Conjugation of Coxsackievirus Types B1-B6 Immunoglobulins with Fluorescein Isothiocyanate by a "Reversed" Dialysis Method

Abstract

Immunoglobulins from antisera to coxsackievirus B types 1–6 were labeled with fluorescein isothiocyanate by a “reverse” dialysis method of conjugation. Conjugates thus obtained were labeled at reproducible ratios of fluorescein to protein with weight-weight ratios ranging from 5.0 to 7.1 and estimated molar ratios ranging from 2.1 to 2.9. Conjugates were tested on cover-slip HeLa cell cultures by the direct method of staining. Two types of specific immunofluorescence were observed: intensely fluorescent perinuclear masses and discrete foci of bright fluorescence scattered throughout the cytoplasm. Titers of homotypic conjugates varied from 1:40 to 1:160. Heterotypic staining, consisting of a diffused hazy fluorescence, was observed only at the highest concentrations of conjugate and not above the 1:10 dilution. This technique proved to be specific and sensitive for the detection of group B coxsackievirus antigens in HeLa cells but failed in specific detection of the same antigens in samples of the myocardium obtained by biopsy of three patients clinically ill with idiopathic myocarditis.