Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies.
We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free alpha subunit of hCG (hCG alpha) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits (E less than 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.
Madersbacher
S
S
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck.
Wolf
H
H
Gerth
R
R
Berger
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005640:Follicle Stimulating Hormone / Q000276:immunology; D015292:Glycoprotein Hormones, alpha Subunit / Q000276:immunology; D006735:Horseradish Peroxidase; D006882:Hydroxyapatites; D007074:Immunoglobulin G; D051379:Mice; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D015292:Glycoprotein Hormones, alpha Subunit; D006882:Hydroxyapatites; D007074:Immunoglobulin G; D005640:Follicle Stimulating Hormone; D017886:Durapatite; D006735:Horseradish Peroxidase