology 24, pages 18-25 (1985) from cells infected with herpes simplex virus (HSV) for the ELISA for detecting anti-HSV antibodies comprises comparable process steps. The procedure for hepatitis A virus (HAV) according to the principle described above is reported by K.-Q. Wang et al., Intervirology 24, 99-107 (1985).
Although cell homogenate (extract) antigens prepared according to the prior art can be used, they contain a very large proportion of cellular proteins which are immunologically irrelevant or even interfere and, during the coating of microtiter plates, compete with the viral proteins and structures required for covering the solid phase, so that the occupation density of the polystyrene surface with virus-specific components is prematurely limited. This competition is also the reason why no further increase in sensitivity (signal) is possible in the ELISA through a more highly concentrated homogenate antigen for coating.
The present invention was therefore based on the object of considerably reducing, with use of a suitable buffer in processing the antigen, the content of cellular proteins present in the cell homogenate in order thus to increase the occupation density for virus-specific components on the solid phase. This is a crucial pre-condition for being able to improve assay properties such as sensitivity and positive predictive value with an unchanged specificity and as far as possible at the same time employing a more economic amount of antigens for coating assay plates.
It has been possible to achieve this object by an isolation process in which the concentration of cellular organelles (especially cell nuclei) and proteins is decreased and that of viral constituents is increased by a differential centrifugation of viru
REFERENCES:
patent: 3514374 (1970-05-01), McAleer et al.
patent: 4100267 (1978-07-01), Shaw
Natalie E. Cremer, Cynthia K. Cossen, Gordon Shell, Janice Diggs, Dana Gallo, and Nathalie J. Schmidt, "Enzyme Immunoassay Versus Plaque Neutralization and Other Methods for Determination of Immune Status to Measles and Varicella-Zoster Viruses and Versus Complement Fixation for Serodiagnosis of Infections with Those Viruses," Journal of Clinical Microbiology, Jun. 1985, pp. 869-874.
Matti Lehtinen, Vesa Koivisto, Tuula Lehtinen, Jorma Paavonen, Pauli Leinikki, "Immunoblotting and Enzyme-Linked Immunosorbent Assay Analysis of Serological Responses in Patients Infected with Herpes Simplex Virus Types 1 and 2," Intervirology 24: 18-25 (1985).
Von Andrea Lindner, H. Liebermann and H. Ambrosius, "Entwicklung eines Enzyme-linked Immunosorbent Assay zum Nachweis von Antikorpern gegen das Bovine Herpesvirus vom Typ 5 (BHV-5) in Schafseren," Deutsche Tierarztliche Wochenschrift, Mai 1993, 100, pp. 390-395.
K.-Q. Wang, C.M. Nielsen, B.F. Vestergaard, "Isolation and Adaptation Characteristics of Hepatitis A Virus in Primary African Green Monkey Kidney Cells: Production of Antigen Useful for ELISA Serology," Intervirology 24: 99-107 (1985).
Behring Diagnostics GmbH
Lankford , Jr. Leon B.
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