1 Research Unit in Developmental Biology, Hospital de Pediatría, Centro Médico Nacional S. XXI, IMSS, 2 Department of Reproductive Biology, Instituto Nacional de la Nutrición SZ, 3 Research Unit in Reproductive Medicine, Hospital de Ginecobstetricia No. 4, IMSS, Mexico DF, Mexico
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Abstract |
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Key words: enzyme-linked immunoassay/FSH/LH
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Introduction |
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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.
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Materials and methods |
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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 ß-subunit of the molecule and was attached to a magnetic particle, whereas the second antibody was directed against the -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 7090 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 6070 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
-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, 2.02.7 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 00014 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 810.5-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 30x1 cm column of polybuffer exchange resin (PBE-118, Pharmacia Biotech), previously equilibrated for 1824 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 7.04.0. 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 ED20ED80 interval.
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Results |
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Discussion |
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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
2,3 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 1025% 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 16, 2000; accepted on August 7, 2000.