A micropreparation of fluorescein conjugates of immunoglobulin G and Fab from serum.
Protein A-Sepharose CL-4B (PAS) was used to isolate rabbit immunoglobulin G from crude anti-herpes simplex virus-1 serum. Papain treatment of the PAS-bound immunoglobulin G released Fab fragments from the solid support, while Fc-containing fragments remained bound to PAS. PAS-immobilized immunoglobulin G was fluoresceinated by reaction with fluorescein isothiocyanate followed by papain cleavage to yield fluorescein-conjugated Fab fragments in solution. These fragments retained activity toward herpes simplex virus-1 infected Vero cells as evaluated by immunofluorescence. This novel procedure represents the fastest and simplest method for preparing Fab or fluoresceinated Fab fragments directly from any volume of immune serum.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008832:Microchemistry; D011817:Rabbits; D013205:Staphylococcal Protein A; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate