Shared reaction in solid-phase immunoassay for estriol determination.
In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.
Università di Pisa, Dipartimento di Anatomia, Biochimica e Fisiologia Veterinaria, Italy.
A
A
Podestà
C J
CJ
Smith
C
C
Villani
G
G
Montagnoli
D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004964:Estriol / Q000032:analysis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology
D012710:Serum Albumin, Bovine; D004964:Estriol