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Reactivity of different LH and FSH standards and preparations in the World Health Organization matched reagents for enzyme-linked immunoassays of gonadotrophins

Reactivity of different LH and FSH standards and preparations in the World Health Organization... Abstract The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system. enzyme-linked immunoassay, FSH, LH Introduction Twenty five years ago, the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization (WHO), established a validation, standardization and distribution programme to provide a number of laboratories with reagents, the so-called matched reagents, for the assay of reproductive hormones in serum, including the pituitary gonadotrophins LH and FSH. The name and concept of the matched reagents originated not only from the necessity for all reagents forming part of an assay system to be matched to each other, but also, from the intention to provide the various WHO collaborative centres with identical, matched reagents for single- or multi-centre clinical trials, the analytical identity of reagents used in various WHO collaborating laboratories being a prerequisite for obtaining reliable and comparable results. As a result of the progress in analytical techniques, the initial assay method (radioimmunoassay) to quantify pituitary gonadotrophins in serum was replaced approximately 5 years ago by enzyme-linked immunoassays (EIA). The change in the assay methodology has brought about some numerical differences in assay results. For example, in the 1994 WHO external quality assessment programme report, the ELISA results were, on average, numerically 30% lower for LH, while those obtained for FSH were 50% higher (WHO Technical Report, 1994). Among the reasons for these numerical discrepancies are the use of different antibodies (e.g. polyclonal antibodies in the radioimmunoassays versus monoclonal antibodies in the EIA) possessing distinct epitope specificities as well as the use of different pituitary preparations for constructing the standard curves, which may be differentially recognized by a determined set of antibodies, particularly by those of monoclonal origin (Petterson et al., 1991; Petterson and Soderholm, 1991; Vermes et al., 1991; Jeffcoate, 1993; Costagliola et al., 1994a,b,c; Martin-Du-Pan et al., 1994; Taylor et al., 1994). In fact, significant variations in molecular composition (mainly determined by the sample source, type of particular oligosaccharide chains attached to the protein core of the molecule and purification methods used to isolate the glycoproteins) have been detected among some of the highly purified pituitary gonadotrophin preparations employed to calibrate the kit standards, which may be distinctly recognized by antibodies and cognate receptors (Chappel et al., 1986; Simoni et al., 1993; Chappel, 1995; Burgon et al., 1997; Lambert et al., 1998). Further, it has been shown that the gonadotrophin glycoforms contained in crude pituitary extracts and recombinant preparations may be either equally or differentially identified depending on the particular antibody configuration of the immunoassay system employed for their quantitative estimation (Zambrano et al., 1996; Oliver et al., 1999). In the present study, we analysed the dose-response curve profiles of various widely employed pituitary and recombinant gonadotrophin preparations in the EIA system provided by the WHO Programme for the Provision of Matched Assay Reagents for the Immunoassay of Hormones (henceforth: the programme), and established their relative potency in this particular immunoassay. In addition, the behaviour of each preparation in the EIA system was correlated with its particular molecular composition as revealed by preparative chromatofocusing. Materials and methods Standards The human LH preparations tested in this study were the partially purified LER-907 standard for radioimmunoassay of gonadotrophins [obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through the National Hormone and Pituitary Program (NHPP), Torrance, CA, USA], highly purified recombinant human LH (coded herein as rhLH for the purpose of the study) produced by Chinese hamster ovary (CHO) cells (kindly provided by Organon International BV, Oss, The Netherlands), the pituitary LH standard (coded herein as WHO INEN) prepared by the WHO Collaborating Center in Cuba (provided by the Cuba–Mexico WHO RIA Reagents Programme, Instituto Nacional de Endocrinología, La Havana, Cuba and Instituto Nacional de la Nutrición SZ, Mexico City, Mexico) and calibrated against the WHO international reference preparation (IRP) of LH 80/552 (Storring and Gaines Das, 1993), the LH standard (coded herein as WHO 80/552C) contained in the LH EIA kit provided by the programme which is calibrated against the WHO IRP 80/552 and the LH standard preparation (coded herein as WHO 68/40C) intended to be used in the former programme's radioimmunoassay system, which was calibrated against the WHO IRP of LH 68/40 (Storring et al., 1978). For FSH, the following were tested: the LER-907 standard, highly purified recombinant human FSH (rhFSH) produced by CHO cells (ORG 32489; Organon International) (Olijve et al., 1996), a FSH standard preparation (WHO INEN) provided by the WHO Collaborating Center in Cuba and calibrated against the WHO IRP of FSH 78/549 (Storring and Gaines Das, 1989), and the FSH standard (coded herein as WHO 78/549C) contained in the FSH EIA kit provided by the programme, which has been calibrated against the WHO IRP 78/549. Immunoassays of LH and FSH Enzyme-linked immunoassays The EIA of LH and FSH were performed employing reagents provided by the WHO Collaborating Centre for Research and Reference Services in the Immunoassay of Hormones in Human Reproduction, London UK, following the instructions provided by the centre. The assays were of an immunometric (`sandwich') design and employed two anti-LH or anti-FSH monoclonal antibodies. In each EIA, the first antibody was directed against the β-subunit of the molecule and was attached to a magnetic particle, whereas the second antibody was directed against the α-subunit, and was labelled with alkaline phosphatase. These LH and FSH EIA systems exhibit ≤0.1% and 0.03% cross-reactivity with highly purified FSH and LH respectively, and undetectable reactivity with growth hormone (GH) and prolactin. The intra- and interassay variables were ≤4.0% and ≤8.0% respectively. Results are expressed as ng or mIU as appropriate according to the particular standard preparation analysed. Radioimmunoassays The radioimmunoassay of LH was performed employing 125I-labelled LH-I3 as the tracer (specific activity 70–90 μCi/μg protein), the reference preparation LER-907 as the standard and the antihuman LH-3, at a final dilution of 1:800 000, as the antiserum (Ropelato et al., 1999). Cross-reactivity of this antiserum with highly purified FSH, GH and prolactin is <0.2%. The sensitivity of the assay was 0.7 IU/l. The FSH radioimmunoassay was performed employing 125I-labelled FSH I-1 as the tracer (specific activity 60–70 μCi/μg protein), the LER-907 preparation as the standard and anti-human FSH-6 at final dilution of 1:250 000, as the antiserum (Timossi et al., 1998). This antiserum exhibits less than 0.1% cross-reactivity with highly purified human LH and prolactin and undetectable reactivity with free α-subunit and GH. In both radioimmunoassay systems, all LH and FSH isoforms displaced either 125I-labelled FSH or LH from the antibody in a parallel fashion when tested at seven to 10 different dilutions; in fact, simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by FSH and LH present in the LER-907 standard and concentrated pools of the several isoforms fractionated by chromatofocusing (Zambrano et al., 1996). All LH and FSH radioimmunoassay reagents were generously provided by the NIDDK through Dr A.F.Parlow from the NHPP. For both radioimmunoassays, the inter- and intra-assay coefficients of variation were <13 and <8% respectively. The results are expressed as ng of the LER-907 standard. Preparative chromatofocusing of LH and FSH Chromatofocusing of each gonadotrophin preparation was performed as described previously (Ulloa-Aguirre et al., 1992; Castro-Fernández et al., 2000) with some modifications. This technique allowed the separation of different gonadotrophin isoforms on the basis of charge, which in all glycoprotein hormones is mainly determined by the amount of terminal sialic acid and/or sulphate residues present in the oligosaccharide structures of the molecule (Ulloa-Aguirre et al., 1995b). Briefly, 2.0–2.7 IU of each WHO standard, ~111 μg of LER-907, ~5.0 μg of rhLH, and ~50 μg of rhFSH were redissolved in phosphate (0.01 mol/l) buffered physiological (0.15 mol/l) saline, transferred to dialysis membrane tubing (molecular weight cut-off, 12 000–14 000; Spectrum Medical Industries, Los Angeles, CA, USA), dialysed at 4°C for 24 h against deionized water and thereafter against 0.01 mol/l ammonium carbonate (pH 7.5) and freeze-dried. Lyophilates were redissolved to one tenth of original volume in Pharmalyte pH 8–10.5-HCl (Pharmacia Biotech, Piscataway, NJ, USA) (1:45 dilution in deonized water, pH 7.0) and the suspension was then applied to the top of a 30×1 cm column of polybuffer exchange resin (PBE-118, Pharmacia Biotech), previously equilibrated for 18–24 h with 25 mmol/l triethylamine-HCl, pH 11.0, and chromatofocused at 4°C. Eluate fractions (2 ml each) were collected at a flow rate of 1 ml/4 min. The pH of each fraction was then measured, and when a limiting pH of 7.0 had been reached the eluent buffer (Pharmalyte-HCl) was changed by Polybuffer-74 (Pharmacia Biotech) diluted 1:8 in deionized water, pH 4.0, to elute proteins bound at pH 7.0–4.0. Proteins bound at the lower limiting pH (pH <3.50; salt peak) were finally recovered by the addition of 1.0 mol/l NaCl to the chromatofocusing column. The pH of each fraction was neutralized to pH 7.0 by the addition of either 200 μl 1.0 mol/l triethylamine-HCl pH 7.0 (to those fractions with an elution pH value of 11.0 to 7.0) or 1.0 mol/l imidazole-HCl pH 7.4 (to fractions recovered within elution pH values of 6.99 to <4.0). Each fraction was stored frozen at –20°C until the day of the LH or FSH radioimmunoassays. All fractions from a single column were assayed in duplicate incubations in the same radioimmunoassay run. Recoveries of immunoactive LH and FSH after chromatofocusing were 75 ± 5% of the total amount applied to the columns. Each gonadotrophin preparation was chromatofocused in three separate runs. Statistical analysis Tests for parallelism among the slopes generated by the different gonadotrophin preparations in the EIA and the LH radioimmunoassay were performed following the method of DeLean et al. (1978). This method allows for simultaneous fitting and analysis of families of sigmoidal dose-response curves and describes the curves in terms of basal and half maximal responses (ED50) and curve shape or steepness. Relative potencies among the various preparations were calculated considering multiple points comprising the ED20–ED80 interval. Results EIA of LH preparations The dose-response curves of the different WHO standards, LER-907 and rhLH, in the LH EIA are shown in Figure 1. The profile of the dose-response curves elicited by the three WHO LH standards tested in the EIA system were superimposable and almost identical; simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards (mean slope values: 1.27 ± 0.07, 1.26 ± 0.07 and 1.13 ± 0.10 for the 68/40C, WHO INEN and 80/552C standards respectively; n = 6 EIA runs for each standard) and LER-907 (mean slope value 1.25 ± 0.07; n = 6). On the contrary, no parallelism was found between the curves generated by rhLH (mean slope value, 0.72 ± 0.08) and the WHO and LER-907 LH standards. This finding contrasted with the dose-response curve elicited by rhLH in the polyclonal antibodies-based radioimmunoassay, which was parallel with that of the LER-907 standard (Figure 2). The relative potency of each preparation tested in this LH EIA system is shown in Table I. The potency of LH contained in the WHO INEN standard was slightly higher than that of the WHO 80/552C standard and lower than that yielded by WHO 68/40C; the immunopotency of these two latter standards was nearly the same. According to this EIA system, the potency of the WHO standards was similar when expressed in terms of the LER-907 preparation and vice versa. EIA of the FSH preparations Representative dose-response curves elicited by the two WHO FSH standards, LER-907 and rhFSH, in the FSH EIA system are shown in Figure 3. Simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by the various preparations tested (mean slope values: 1.05 ± 0.05, 1.07 ± 0.06, 1.04 ± 0.04 and 1.12 ± 0.03, for the 78/549C, WHO INEN, rhFSH and LER-907 standards respectively; n = 6 EIA runs for each preparation). On a weight-to-weight basis, rhFSH was approximately four times more potent that LER-907; the immunopotency of the FSH 78/549C kit standard was slightly higher than that of the WHO INEN standard when compared on a unit-to-unit basis (Table II). Chromatofocusing of the LH and FSH standards and preparations Figures 4 and 5 show the pH distribution pattern of immunoreactive LH and FSH after chromatofocusing of the various preparations analysed. As shown, each preparation contained two or more charge isoforms recovered within a pH window of 9.0 to 3.50 and 6.50 to 3.50 for the LH and FSH containing preparations respectively. In addition, all standards and preparations showed variable amounts of LH and FSH immunoactivity in fractions corresponding to molecules bound at the lower limiting pH, which were recovered after the addition of a highly concentrated NaCl solution to the chromatofocusing column (salt peak). As depicted in Figure 4, the WHO 68/40C and 80/552C LH kit standards exhibited very similar charge distribution profiles; both standards contained negligible amounts (<3% of the total LH recovered) of LH molecules within the salt peak. In contrast, the WHO INEN, rhLH and LER-907 preparations presented a higher (6–16%) content of this strongly acidic LH species. Although the bulk (71–75% of total) of LH immunoreactivity in the WHO INEN standard and rhLH preparation was recovered at pH values <7.0, significant amounts of immunoactivity (15–22% of total) were additionally detected at higher pH values; in fact, these two preparations presented the highest degree of charge heterogeneity (in terms of total number of LH components resolved by the charge-based separation technique employed). In general, LH contained in LER-907, rhLH and WHO INEN was more acidic than that present in the remaining WHO LH standards. In the chromatofocusing system employed (pH window from 10.0 to <3.5), the bulk (<80% of total) of immunoreactive FSH contained in the preparations analysed was recovered at pH values 5.20 to 3.50 and in the salt peak. Negligible amounts (<5%) of immunoreactive FSH material eluting at pH values 8.73 to 8.27 was consistently detected in the LER-907 standard (Figure 5). This latter preparation contained three major FSH charge isoforms that focused at pH values similar or close to those of the isoforms present in the WHO 78/549C standard. The pH elution profiles of rhFSH and the WHO INEN FSH standard were similar; both preparations exhibited a single broad FSH peak with a median pH value of 4.53–4.60 and a second large component recovered within the salt peak. Discussion Analysis of the dose-response curves of several LH standards and preparations in the EIA system, developed for the WHO Special Programme of Research in Human Reproduction, revealed a close similarity among the curves elicited by the WHO LH 68/40C, 80/552C and INEN standards. This agreement in dose-response curve profiles among the various WHO LH standards tested was in marked contrast with that exhibited by the rhLH preparation produced in CHO cells, whose absence of parallelism with the WHO and LER-907 curves was clearly noticeable. The non-parallelism exhibited by this particular preparation may be due either to vulnerability to matrix effects at low doses or to differences in molecular specificity of the matched monoclonal antibodies employed in this EIA system for the mixture of rhLH glycoforms contained in this particular preparation and that of the naturally occurring variants present in the preparations of pituitary origin. In fact, several differences in glycosylation exist between rhLH produced by CHO cells and pituitary LH: oligosaccharides in CHO cell-derived gonadotrophins are sialylated only in an α2–3 conformation, do not contain bisecting N-acetyl glucosamine moieties and sulphated terminal glycosylation is missing (Hård et al., 1990; Rafferty et al., 1995; Amoresano et al., 1996; Talbot et al., 1996). In addition, it is known that CHO cells have a limited ability to introduce complex branched carbohydrate structures onto the protein core in comparison with the naturally producing cells, the gonadotrophs (Hård et al., 1990; Harris et al., 1998). Several lines of evidence indicate that differences in glycosylation may affect the reactivity of glycoprotein towards certain antibodies (Papandreou et al., 1990, 1991; Labbe-Jullie et al., 1992; Zerfaoui and Ronin, 1996). For example, removal of sialic acid from pituitary thyrotropin specifically abolished the binding of this natural glycoprotein to anti-β monoclonal antibodies (Zerfaoui and Ronin, 1996) and its deglycosylation led to a five-fold reduction in immunoreactivity toward certain anti-β polyclonal antibodies (Papandreou et al., 1990). In the present study, the behaviour of rhLH in the WHO EIA contrasted with that exhibited in the radioimmunoassay, in which rhLH displaced 125I-labelled LH from the antibody in a fashion similar to that shown by the pituitary-derived LER-907 standard, thus emphasizing the importance of the particular antibody configuration employed for determining the immunoreactivity of recombinant DNA-derived preparations. The overall data indicate that recombinant human LH produced by CHO cells is not a suitable preparation for use as the calibration standard in this particular monoclonal antibody-based EIA system and that all other WHO LH standard preparations tested may be indistinctly employed without significant variations in the final concentration results of the unknown samples. In contrast to the set of LH preparations tested in the EIA system, the dose-response curves elicited by both the recombinant and pituitary FSH preparations were parallel to each other. Thus, differences in glycosylation between the CHO cell-derived preparation and the pituitary standards did not apparently influence to a considerable extent the reactivity of the former preparation toward the set of monoclonal antibodies employed in this FSH EIA system. Although the present study does not unambiguously resolve whether the monoclonal antibodies employed reacted equally with all FSH preparations tested, the profile of the dose-response curves elicited by the four preparations indicates that all preparations may be used as calibration standards at least in this particular human FSH EIA. All LH preparations exhibited a high degree of charge heterogeneity. In particular, the pH distribution profile exhibited by rhLH, LER-907 and the WHO INEN LH preparations was more acidic than that of the WHO 68/40C and 80/552C standards; these differences may be due to selective removal of the more or less basic isoforms during the purification procedures as well as to cell-specific (gonadotrophs and CHO cells) particularities in post-translational processing of the gonadotrophin molecule (Ulloa-Aguirre et al., 1995a). Interestingly, despite the marked differences in charge distribution between the WHO 68/40C and 80/552C preparations and the WHO INEN standard, their behaviour in the EIA system was remarkably similar as disclosed by their superimposed dose-response curves. This observation suggests that variability in distribution and/or conformation of terminally charged sugars in the isoforms contained in these pituitary preparations does not greatly influence their reactivity towards the particular antibodies employed by the EIA system. On the contrary, it appears that the high abundance of sialic acid attached exclusively in α2,3 position and the complete absence of sulphate residues in the acidic rhLH molecules (which conform the bulk of immunoreactive material present in this preparation) influenced the reactivity of the monoclonal antibodies for this compound. As previously reported, all FSH preparations tested exhibited a predominantly acidic pH distribution profile with the bulk of FSH immunoactivity recovered at elution pH values ≤5.0. The charge distribution profile of all these FSH preparations varied considerably from that previously reported for FSH in crude pituitary extracts and serum, in which 10–25% of total FSH may be recovered at elution pH values >4.50 (Simoni et al., 1994; Ulloa-Aguirre et al., 1995a,b; Zambrano et al., 1995; Anobile et al., 1998). In a previous study employing this particular EIA system, it was observed that the intrapituitary FSH isoforms with pH values 6.60 to <3.80 elicited parallel dose-response curves whereas the less acidic/sialylated form (pH >7.10) could not be accurately quantified due to significant non-concordance between this isoform and the corresponding standard curve (LER-907) (Zambrano et al., 1996). How this reduced reactivity of the monoclonal antibodies of this EIA toward the less acidic FSH isoforms may affect the accuracy of the concentrations of FSH in serum is not known. It must be emphasized, however, that the abundance of this particular isoform in serum is extremely low and thus its presence in the mixture of variants released from the pituitary may be irrelevant at least in physiological conditions. In summary, with the exception of rhLH produced in CHO cells, all LH and FSH preparations tested in the EIA systems provided by the WHO Programme for the Provision of Matched Assay Reagents for the Immunoassay of Hormones (currently distributed through the non-profit organization Immunometrics Ltd, London, UK) elicited dose-response curves which were parallel to each other across a wide range of concentration values. Although variations in purity and concentration units of the various gonadotrophin preparations do not allow accurate establishment of the exact degree of cross-reactivity of the monoclonal antibodies employed in these EIA kits toward the standards tested, all the pituitary-derived preparations as well as rhFSH produced by CHO cells seem appropriate for using as calibration standards in this particular immunometric system. The impact of the differences in mixture of gonadotrophin isoforms between these particular preparations and the human serum on the accuracy of measurements of LH and FSH in serum samples by this and other immunometric assay systems is an issue that deserves to be scrutinized carefully. Table I. Relative potencies (means and 95% confidence limits) of the different LH standards tested in the World Health Organization (WHO) LH enzyme-linked immunoassay (EIA) system LH standard  68/40C (IU)  80/552C (IU)  INEN (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 68/40C  1  0.99 (0.92, 1.05)  1.16 (1.10, 1.20)  22.2 (21.6, 22.6)  1 IU 80/552C  1.04 (1.10, 0.97)a  1  0.84  25.5 (24.2, 26.7)  1 IU INEN  0.882 (0.840, 0.910)  1.21 (1.15, 1.27)  1  23.5 (22.3, 24.7)  1 μg LER-907  0.045 (0.044, 0.046)  0.040 (0.038, 0.042)  0.044 (0.041, 0.046)  1  LH standard  68/40C (IU)  80/552C (IU)  INEN (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 68/40C  1  0.99 (0.92, 1.05)  1.16 (1.10, 1.20)  22.2 (21.6, 22.6)  1 IU 80/552C  1.04 (1.10, 0.97)a  1  0.84  25.5 (24.2, 26.7)  1 IU INEN  0.882 (0.840, 0.910)  1.21 (1.15, 1.27)  1  23.5 (22.3, 24.7)  1 μg LER-907  0.045 (0.044, 0.046)  0.040 (0.038, 0.042)  0.044 (0.041, 0.046)  1  View Large Table II. Relative potencies (means and 95% confidence limits) of the different FSH standards and preparations tested in the WHO FSH EIA system FSH standard or preparation  78/549C (IU)  INEN (IU)  rhFSH (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 78/549C  1  1.29 (1.21, 1.36)  9.86 (9.52, 10.20)  48.81 (47.20, 50.40)  1 IU INEN  0.80 (0.75, 0.84)a  1  8.28 (7.85, 8.70)  35.86 (33.80, 37.90)  1 μg rhFSH  0.102 (0.099, 0.106)  0.124 (0.117, 0.130)  1  4.33 (4.16, 4.49)  1 μg LER-907  0.021 (0.020, 0.022)  0.029 (0.027, 0.030)  0.234 (0.225, 0.243)  1  FSH standard or preparation  78/549C (IU)  INEN (IU)  rhFSH (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 78/549C  1  1.29 (1.21, 1.36)  9.86 (9.52, 10.20)  48.81 (47.20, 50.40)  1 IU INEN  0.80 (0.75, 0.84)a  1  8.28 (7.85, 8.70)  35.86 (33.80, 37.90)  1 μg rhFSH  0.102 (0.099, 0.106)  0.124 (0.117, 0.130)  1  4.33 (4.16, 4.49)  1 μg LER-907  0.021 (0.020, 0.022)  0.029 (0.027, 0.030)  0.234 (0.225, 0.243)  1  View Large Figure 1. View largeDownload slide Representative dose-response curves of the different World Health Organization (WHO) LH standards, LER-907 and rhLH produced in Chinese hamster ovary (CHO) cells in the WHO LH enzyme-linked immunoassay (EIA) system. OD = optical density. Figure 1. View largeDownload slide Representative dose-response curves of the different World Health Organization (WHO) LH standards, LER-907 and rhLH produced in Chinese hamster ovary (CHO) cells in the WHO LH enzyme-linked immunoassay (EIA) system. OD = optical density. Figure 2. View largeDownload slide Representative dose-response curves elicited by rhLH produced in CHO cells and LER-907 in the LH radioimmunoassay. B/B0 = Bound/Bound at dose zero. Figure 2. View largeDownload slide Representative dose-response curves elicited by rhLH produced in CHO cells and LER-907 in the LH radioimmunoassay. B/B0 = Bound/Bound at dose zero. Figure 3. View largeDownload slide Representative dose-response curves of the different WHO FSH standards, LER-907 and rhFSH produced in CHO cells in the WHO EIA system. Figure 3. View largeDownload slide Representative dose-response curves of the different WHO FSH standards, LER-907 and rhFSH produced in CHO cells in the WHO EIA system. Figure 4. View largeDownload slide Representative profiles of pH distribution of immunoreactive LH after chromatofocusing of different LH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 4. View largeDownload slide Representative profiles of pH distribution of immunoreactive LH after chromatofocusing of different LH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 5. View largeDownload slide Representative profiles of pH distribution of immunoreactive FSH after chromatofocusing of different FSH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 5. View largeDownload slide Representative profiles of pH distribution of immunoreactive FSH after chromatofocusing of different FSH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. 4 To whom correspondence should be addressed at: Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, Apdo. Postal 99–065, Unidad Independencia, Mexico 10101, DF, Mexico. E-mail: [email protected] The authors are grateful to Dr A.F.Parlow and the NIDDK National Hormone and Pituitary Program for the human FSH and LH radioimmunoassay reagents and to the WHO Centre for Research and Reference Services in the Immunoassay of Hormones in Human Reproduction, London, UK, for providing the LH and FSH EIA reagents. This study received financial support from the Laboratory Methods Group, Human Reproduction Programme, WHO, Geneva, Switzerland and from Consejo Nacional de Ciencia y Technologia (CONACyT), grant Z8589N), Mexico, D.F., Mexico. References Amoresano, A., Siciliano, R., Orru, S. et al. ( 1996) Structural characterisation of human recombinant glycohormones follitropin, lutropin and choriogonadotropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. , 242, 608–618. Google Scholar Anobile, C.J., Talbot, J.A., McCann, S.J. et al. ( 1998) Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state. Mol. Hum. Reprod. , 4, 631–639. Google Scholar Burgon, P.G., Stanton, P.G., Petterson, K. and Robertson, D.M. ( 1997) Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vivo, radioreceptor activity and immunoactivity. Reprod. Fertil. Dev. , 9, 501–508. Google Scholar Castro-Fernández, C., Olivares, A., Söderlund, D. et al. (2000) A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological-to-immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone (LH) in obese men. J. Clin. Endocrinol. Metab., in press. Google Scholar Chappel, S.C. ( 1995) Heterogeneity of follicle stimulating hormone: control and physiological function. Hum. Reprod. Update , 1, 479–487. Google Scholar Chappel, S.C., Bashey, H.M. and Snyder, P.J. ( 1986) Similar isoelectric profiles of FSH from gonadotroph cell adenomas and non-adenomatous pituitaries. Acta Endocrinol. (Copenh.) , 113, 311–316. Google Scholar Costagliola, S., Niccoli, P. and Carayon, P. ( 1994a) Glycoprotein hormone isomorphism and assay discrepancy. J. Endocrinol. Invest. , 17, 291–299. Google Scholar Costagliola, S., Niccoli, P., Florentino, M. et al. ( 1994b) European collaborative study of luteinizing hormone assay: 1. Epitope specificity of luteinizing hormone monoclonal antibodies and surface mapping of pituitary and urinary luteinizing hormone. J. Endocrinol. Invest. , 17, 397–406. Google Scholar Costagliola, S., Niccoli, P., Florentino, M. et al. ( 1994c) European collaborative study on luteinizing hormone assay: 2. Discrepancy among assay kits is related to variation both in standard curve calibration and epitope specificity of kit monoclonal antibodies. J. Endocrinol. Invest. , 17, 407–416. Google Scholar De Lean, A.P., Munson, P.J. and Rodbard, R. ( 1978) Simultaneous analysis of families of sigmodal curves: application to bioassay, radioligand assay and physiological dose-response curves. Am. J. Physiol. , 235, E97–E102. Google Scholar Hård, K., Mekking, A., Damm, J.B.L. et al. ( 1990) Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. , 193, 263–271. Google Scholar Harris, S.D., Anobile, C.J., McLoughlin, J.D. et al. ( 1998) Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon®, Org 32489): a comparison with Metrodin and Metrodin-HP. Mol. Hum. Reprod. , 2, 807–811. Google Scholar Jeffcoate, S.L. ( 1993) Analytical and clinical significance of peptide hormone heterogeneity with particular reference to growth hormone and luteinizing hormone in serum. Clin. Endocrinol. , 38, 113–121. Google Scholar Labbe-Jullie, C., Sergi, I., Canonne, C. et al. ( 1992) Comparative mapping of human thyrotropin, gonadotropin and free subunits with antipeptide antibodies. Endocrinology , 131, 1999–2009. Google Scholar Lambert, A., Talbot, J.A., Anobile, C.J. and Robertson, W.R. ( 1998) Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction. Mol. Hum. Reprod. , 4, 619–629. Google Scholar Martin-Du-Pan, R.C., Horak, M. and Bischof, P. ( 1994) Clinical significance of invisible or partially visible luteinizing hormone. Hum. Reprod. , 9, 1987–1990. Google Scholar Olijve, W., de Boer, W., Mulders, J.W.M. et al. ( 1996) Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon®). Mol. Hum. Reprod. , 2, 371–382. Google Scholar Oliver, R.L., Kane, J.W., Waite, A. et al. ( 1999) Do immunoassays differentially detect different acidity glycoforms of FSH? Clin. Endocrinol. (Oxf.) , 51, 681–686. Google Scholar Papandreou, M., Sergi, I., Benkirane, M. et al. ( 1990) Carbohydrate dependent epitope mapping of human thyrotropin. Mol. Cell. Endocrinol. , 73, 15–26. Google Scholar Papandreou, M., Sergi, I., Medri, C. et al. ( 1991) Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin. Mol. Cell. Endocrinol. , 78, 137–150. Google Scholar Petterson, K. and Soderholm, R.M.J. ( 1991) Individual differences in lutropin immunoreactivity revealed by monoclonal antibodies. Clin. Chem. , 37, 333–340. Google Scholar Petterson, K., Ding, Y-Q. and Huhtaniemi, I. ( 1991) Monoclonal antibody-based discrepancies between two-sites immunometric test for lutropin. Clin. Chem. , 37, 1745–1748. Google Scholar Rafferty, B., Mower, J.A., Ward, H.L. et al. ( 1995) Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems. J. Endocrinol. , 145, 527–533. Google Scholar Ropelato, M.G., Garcia-Rudaz, M.C., Castro-Fernández, C. et al. ( 1999) A preponderance of basic luteinizing hormone (LH) isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian syndrome . J. Clin. Endocrinol. Metab. , 84, 4629–4636. Google Scholar Simoni, M., Jockenhövel, F. and Nieschlag, E. ( 1993) Biological and immunological properties of the international standard FSH 83/575: isoelectrofocusing profile and comparison with other FSH preparations. Acta Endocrinol. (Copenh.) , 128, 281–288. Google Scholar Simoni, M., Jockenhövel, F. and Nieschlag, E. ( 1994) Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species. J. Endocrinol. , 141, 359–367. Google Scholar Storring, P.L. and Gaines Das, R.E. ( 1989) The international standard for pituitary FSH: collaborative study of the standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems. J. Endocrinol. , 123, 275–293. Google Scholar Storring, P.L. and Gaines Das, R.E. ( 1993) The second international standard for human pituitary LH: its collaborative study by bioassays and immunoassays. J. Endocrinol. , 138, 345–359. Google Scholar Storring, P.L., Bangham, D.R., Cotes, P.M. et al. ( 1978) The international reference preparation of human pituitary luteinizing hormone for immunoassay. Acta Endocrinol. (Copenh.) , 88, 250–259. Google Scholar Talbot, J.A., Mitchell, R., Hoy, A.M. et al. ( 1996) Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization. Mol. Hum. Reprod. , 2, 799–806. Google Scholar Taylor, A.E., Khoury, R.H. and Crowley, W.F. ( 1994) A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation. J. Clin. Endocrinol. Metab. , 79, 240–247. Google Scholar Timossi, C., Barrios de Tomasi, J., Zambrano, E. et al. ( 1998) A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro. Neuroendocrinology , 67, 153–163. Google Scholar Ulloa-Aguirre, A., Cravioto, A., Damián-Matsumura, P. et al. ( 1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle-stimulating hormone. Hum. Reprod. , 7, 23–30. Google Scholar Ulloa-Aguirre, A., Zambrano, E., Timossi, C. et al. ( 1995a) On the nature of the follicle-stimulating signal delivered to the ovary during exogenously controlled follicular maturation. A search into the immunological and biological attributes and the molecular composition of two preparations of urofollitropin. Arch. Med. Res. , 26S, 219–230. Google Scholar Ulloa-Aguirre A., Midgely, A.R. Jr, Beitins, I.Z. et al. ( 1995b) Follicle-stimulating isohormones: Characterization and physiological relevance. Endocr. Rev. , 16, 765–787. Google Scholar Vermes, I., Bonte, H.A., Sluijs Veer, G. and Schoemaker, J. ( 1991) Interpretations of five monoclonal immunoassays of lutropin and follitropin: effects of normalization with WHO standard. Clin. Chem. , 37, 415–421. Google Scholar WHO Collaborating Centre for Research and References Services in the Immunoassay of Hormones in Human Reproduction (1994) Annual Technical Report to the Human Reproduction Programme. WHO, Geneva. Google Scholar Zambrano, E., Olivares, A., Mendez, J.P. et al. ( 1995) Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution throughout the human menstrual cycle . J. Clin. Endocrinol. Metab. , 80, 1647–1656. Google Scholar Zambrano, E., Barrios-de-Tomasi, J., Cárdenas, M. et al. ( 1996) Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle-stimulating hormone. Mol. Hum. Reprod. , 2, 463–571. Google Scholar Zerfaoui, M. and Ronin, C. ( 1996) Glycosylation is the structural basis for changes in polymorphism and immunoreactivity of pituitary glycoprotein hormones. Eur. J. Clin. Chem. Clin. Biochem. , 34, 749–753. Google Scholar © European Society of Human Reproduction and Embryology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Reactivity of different LH and FSH standards and preparations in the World Health Organization matched reagents for enzyme-linked immunoassays of gonadotrophins

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Abstract

Abstract The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system. enzyme-linked immunoassay, FSH, LH Introduction Twenty five years ago, the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization (WHO), established a validation, standardization and distribution programme to provide a number of laboratories with reagents, the so-called matched reagents, for the assay of reproductive hormones in serum, including the pituitary gonadotrophins LH and FSH. The name and concept of the matched reagents originated not only from the necessity for all reagents forming part of an assay system to be matched to each other, but also, from the intention to provide the various WHO collaborative centres with identical, matched reagents for single- or multi-centre clinical trials, the analytical identity of reagents used in various WHO collaborating laboratories being a prerequisite for obtaining reliable and comparable results. As a result of the progress in analytical techniques, the initial assay method (radioimmunoassay) to quantify pituitary gonadotrophins in serum was replaced approximately 5 years ago by enzyme-linked immunoassays (EIA). The change in the assay methodology has brought about some numerical differences in assay results. For example, in the 1994 WHO external quality assessment programme report, the ELISA results were, on average, numerically 30% lower for LH, while those obtained for FSH were 50% higher (WHO Technical Report, 1994). Among the reasons for these numerical discrepancies are the use of different antibodies (e.g. polyclonal antibodies in the radioimmunoassays versus monoclonal antibodies in the EIA) possessing distinct epitope specificities as well as the use of different pituitary preparations for constructing the standard curves, which may be differentially recognized by a determined set of antibodies, particularly by those of monoclonal origin (Petterson et al., 1991; Petterson and Soderholm, 1991; Vermes et al., 1991; Jeffcoate, 1993; Costagliola et al., 1994a,b,c; Martin-Du-Pan et al., 1994; Taylor et al., 1994). In fact, significant variations in molecular composition (mainly determined by the sample source, type of particular oligosaccharide chains attached to the protein core of the molecule and purification methods used to isolate the glycoproteins) have been detected among some of the highly purified pituitary gonadotrophin preparations employed to calibrate the kit standards, which may be distinctly recognized by antibodies and cognate receptors (Chappel et al., 1986; Simoni et al., 1993; Chappel, 1995; Burgon et al., 1997; Lambert et al., 1998). Further, it has been shown that the gonadotrophin glycoforms contained in crude pituitary extracts and recombinant preparations may be either equally or differentially identified depending on the particular antibody configuration of the immunoassay system employed for their quantitative estimation (Zambrano et al., 1996; Oliver et al., 1999). In the present study, we analysed the dose-response curve profiles of various widely employed pituitary and recombinant gonadotrophin preparations in the EIA system provided by the WHO Programme for the Provision of Matched Assay Reagents for the Immunoassay of Hormones (henceforth: the programme), and established their relative potency in this particular immunoassay. In addition, the behaviour of each preparation in the EIA system was correlated with its particular molecular composition as revealed by preparative chromatofocusing. Materials and methods Standards The human LH preparations tested in this study were the partially purified LER-907 standard for radioimmunoassay of gonadotrophins [obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through the National Hormone and Pituitary Program (NHPP), Torrance, CA, USA], highly purified recombinant human LH (coded herein as rhLH for the purpose of the study) produced by Chinese hamster ovary (CHO) cells (kindly provided by Organon International BV, Oss, The Netherlands), the pituitary LH standard (coded herein as WHO INEN) prepared by the WHO Collaborating Center in Cuba (provided by the Cuba–Mexico WHO RIA Reagents Programme, Instituto Nacional de Endocrinología, La Havana, Cuba and Instituto Nacional de la Nutrición SZ, Mexico City, Mexico) and calibrated against the WHO international reference preparation (IRP) of LH 80/552 (Storring and Gaines Das, 1993), the LH standard (coded herein as WHO 80/552C) contained in the LH EIA kit provided by the programme which is calibrated against the WHO IRP 80/552 and the LH standard preparation (coded herein as WHO 68/40C) intended to be used in the former programme's radioimmunoassay system, which was calibrated against the WHO IRP of LH 68/40 (Storring et al., 1978). For FSH, the following were tested: the LER-907 standard, highly purified recombinant human FSH (rhFSH) produced by CHO cells (ORG 32489; Organon International) (Olijve et al., 1996), a FSH standard preparation (WHO INEN) provided by the WHO Collaborating Center in Cuba and calibrated against the WHO IRP of FSH 78/549 (Storring and Gaines Das, 1989), and the FSH standard (coded herein as WHO 78/549C) contained in the FSH EIA kit provided by the programme, which has been calibrated against the WHO IRP 78/549. Immunoassays of LH and FSH Enzyme-linked immunoassays The EIA of LH and FSH were performed employing reagents provided by the WHO Collaborating Centre for Research and Reference Services in the Immunoassay of Hormones in Human Reproduction, London UK, following the instructions provided by the centre. The assays were of an immunometric (`sandwich') design and employed two anti-LH or anti-FSH monoclonal antibodies. In each EIA, the first antibody was directed against the β-subunit of the molecule and was attached to a magnetic particle, whereas the second antibody was directed against the α-subunit, and was labelled with alkaline phosphatase. These LH and FSH EIA systems exhibit ≤0.1% and 0.03% cross-reactivity with highly purified FSH and LH respectively, and undetectable reactivity with growth hormone (GH) and prolactin. The intra- and interassay variables were ≤4.0% and ≤8.0% respectively. Results are expressed as ng or mIU as appropriate according to the particular standard preparation analysed. Radioimmunoassays The radioimmunoassay of LH was performed employing 125I-labelled LH-I3 as the tracer (specific activity 70–90 μCi/μg protein), the reference preparation LER-907 as the standard and the antihuman LH-3, at a final dilution of 1:800 000, as the antiserum (Ropelato et al., 1999). Cross-reactivity of this antiserum with highly purified FSH, GH and prolactin is <0.2%. The sensitivity of the assay was 0.7 IU/l. The FSH radioimmunoassay was performed employing 125I-labelled FSH I-1 as the tracer (specific activity 60–70 μCi/μg protein), the LER-907 preparation as the standard and anti-human FSH-6 at final dilution of 1:250 000, as the antiserum (Timossi et al., 1998). This antiserum exhibits less than 0.1% cross-reactivity with highly purified human LH and prolactin and undetectable reactivity with free α-subunit and GH. In both radioimmunoassay systems, all LH and FSH isoforms displaced either 125I-labelled FSH or LH from the antibody in a parallel fashion when tested at seven to 10 different dilutions; in fact, simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by FSH and LH present in the LER-907 standard and concentrated pools of the several isoforms fractionated by chromatofocusing (Zambrano et al., 1996). All LH and FSH radioimmunoassay reagents were generously provided by the NIDDK through Dr A.F.Parlow from the NHPP. For both radioimmunoassays, the inter- and intra-assay coefficients of variation were <13 and <8% respectively. The results are expressed as ng of the LER-907 standard. Preparative chromatofocusing of LH and FSH Chromatofocusing of each gonadotrophin preparation was performed as described previously (Ulloa-Aguirre et al., 1992; Castro-Fernández et al., 2000) with some modifications. This technique allowed the separation of different gonadotrophin isoforms on the basis of charge, which in all glycoprotein hormones is mainly determined by the amount of terminal sialic acid and/or sulphate residues present in the oligosaccharide structures of the molecule (Ulloa-Aguirre et al., 1995b). Briefly, 2.0–2.7 IU of each WHO standard, ~111 μg of LER-907, ~5.0 μg of rhLH, and ~50 μg of rhFSH were redissolved in phosphate (0.01 mol/l) buffered physiological (0.15 mol/l) saline, transferred to dialysis membrane tubing (molecular weight cut-off, 12 000–14 000; Spectrum Medical Industries, Los Angeles, CA, USA), dialysed at 4°C for 24 h against deionized water and thereafter against 0.01 mol/l ammonium carbonate (pH 7.5) and freeze-dried. Lyophilates were redissolved to one tenth of original volume in Pharmalyte pH 8–10.5-HCl (Pharmacia Biotech, Piscataway, NJ, USA) (1:45 dilution in deonized water, pH 7.0) and the suspension was then applied to the top of a 30×1 cm column of polybuffer exchange resin (PBE-118, Pharmacia Biotech), previously equilibrated for 18–24 h with 25 mmol/l triethylamine-HCl, pH 11.0, and chromatofocused at 4°C. Eluate fractions (2 ml each) were collected at a flow rate of 1 ml/4 min. The pH of each fraction was then measured, and when a limiting pH of 7.0 had been reached the eluent buffer (Pharmalyte-HCl) was changed by Polybuffer-74 (Pharmacia Biotech) diluted 1:8 in deionized water, pH 4.0, to elute proteins bound at pH 7.0–4.0. Proteins bound at the lower limiting pH (pH <3.50; salt peak) were finally recovered by the addition of 1.0 mol/l NaCl to the chromatofocusing column. The pH of each fraction was neutralized to pH 7.0 by the addition of either 200 μl 1.0 mol/l triethylamine-HCl pH 7.0 (to those fractions with an elution pH value of 11.0 to 7.0) or 1.0 mol/l imidazole-HCl pH 7.4 (to fractions recovered within elution pH values of 6.99 to <4.0). Each fraction was stored frozen at –20°C until the day of the LH or FSH radioimmunoassays. All fractions from a single column were assayed in duplicate incubations in the same radioimmunoassay run. Recoveries of immunoactive LH and FSH after chromatofocusing were 75 ± 5% of the total amount applied to the columns. Each gonadotrophin preparation was chromatofocused in three separate runs. Statistical analysis Tests for parallelism among the slopes generated by the different gonadotrophin preparations in the EIA and the LH radioimmunoassay were performed following the method of DeLean et al. (1978). This method allows for simultaneous fitting and analysis of families of sigmoidal dose-response curves and describes the curves in terms of basal and half maximal responses (ED50) and curve shape or steepness. Relative potencies among the various preparations were calculated considering multiple points comprising the ED20–ED80 interval. Results EIA of LH preparations The dose-response curves of the different WHO standards, LER-907 and rhLH, in the LH EIA are shown in Figure 1. The profile of the dose-response curves elicited by the three WHO LH standards tested in the EIA system were superimposable and almost identical; simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards (mean slope values: 1.27 ± 0.07, 1.26 ± 0.07 and 1.13 ± 0.10 for the 68/40C, WHO INEN and 80/552C standards respectively; n = 6 EIA runs for each standard) and LER-907 (mean slope value 1.25 ± 0.07; n = 6). On the contrary, no parallelism was found between the curves generated by rhLH (mean slope value, 0.72 ± 0.08) and the WHO and LER-907 LH standards. This finding contrasted with the dose-response curve elicited by rhLH in the polyclonal antibodies-based radioimmunoassay, which was parallel with that of the LER-907 standard (Figure 2). The relative potency of each preparation tested in this LH EIA system is shown in Table I. The potency of LH contained in the WHO INEN standard was slightly higher than that of the WHO 80/552C standard and lower than that yielded by WHO 68/40C; the immunopotency of these two latter standards was nearly the same. According to this EIA system, the potency of the WHO standards was similar when expressed in terms of the LER-907 preparation and vice versa. EIA of the FSH preparations Representative dose-response curves elicited by the two WHO FSH standards, LER-907 and rhFSH, in the FSH EIA system are shown in Figure 3. Simultaneous curve fitting of the dose-response curves revealed no significant differences among the slopes generated by the various preparations tested (mean slope values: 1.05 ± 0.05, 1.07 ± 0.06, 1.04 ± 0.04 and 1.12 ± 0.03, for the 78/549C, WHO INEN, rhFSH and LER-907 standards respectively; n = 6 EIA runs for each preparation). On a weight-to-weight basis, rhFSH was approximately four times more potent that LER-907; the immunopotency of the FSH 78/549C kit standard was slightly higher than that of the WHO INEN standard when compared on a unit-to-unit basis (Table II). Chromatofocusing of the LH and FSH standards and preparations Figures 4 and 5 show the pH distribution pattern of immunoreactive LH and FSH after chromatofocusing of the various preparations analysed. As shown, each preparation contained two or more charge isoforms recovered within a pH window of 9.0 to 3.50 and 6.50 to 3.50 for the LH and FSH containing preparations respectively. In addition, all standards and preparations showed variable amounts of LH and FSH immunoactivity in fractions corresponding to molecules bound at the lower limiting pH, which were recovered after the addition of a highly concentrated NaCl solution to the chromatofocusing column (salt peak). As depicted in Figure 4, the WHO 68/40C and 80/552C LH kit standards exhibited very similar charge distribution profiles; both standards contained negligible amounts (<3% of the total LH recovered) of LH molecules within the salt peak. In contrast, the WHO INEN, rhLH and LER-907 preparations presented a higher (6–16%) content of this strongly acidic LH species. Although the bulk (71–75% of total) of LH immunoreactivity in the WHO INEN standard and rhLH preparation was recovered at pH values <7.0, significant amounts of immunoactivity (15–22% of total) were additionally detected at higher pH values; in fact, these two preparations presented the highest degree of charge heterogeneity (in terms of total number of LH components resolved by the charge-based separation technique employed). In general, LH contained in LER-907, rhLH and WHO INEN was more acidic than that present in the remaining WHO LH standards. In the chromatofocusing system employed (pH window from 10.0 to <3.5), the bulk (<80% of total) of immunoreactive FSH contained in the preparations analysed was recovered at pH values 5.20 to 3.50 and in the salt peak. Negligible amounts (<5%) of immunoreactive FSH material eluting at pH values 8.73 to 8.27 was consistently detected in the LER-907 standard (Figure 5). This latter preparation contained three major FSH charge isoforms that focused at pH values similar or close to those of the isoforms present in the WHO 78/549C standard. The pH elution profiles of rhFSH and the WHO INEN FSH standard were similar; both preparations exhibited a single broad FSH peak with a median pH value of 4.53–4.60 and a second large component recovered within the salt peak. Discussion Analysis of the dose-response curves of several LH standards and preparations in the EIA system, developed for the WHO Special Programme of Research in Human Reproduction, revealed a close similarity among the curves elicited by the WHO LH 68/40C, 80/552C and INEN standards. This agreement in dose-response curve profiles among the various WHO LH standards tested was in marked contrast with that exhibited by the rhLH preparation produced in CHO cells, whose absence of parallelism with the WHO and LER-907 curves was clearly noticeable. The non-parallelism exhibited by this particular preparation may be due either to vulnerability to matrix effects at low doses or to differences in molecular specificity of the matched monoclonal antibodies employed in this EIA system for the mixture of rhLH glycoforms contained in this particular preparation and that of the naturally occurring variants present in the preparations of pituitary origin. In fact, several differences in glycosylation exist between rhLH produced by CHO cells and pituitary LH: oligosaccharides in CHO cell-derived gonadotrophins are sialylated only in an α2–3 conformation, do not contain bisecting N-acetyl glucosamine moieties and sulphated terminal glycosylation is missing (Hård et al., 1990; Rafferty et al., 1995; Amoresano et al., 1996; Talbot et al., 1996). In addition, it is known that CHO cells have a limited ability to introduce complex branched carbohydrate structures onto the protein core in comparison with the naturally producing cells, the gonadotrophs (Hård et al., 1990; Harris et al., 1998). Several lines of evidence indicate that differences in glycosylation may affect the reactivity of glycoprotein towards certain antibodies (Papandreou et al., 1990, 1991; Labbe-Jullie et al., 1992; Zerfaoui and Ronin, 1996). For example, removal of sialic acid from pituitary thyrotropin specifically abolished the binding of this natural glycoprotein to anti-β monoclonal antibodies (Zerfaoui and Ronin, 1996) and its deglycosylation led to a five-fold reduction in immunoreactivity toward certain anti-β polyclonal antibodies (Papandreou et al., 1990). In the present study, the behaviour of rhLH in the WHO EIA contrasted with that exhibited in the radioimmunoassay, in which rhLH displaced 125I-labelled LH from the antibody in a fashion similar to that shown by the pituitary-derived LER-907 standard, thus emphasizing the importance of the particular antibody configuration employed for determining the immunoreactivity of recombinant DNA-derived preparations. The overall data indicate that recombinant human LH produced by CHO cells is not a suitable preparation for use as the calibration standard in this particular monoclonal antibody-based EIA system and that all other WHO LH standard preparations tested may be indistinctly employed without significant variations in the final concentration results of the unknown samples. In contrast to the set of LH preparations tested in the EIA system, the dose-response curves elicited by both the recombinant and pituitary FSH preparations were parallel to each other. Thus, differences in glycosylation between the CHO cell-derived preparation and the pituitary standards did not apparently influence to a considerable extent the reactivity of the former preparation toward the set of monoclonal antibodies employed in this FSH EIA system. Although the present study does not unambiguously resolve whether the monoclonal antibodies employed reacted equally with all FSH preparations tested, the profile of the dose-response curves elicited by the four preparations indicates that all preparations may be used as calibration standards at least in this particular human FSH EIA. All LH preparations exhibited a high degree of charge heterogeneity. In particular, the pH distribution profile exhibited by rhLH, LER-907 and the WHO INEN LH preparations was more acidic than that of the WHO 68/40C and 80/552C standards; these differences may be due to selective removal of the more or less basic isoforms during the purification procedures as well as to cell-specific (gonadotrophs and CHO cells) particularities in post-translational processing of the gonadotrophin molecule (Ulloa-Aguirre et al., 1995a). Interestingly, despite the marked differences in charge distribution between the WHO 68/40C and 80/552C preparations and the WHO INEN standard, their behaviour in the EIA system was remarkably similar as disclosed by their superimposed dose-response curves. This observation suggests that variability in distribution and/or conformation of terminally charged sugars in the isoforms contained in these pituitary preparations does not greatly influence their reactivity towards the particular antibodies employed by the EIA system. On the contrary, it appears that the high abundance of sialic acid attached exclusively in α2,3 position and the complete absence of sulphate residues in the acidic rhLH molecules (which conform the bulk of immunoreactive material present in this preparation) influenced the reactivity of the monoclonal antibodies for this compound. As previously reported, all FSH preparations tested exhibited a predominantly acidic pH distribution profile with the bulk of FSH immunoactivity recovered at elution pH values ≤5.0. The charge distribution profile of all these FSH preparations varied considerably from that previously reported for FSH in crude pituitary extracts and serum, in which 10–25% of total FSH may be recovered at elution pH values >4.50 (Simoni et al., 1994; Ulloa-Aguirre et al., 1995a,b; Zambrano et al., 1995; Anobile et al., 1998). In a previous study employing this particular EIA system, it was observed that the intrapituitary FSH isoforms with pH values 6.60 to <3.80 elicited parallel dose-response curves whereas the less acidic/sialylated form (pH >7.10) could not be accurately quantified due to significant non-concordance between this isoform and the corresponding standard curve (LER-907) (Zambrano et al., 1996). How this reduced reactivity of the monoclonal antibodies of this EIA toward the less acidic FSH isoforms may affect the accuracy of the concentrations of FSH in serum is not known. It must be emphasized, however, that the abundance of this particular isoform in serum is extremely low and thus its presence in the mixture of variants released from the pituitary may be irrelevant at least in physiological conditions. In summary, with the exception of rhLH produced in CHO cells, all LH and FSH preparations tested in the EIA systems provided by the WHO Programme for the Provision of Matched Assay Reagents for the Immunoassay of Hormones (currently distributed through the non-profit organization Immunometrics Ltd, London, UK) elicited dose-response curves which were parallel to each other across a wide range of concentration values. Although variations in purity and concentration units of the various gonadotrophin preparations do not allow accurate establishment of the exact degree of cross-reactivity of the monoclonal antibodies employed in these EIA kits toward the standards tested, all the pituitary-derived preparations as well as rhFSH produced by CHO cells seem appropriate for using as calibration standards in this particular immunometric system. The impact of the differences in mixture of gonadotrophin isoforms between these particular preparations and the human serum on the accuracy of measurements of LH and FSH in serum samples by this and other immunometric assay systems is an issue that deserves to be scrutinized carefully. Table I. Relative potencies (means and 95% confidence limits) of the different LH standards tested in the World Health Organization (WHO) LH enzyme-linked immunoassay (EIA) system LH standard  68/40C (IU)  80/552C (IU)  INEN (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 68/40C  1  0.99 (0.92, 1.05)  1.16 (1.10, 1.20)  22.2 (21.6, 22.6)  1 IU 80/552C  1.04 (1.10, 0.97)a  1  0.84  25.5 (24.2, 26.7)  1 IU INEN  0.882 (0.840, 0.910)  1.21 (1.15, 1.27)  1  23.5 (22.3, 24.7)  1 μg LER-907  0.045 (0.044, 0.046)  0.040 (0.038, 0.042)  0.044 (0.041, 0.046)  1  LH standard  68/40C (IU)  80/552C (IU)  INEN (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 68/40C  1  0.99 (0.92, 1.05)  1.16 (1.10, 1.20)  22.2 (21.6, 22.6)  1 IU 80/552C  1.04 (1.10, 0.97)a  1  0.84  25.5 (24.2, 26.7)  1 IU INEN  0.882 (0.840, 0.910)  1.21 (1.15, 1.27)  1  23.5 (22.3, 24.7)  1 μg LER-907  0.045 (0.044, 0.046)  0.040 (0.038, 0.042)  0.044 (0.041, 0.046)  1  View Large Table II. Relative potencies (means and 95% confidence limits) of the different FSH standards and preparations tested in the WHO FSH EIA system FSH standard or preparation  78/549C (IU)  INEN (IU)  rhFSH (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 78/549C  1  1.29 (1.21, 1.36)  9.86 (9.52, 10.20)  48.81 (47.20, 50.40)  1 IU INEN  0.80 (0.75, 0.84)a  1  8.28 (7.85, 8.70)  35.86 (33.80, 37.90)  1 μg rhFSH  0.102 (0.099, 0.106)  0.124 (0.117, 0.130)  1  4.33 (4.16, 4.49)  1 μg LER-907  0.021 (0.020, 0.022)  0.029 (0.027, 0.030)  0.234 (0.225, 0.243)  1  FSH standard or preparation  78/549C (IU)  INEN (IU)  rhFSH (IU)  LER-907 (μg)  a95% confidence limits.  1 IU 78/549C  1  1.29 (1.21, 1.36)  9.86 (9.52, 10.20)  48.81 (47.20, 50.40)  1 IU INEN  0.80 (0.75, 0.84)a  1  8.28 (7.85, 8.70)  35.86 (33.80, 37.90)  1 μg rhFSH  0.102 (0.099, 0.106)  0.124 (0.117, 0.130)  1  4.33 (4.16, 4.49)  1 μg LER-907  0.021 (0.020, 0.022)  0.029 (0.027, 0.030)  0.234 (0.225, 0.243)  1  View Large Figure 1. View largeDownload slide Representative dose-response curves of the different World Health Organization (WHO) LH standards, LER-907 and rhLH produced in Chinese hamster ovary (CHO) cells in the WHO LH enzyme-linked immunoassay (EIA) system. OD = optical density. Figure 1. View largeDownload slide Representative dose-response curves of the different World Health Organization (WHO) LH standards, LER-907 and rhLH produced in Chinese hamster ovary (CHO) cells in the WHO LH enzyme-linked immunoassay (EIA) system. OD = optical density. Figure 2. View largeDownload slide Representative dose-response curves elicited by rhLH produced in CHO cells and LER-907 in the LH radioimmunoassay. B/B0 = Bound/Bound at dose zero. Figure 2. View largeDownload slide Representative dose-response curves elicited by rhLH produced in CHO cells and LER-907 in the LH radioimmunoassay. B/B0 = Bound/Bound at dose zero. Figure 3. View largeDownload slide Representative dose-response curves of the different WHO FSH standards, LER-907 and rhFSH produced in CHO cells in the WHO EIA system. Figure 3. View largeDownload slide Representative dose-response curves of the different WHO FSH standards, LER-907 and rhFSH produced in CHO cells in the WHO EIA system. Figure 4. View largeDownload slide Representative profiles of pH distribution of immunoreactive LH after chromatofocusing of different LH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 4. View largeDownload slide Representative profiles of pH distribution of immunoreactive LH after chromatofocusing of different LH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 5. View largeDownload slide Representative profiles of pH distribution of immunoreactive FSH after chromatofocusing of different FSH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. Figure 5. View largeDownload slide Representative profiles of pH distribution of immunoreactive FSH after chromatofocusing of different FSH standards and preparations. The elution pH value of the several isoforms present in each preparation are indicated. The arrowhead marks the addition of 1 mol/l NaCl to the chromatofocusing column. 4 To whom correspondence should be addressed at: Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, Apdo. Postal 99–065, Unidad Independencia, Mexico 10101, DF, Mexico. E-mail: [email protected] The authors are grateful to Dr A.F.Parlow and the NIDDK National Hormone and Pituitary Program for the human FSH and LH radioimmunoassay reagents and to the WHO Centre for Research and Reference Services in the Immunoassay of Hormones in Human Reproduction, London, UK, for providing the LH and FSH EIA reagents. This study received financial support from the Laboratory Methods Group, Human Reproduction Programme, WHO, Geneva, Switzerland and from Consejo Nacional de Ciencia y Technologia (CONACyT), grant Z8589N), Mexico, D.F., Mexico. References Amoresano, A., Siciliano, R., Orru, S. et al. ( 1996) Structural characterisation of human recombinant glycohormones follitropin, lutropin and choriogonadotropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. , 242, 608–618. Google Scholar Anobile, C.J., Talbot, J.A., McCann, S.J. et al. ( 1998) Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state. Mol. Hum. Reprod. , 4, 631–639. Google Scholar Burgon, P.G., Stanton, P.G., Petterson, K. and Robertson, D.M. ( 1997) Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vivo, radioreceptor activity and immunoactivity. Reprod. Fertil. Dev. , 9, 501–508. Google Scholar Castro-Fernández, C., Olivares, A., Söderlund, D. et al. (2000) A preponderance of circulating basic isoforms is associated with decreased plasma half-life and biological-to-immunological ratio of gonadotropin-releasing hormone-releasable luteinizing hormone (LH) in obese men. J. Clin. Endocrinol. Metab., in press. Google Scholar Chappel, S.C. ( 1995) Heterogeneity of follicle stimulating hormone: control and physiological function. Hum. Reprod. Update , 1, 479–487. Google Scholar Chappel, S.C., Bashey, H.M. and Snyder, P.J. ( 1986) Similar isoelectric profiles of FSH from gonadotroph cell adenomas and non-adenomatous pituitaries. Acta Endocrinol. (Copenh.) , 113, 311–316. Google Scholar Costagliola, S., Niccoli, P. and Carayon, P. ( 1994a) Glycoprotein hormone isomorphism and assay discrepancy. J. Endocrinol. Invest. , 17, 291–299. Google Scholar Costagliola, S., Niccoli, P., Florentino, M. et al. ( 1994b) European collaborative study of luteinizing hormone assay: 1. Epitope specificity of luteinizing hormone monoclonal antibodies and surface mapping of pituitary and urinary luteinizing hormone. J. Endocrinol. Invest. , 17, 397–406. Google Scholar Costagliola, S., Niccoli, P., Florentino, M. et al. ( 1994c) European collaborative study on luteinizing hormone assay: 2. Discrepancy among assay kits is related to variation both in standard curve calibration and epitope specificity of kit monoclonal antibodies. J. Endocrinol. Invest. , 17, 407–416. Google Scholar De Lean, A.P., Munson, P.J. and Rodbard, R. ( 1978) Simultaneous analysis of families of sigmodal curves: application to bioassay, radioligand assay and physiological dose-response curves. Am. J. Physiol. , 235, E97–E102. Google Scholar Hård, K., Mekking, A., Damm, J.B.L. et al. ( 1990) Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells. Eur. J. Biochem. , 193, 263–271. Google Scholar Harris, S.D., Anobile, C.J., McLoughlin, J.D. et al. ( 1998) Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon®, Org 32489): a comparison with Metrodin and Metrodin-HP. Mol. Hum. Reprod. , 2, 807–811. Google Scholar Jeffcoate, S.L. ( 1993) Analytical and clinical significance of peptide hormone heterogeneity with particular reference to growth hormone and luteinizing hormone in serum. Clin. Endocrinol. , 38, 113–121. Google Scholar Labbe-Jullie, C., Sergi, I., Canonne, C. et al. ( 1992) Comparative mapping of human thyrotropin, gonadotropin and free subunits with antipeptide antibodies. Endocrinology , 131, 1999–2009. Google Scholar Lambert, A., Talbot, J.A., Anobile, C.J. and Robertson, W.R. ( 1998) Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction. Mol. Hum. Reprod. , 4, 619–629. Google Scholar Martin-Du-Pan, R.C., Horak, M. and Bischof, P. ( 1994) Clinical significance of invisible or partially visible luteinizing hormone. Hum. Reprod. , 9, 1987–1990. Google Scholar Olijve, W., de Boer, W., Mulders, J.W.M. et al. ( 1996) Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon®). Mol. Hum. Reprod. , 2, 371–382. Google Scholar Oliver, R.L., Kane, J.W., Waite, A. et al. ( 1999) Do immunoassays differentially detect different acidity glycoforms of FSH? Clin. Endocrinol. (Oxf.) , 51, 681–686. Google Scholar Papandreou, M., Sergi, I., Benkirane, M. et al. ( 1990) Carbohydrate dependent epitope mapping of human thyrotropin. Mol. Cell. Endocrinol. , 73, 15–26. Google Scholar Papandreou, M., Sergi, I., Medri, C. et al. ( 1991) Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin. Mol. Cell. Endocrinol. , 78, 137–150. Google Scholar Petterson, K. and Soderholm, R.M.J. ( 1991) Individual differences in lutropin immunoreactivity revealed by monoclonal antibodies. Clin. Chem. , 37, 333–340. Google Scholar Petterson, K., Ding, Y-Q. and Huhtaniemi, I. ( 1991) Monoclonal antibody-based discrepancies between two-sites immunometric test for lutropin. Clin. Chem. , 37, 1745–1748. Google Scholar Rafferty, B., Mower, J.A., Ward, H.L. et al. ( 1995) Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems. J. Endocrinol. , 145, 527–533. Google Scholar Ropelato, M.G., Garcia-Rudaz, M.C., Castro-Fernández, C. et al. ( 1999) A preponderance of basic luteinizing hormone (LH) isoforms accompanies inappropriate hypersecretion of both basal and pulsatile LH in adolescents with polycystic ovarian syndrome . J. Clin. Endocrinol. Metab. , 84, 4629–4636. Google Scholar Simoni, M., Jockenhövel, F. and Nieschlag, E. ( 1993) Biological and immunological properties of the international standard FSH 83/575: isoelectrofocusing profile and comparison with other FSH preparations. Acta Endocrinol. (Copenh.) , 128, 281–288. Google Scholar Simoni, M., Jockenhövel, F. and Nieschlag, E. ( 1994) Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species. J. Endocrinol. , 141, 359–367. Google Scholar Storring, P.L. and Gaines Das, R.E. ( 1989) The international standard for pituitary FSH: collaborative study of the standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems. J. Endocrinol. , 123, 275–293. Google Scholar Storring, P.L. and Gaines Das, R.E. ( 1993) The second international standard for human pituitary LH: its collaborative study by bioassays and immunoassays. J. Endocrinol. , 138, 345–359. Google Scholar Storring, P.L., Bangham, D.R., Cotes, P.M. et al. ( 1978) The international reference preparation of human pituitary luteinizing hormone for immunoassay. Acta Endocrinol. (Copenh.) , 88, 250–259. Google Scholar Talbot, J.A., Mitchell, R., Hoy, A.M. et al. ( 1996) Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization. Mol. Hum. Reprod. , 2, 799–806. Google Scholar Taylor, A.E., Khoury, R.H. and Crowley, W.F. ( 1994) A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation. J. Clin. Endocrinol. Metab. , 79, 240–247. Google Scholar Timossi, C., Barrios de Tomasi, J., Zambrano, E. et al. ( 1998) A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro. Neuroendocrinology , 67, 153–163. Google Scholar Ulloa-Aguirre, A., Cravioto, A., Damián-Matsumura, P. et al. ( 1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle-stimulating hormone. Hum. Reprod. , 7, 23–30. Google Scholar Ulloa-Aguirre, A., Zambrano, E., Timossi, C. et al. ( 1995a) On the nature of the follicle-stimulating signal delivered to the ovary during exogenously controlled follicular maturation. A search into the immunological and biological attributes and the molecular composition of two preparations of urofollitropin. Arch. Med. Res. , 26S, 219–230. Google Scholar Ulloa-Aguirre A., Midgely, A.R. Jr, Beitins, I.Z. et al. ( 1995b) Follicle-stimulating isohormones: Characterization and physiological relevance. Endocr. Rev. , 16, 765–787. Google Scholar Vermes, I., Bonte, H.A., Sluijs Veer, G. and Schoemaker, J. ( 1991) Interpretations of five monoclonal immunoassays of lutropin and follitropin: effects of normalization with WHO standard. Clin. Chem. , 37, 415–421. Google Scholar WHO Collaborating Centre for Research and References Services in the Immunoassay of Hormones in Human Reproduction (1994) Annual Technical Report to the Human Reproduction Programme. WHO, Geneva. Google Scholar Zambrano, E., Olivares, A., Mendez, J.P. et al. ( 1995) Dynamics of basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution throughout the human menstrual cycle . J. Clin. Endocrinol. Metab. , 80, 1647–1656. Google Scholar Zambrano, E., Barrios-de-Tomasi, J., Cárdenas, M. et al. ( 1996) Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle-stimulating hormone. Mol. Hum. Reprod. , 2, 463–571. Google Scholar Zerfaoui, M. and Ronin, C. ( 1996) Glycosylation is the structural basis for changes in polymorphism and immunoreactivity of pituitary glycoprotein hormones. Eur. J. Clin. Chem. Clin. Biochem. , 34, 749–753. Google Scholar © European Society of Human Reproduction and Embryology

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Human Reproduction – Oxford University Press

Published: Nov 1, 2000

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