TECHNICAL CONSIDERATIONS IN THE PREPARATION OF FLUORESCENT-ANTIBODY CONJUGATES.
A comparison was made of (NH(4))(2)SO(4), HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.
D000818:Animals; D001797:Blood Protein Electrophoresis; D003353:Corynebacterium diphtheriae; D004926:Escherichia coli; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera; D007202:Indicators and Reagents; D007709:Klebsiella; D007793:Lagomorpha; D011506:Proteins; D011817:Rabbits; D012106:Research; D013053:Spectrophotometry; D013861:Thiocyanates
D005452:Fluoresceins; D007106:Immune Sera; D007202:Indicators and Reagents; D011506:Proteins; D013861:Thiocyanates
BLOOD PROTEIN ELECTROPHORESIS; CORYNEBACTERIUM DIPHTHERIAE; ESCHERICHIA COLI; EXPERIMENTAL LAB STUDY; FLUORESCEINS; FLUORESCENT ANTIBODY TECHNIC; IMMUNE SERUMS; KLEBSIELLA; RABBITS; SPECTROPHOTOMETRY; THIOCYANATES