Graduate Thesis Or Dissertation

 

Development and optimization of a kinetics-based enzyme-linked immunosorbent assay (KELA) for serologic diagnosis of bovine respiratory syncytial virus infection Public Deposited

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  • A kinetics-based enzyme-linked immunosorbent assay (KELA) for detection of serum IgG antibody against bovine respiratory syncytial virus (BRSV) was developed and optimized. Optimization factors tested included brand of microELISA plate, length of incubation periods at various temperatures, type of conjugate, purified versus precipitated antigen preparations, type of dilution buffer as well as various blocking regimes. Factors crucial to good, reproducible KELA results were careful titration of reagents, antigen adsorption time of 72 hours, use of Tween in serum and conjugate dilution buffers, along with washing procedures using a Tween-free wash solution. Fifty per cent endpoints were determined for 32 sera tested by KELA and were converted to a "serum-virus neutralization-equivalent 50% endpoint". These KELA endpoints were then compared to 50% endpoints of neutralization determined by the serum-virus neutralization assay. Coefficient of correlation (R²) was equal to 0.58. Sensitivity of KELA was 100%. A discussion of the factors affecting the correlation between the two is presented as well as the feasibility of KELA in the clinical laboratory.
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