Development of an EIA for pLH A first, required step to study the secretory response of porcine gonadotrophs in culture was the development and validation of a quantitative EIA for the reliable measurement of pLH. For this purpose, a careful optimization process was carried out by changing the concentration of pairs of the parameters employed (pLH for coating, primary and secondary antisera, ABC, OPD, and H202) while maintaining the rest of them constant (data not shown). Likewise, optimal length and temperature for coating and incubations were determined previously. buy antibiotics online
As illustrated in Figure la, the optimal values obtained (detailed in Materials and Methods) provided standard curves of appropriate shape. Next, we assessed the validity of the developed assay by testing four parameters: reproducibility, sensitivity, specificity, and parallelism. Reproducibility of the assay was indicated by the small size of the standard errors of the average curve shown in Figure la. This was corroborated by calculating the intraassay (8.5%, n = 10) and interassay (10.5%, n = 6) coefficients of variation at 50% binding. Sensitivity of the EIA was demonstrated by the value of the minimum detectable dose of pLH (0.98 ±0.13 ng/ml; mean ± SEM, n = 6), calculated according to Signorella and Hymer. Effective doses were ED2o = 40.88 ± 4.72, ED50 = 14.35 ± 1.30, and ED80 = 5.11 ± 0.76 ng/ml (n = 6). That the assay was specific for pLH was verified by the low (1.8%) cross-reactivity of the primary antiserum (anti-pLH) with the structurally and genetically related antigen pFSH. Finally, we tested the parallelism between the pLH used as standard and that contained in samples from cultured gonadotrophs. Figure lb illustrates the existence of such a parallelism between the lines obtained after transforming the corresponding displacement curves. Thus, the EIA developed in the present study enables the measurement of the amount of pLH secreted and/or contained by cultured gonadotrophs in a simple, specific, reproducible, and sensitive manner. FIG. 1. Development of an EIA for pLH. a) Average standard curve for pLH EIA obtained from serial dilutions of the hormone standard (250-1.9 ng/ml). Each point represents the average of 6 experiments ± SEM. b) Parallelism of dilutions of culture medium and cellular homogenates compared to those of the pLH standard.