Date of Award:

5-1-1984

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Life Sciences:Biology

Committee Chair(s)

Bill B. Barnett

Committee

Bill B. Barnett

Committee

Rex S. Spendlove

Abstract

An enzyme labeled immunosorbent assay (ELISA) which uses the enzyme acetylcholinesterase (AchE) as the immunoreactant label has been developed. The AchE was detected by a reaction between the hydrolysed substrate acetylthiocholine (AchS) and 5:5-dithiobis-2-nitrobenzoate ion. This reaction was spectrophotometrically followed at a wavelength of 405 nm. The exposed thiol group will also react with a thiol-specific fluorochrome, monobromobimane, to produce fluorescence. AchE was conjugated to immunoglobulin using glutaraldehyde without loss of enzyme or antibody activity. The AchE-ELISA was shown to be equal in activity and sensitivity to two widely used commercial ELISA's at an 8-fold increase in the dilution of the AchE conjugate. Also, the AchE-ELISA used 33% less antibody for conjugation than some ELISA procedures and had equal sensitivity. The fluorogenic AchE-ELISA was 4-fold more sensitive than the chromagenic AchE-ELISA. The AchE-ELISA was used to study murine rotaviral (MRV) antigen development in the intestines of infant mice at varying times following gavage viral inoculations and was also used to study the subsequent immune response of the infected infant mice. MRV antigen was detected 24 h post-inoculation and persisted until day 7 with peak antigen concentration occurring on day 4. Specific anti-MRV globulins were detectable in the infected infant mice 13 days post-inoculation.

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