Measurement of reproductive hormones in urine is a practical way of obtaining large amounts of information; however, there is still controversy on how to overcome problems derived from volume fluctuations between samples. Creatinine adjustment is a widely accepted solution, however, it introduces an extra cost, and large studies involving multiple sequential determinations would benefit from more economical solutions. We determined the value of creatinine adjustment, and compared it with a mathematical method that uses the smoothed profile of pregnanediol (PdG) as a reference to adjust other hormonal markers. To do this, we investigated the effects on three major urinary reproductive hormonal markers (luteinizing hormone (LH), estrone 3-glucuronide (E1G) and PdG) in 17 complete menstrual cycles. Detection of the day of LH peak did not differ between raw and adjusted data. Creatinine adjustment reduced variation in pre-ovulatory E1G levels between individuals, though the effect was negligible within individuals. No significant differences were found regarding post-ovulatory PdG rise. Although creatinine adjustment significantly reduces variability, producing smoother profiles, an equivalent degree of smoothness is obtained using the PdG adjustment. We conclude that under the current technology, for the retrospective study of urinary hormonal profiles in the human menstrual cycle, PdG adjustment is a valid alternative to creatinine.

This paper describes the application of two novel screening technologies, i.e. Domain Scan (24- and 30-mer peptides) and Matrix Scan (24-mer peptides) technology, in the mapping of a discontinuous epitope on FSH-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-loop of FSH in the Domain and/or Matrix Scan. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70, D71, and L73 form the core of the epitope. A Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.

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Spleen cells from Balb/c mice immunized with HCG were subjected to the normal hybridoma procedures. The resulting MCA's (from 78 clones) were evaluated in radioimmunoassay, haemagglutination and enzyme immunoassay with whole HCG. The RIA analysis was extended to include HCG subunits and intact LH. The alpha-chain of HCG was found to be strongly immunodominant, as shown by the very high frequency (46 of 78) of MCA's directed at the alpha-chain epitopes. These antibodies would bind luteinizing hormone as well as HCG. Only 1 of the 78 clones was specific for the B subunit of HCG, in RIA. This clone was later found to be positive for LH in EIA, thus making the derivation of antibodies specific for this B-epitope extremely difficult. Most MCA's were found to be applicable in the three types of assay systems (48 of 78), but some were compatible in just one or two of the systems. These differences are believed to be due to epitope masking resulting from the way in which the antigen was handled and presented in the different systems.