Date of Award:

5-1-1983

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Life Sciences:Biology

Committee Chair(s)

Rex S. Spendlove

Committee

Rex S. Spendlove

Committee

J. Clifton Spendlove

Committee

D. Jack Adams

Committee

A. Jeff Mohr

Abstract

Monoclonal antibodies used in serological procedures have the potential to recognize and identify one single antigenic determinant on a virus that is not shared by closely related viruses. This procedure would have the advantage of reducing non-specific background and would allow differentiation between a mixture of viruses. In this investigation, a monoclonal antibody to reovirus type 1 was produced and compared with polyclonal antibody for immunoassay detection of virus. The tests compared were the enzyme-linked immunosorbent assay (ELISA), the enzyme-linked fluorescent assay (ELFA), and the immunofluorescent cell count assay. A comparison of monoclonal antibody to polyclonal antibody in an immunofluorescent cell count assay showed the polyclonal antibody to be superior. Background levels for both antibodies were minimal, but the intensity of fluorescent staining was greater with the polyclonal antibody. The ELISA response of monoclonal and polyclonal antibody to detect limiting dilutions of reovirus indicates that the monoclonal antibody is comparable to polyclonal antibody; both could detect 60 ng/ml purified reovirus and 5x106 infectious fluorescent cell count units (ICU)/ml. When the fluorogenic substrate (ELFA, 4-methylumbilliferyl phosphate) was substituted for the chromogenic substrate (ELISA, p-nitrophenyl phosphate) the detection limits with monoclonal antibody and purified reovirus increased sixteen-fold (4 ng / ml). The ELFA detection limit of polyclonal antibody and purified virus increased only two-fold (30 ng/ml). Monoclonal and polyclonal antibodies were used in ELISA and ELFA immunoassays to detect aerosolized virus. Using the immunofluorescent cell count assay, 69% of the samples could be detected positive for infectious reovirus. The monoclonal antibody used in an ELISA was positive for 63% of the samples, while the polyclonal antibody in ELISA was only positive with 44% of the samples. The ELFA response was lower than ELISA, with the monoclonal and polyclonal antibodies positive for 50% and 23% of the samples, respectively. Monoclonal antibodies can be used for detection and identification of reovirus. Monoclonal antibodies have the advantage of a virtually unlimited supply of homologous reagent quality antibody. ELISA detection of virus has the advantage of being a rapid, specific test without the requirement of tissue culture technique and equipment. This method has the possibility of developing into a field assay capability for rapid identification of viruses in environmental conditions.

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