Standardization of the Fluorescent Antibody Technique for the Detection of Avian Respiratory Viruses

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Abstract
Each phase of the fluorescent antibody (FA) technique was investigated. It was found that gamma globulin solutions prepared from specific chicken antiserums against Newcastle Disease virus, infectious bronchitis virus, and infectious laryngotracheitis having neutralization indices of 3.5 or greater were satisfactory for conjugation. The optimum conjugation ratio of fluorescein isothiocyanate to protein was found to be 0.03 mg dye per mg protein at a conjugation time of 12 hr. Purification of conjugates was achieved on columns of Sephadex G-25. Reactions of a high fluorescent intensity were observed in infected tracheal smear preparations which were fixed in acetone at[long dash]20 CC for 10 min. and reacted for 30 min. with conjugates adjusted to contain twice the protein concentration of the FA titration endpoint at a pH of 7.25. A washing time of 20 min. or more in phosphate-buffered saline (pH 7.25) did not affect the intensity of specific reactions. Infected tracheas could be stored at 4[degree]C under moist conditions for 72 hr. and retain their reactivity.