Immunochemical criteria for successful matching of monoclonal antibodies to immunoassays of peptide hormones for assessment of pregnancy and ovulation.
The development of simple, robust enzyme immunoassay (EIA) systems for measurement of levels of hCG and LH, suitable for use both in the home and in the clinic, imposes a number of constraints on the selection of antibody for the assay. Important criteria must be met with regard to specificity, sensitivity, and stability of the materials involved in the chemistries of solid-phase and enzyme coupling. Furthermore, pairing of antibodies that react with distinct epitopes, thereby allowing maximum sensitivity with minimum non-specific interaction with related analytes, places stringent demands on the selection of antibody-producing clones. These requirements are exemplified by the characteristics of a number of clones which provide antibodies with specificities for epitopes on alpha subunits, beta subunits, and intact hormone, and which were selected from several hundred potential clones. An optimized, two-site, sandwich assay was developed from a matrix study of solid-phase and enzyme-conjugate reagents. The assay was then converted to a simpler reaction format in which the chosen solid phase and the enzyme conjugate were organized to react simultaneously with analyte. A procedure for overcoming any "hook effect" due to high levels of hormone was also developed.
Porter
P
P
Department of Immunology, Unilever Research, Bedford UK.
Coley
J
J
Gani
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007986:Luteinizing Hormone / Q000652:urine*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010060:Ovulation; D011247:Pregnancy; D011259:Pregnancy Tests, Immunologic / Q000379:methods*; D011863:Radioimmunoassay / Q000379:methods
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D000939:Epitopes; D007986:Luteinizing Hormone