Enzymeimmunoassay for Progesterone and Oestradiol: A Study of Factors Influencing Sensitivity

摘要

Enzymeimmunoassays for progesterone and oestradiol have been developed at the Tenovus Institute using horseradish peroxidase conjugates and a solid phase system for separation of free and bound steroid. The sensitivity of these assays was inadequate and precluded their use for the assay of low titre samples. Attempts to increase the sensitivity of the assay by reducing the affinity of the conjugate for the antiserum by altering the bridge groups were unsuccessful. Decrease in the molar incorporation of the steroid into the enzyme conjugate effected a significant improvement, increasing the sensitivity in the progesterone assay by 50% and in that of oestradiol by 70%. Initial attempts to replace the solid phase separation technique by a second antibody procedure based on a sheep, antirabbit IgG serum were of limited success. Increased sensitivity in the progesterone radioimmunoassay (RIA) was achieved but severe problems with high non-specific binding remained. The apparent specific binding in the oestradiol assay did not exceed 30% and a viable system could not therefore be established. An improvement second antibody produced in guinea pigs to a wider spectrum of immunoglobulins, when used in the EIA of progesterone increased sensitivity 50 fold c.f. the solid phase assay. The sensitivity of the oestradiol assay was increased 8 fold c.f. the solid phase system, and is now comparable with that of the RIA (2.0 pg/0.7 pg). The significant improvement in sensitivity in enzymeimmunoassay associated with the use of double antibody separation procedures, and the importance of tailoring the affinity of the second affinity to the immunoglobulin profile of the first antibody are emphasised. (Atomindex citation 09:389293)