A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG.
An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102).
D000818:Animals; D000936:Antigen-Antibody Complex; D003429:Cross Reactions; D006168:Guinea Pigs; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000032:analysis*; D007330:Insulin Antibodies / Q000032:analysis*; D008832:Microchemistry; D011817:Rabbits / Q000276:immunology; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007330:Insulin Antibodies; D012710:Serum Albumin, Bovine