Identification of Poliovirus Isolates with Fluorescent Antibody

Abstract

Isolation and identification of enteroviruses from clinical specimens by routine methods generally require 2 or more weeks. Fluorescent antibody procedures, which have been applied to rapid diagnosis of influenza by Liu (1) and to demonstration of several viruses in tissue culture(2,3), also appear to offer the possibility of more rapid identification of enteroviruses isolated from clinical material. This report presents data concerning use of fluorescent antibody for identification of poliovirus isolates. A preliminary report of some of these data has already been made(4).

Materials and methods. Hyperimmune monkey poliovirus typing sera prepared by Wenner et al.(5) were used for preparation of fluorescent antibodies. Trypsinized rhesus monkey kidney cells were employed for isolation of viruses. Buffered saline consisted of 0.14 M NaCl, 0.01 M NaH₂PO₄ · H₂O, with the final pH adjusted to 7.0-7.2 by addition of 40% NaOH solution.

Globulins were precipitated from monkey antisera by 50% saturation with (NH₄)₂SO₄ and labeled with either fluorescein isocyanate (6) or fluorescein isothiocyanate (7). The latter compound was added in powdered form to globulin solutions at pH 9 with stirring in an ice bath (personal communication, Riggs. 1958). Before use, conjugates were absorbed twice with monkey liver powder(8) and once with a suspension of monkey kidney cells.

For fluorescent antibody study. 85 stools previously tested by routine methods were selected from storage and given to 2 of the authors (MHH and GWA) who had no knowledge of the earlier results. The specimens were then processed entirely as unknowns. Ten to 20% stool suspensions were prepared. penicillin and streptomycin added to final concentrations of 4000 units/ml and 2000 μg/ml, respectively, and 0.1 or 0.2 ml portions inoculated into tubes of monkey kidney cells. As soon as a 1 to 2+ cytopathic effect (CPE) was observed, maintenance fluid was poured off and cells rinsed with 1 ml of 0.02% versene in buffered saline.