Jun 11, 1990 - Roger Ekins (Middlesex Hospital,. London, U.K.). All antibodies except two were obtained in purified form; those two were in ascitic fluid and.
CLIN.CHEM.37/3, 333-340 (1991)
Individual Differences in Lutropin Immunoreactivity Revealed by Monoclonal Antibodies Kim S. I. Petterssonand JuanItaR.-M.SOderhoim We screened 11 monoclonal anti-lutropin (LH) antibodies for use in two-site immunometric combinations, with timeresolved fluorescence as the detection principle. Seven immunofluorometric assays (IFMA), six detecting only the intact hormone and one detecting both the intact hormone and freebeta subunit, were compared with each other and witha conventional radioimmunoassay involving a polyclonal antiserum. Two monoclonal antibodies that exclusively reactedwith intact LH showed restricted reactivity with LH in 25% ofthesamplestested. The LH of one individual was not detected at all by one of the antibodies. The existenceof two distinct populations suggests the existence of a genetic variant of LH. A comparison of the results obtained by IFMAS with those by radioimmunoassay showed marked discrepancies in the low range, with IFMAS measuring much lowervalues.The excellent correlation between the various monoclonal IFMASand between these and a poly/oligoclonal IFMA strongly suggests that the discrepancies seen are caused by the insufficient sensitivity
and matrix effects of the radioimmunoassay.
The main clinical use of immunoassays of lutropin (luteinizing hormone, LH) has been to measure normal and above-normal concentrations of the hormone.’ Attempts have also been made to measure low concentrations of it, e.g., in the prepubertal state (1, 2), but inadequatesensitivity of competitive-type immunoassays has rendered these results questionable. With the advent of the “sandwich technique” or the two-site immunoassay principle (3-5), a new generation ofimmunoassays, generally referred to as immunometric assays, has emerged, with highly improved analytical sensitivities. Comparisons of the patients’ results obtained by these two types of immunoassay designs revealstriking differences, with immunometric assays measuring very low LH concentrations (6-B). Because the emergence of immunometric assays closely coincided with the introduction of monoclonal antibodies and because of the well-documented molecular heterogeneity of the glycoprotein hormones, the above discrepancy in results might be attributable to the restricted reactivity of the monoclonal reagents with the circulating hormone. Are perhaps the monoclonal antibodies too specific? i.e., do they “see” only parts of the LH heterogeneity, whereas polyclonal antisera react with all forms of the hormone? Wallac Immunodiagnostic Research Laboratory, P.O. Box 10, SF-20101 Turku 10, Finland. ‘Nonstandard abbreviations: LII, lutropin; FSH, follitropin; TSH, thyrotropin; hCG, human choriogonadotropin; IFMA, immunofluorometric assay; Mab, monoclonal antibody; and IRP, Inter-
national Reference Preparation. Received June 11, 1990; accepted November
7, 1990.
To investigate the causes of discrepancies between competitive radioimmunoassays and immunometric assays, we screened 11 monoclonal antibodies against LH foruse in two-site immunometric combinations and compared these with each other and with a conventional polyclonal radioimmunoassay. Materials and Methods Reagents Eleven monoclonal antibodies (Mabs) against LH were obtainedfrom various sources. Two Mabs were from Medix Biochemica (Kauniainen, Finland), three from Biosoft (Paris, France), four from Diagnostika (Falkenberg, Sweden), and one from Bioprobes Unit Research (Torino, Italy). One Mab was provided noncommercially by Prof. Roger Ekins (Middlesex Hospital, London, U.K.). All antibodies except two were obtained in purified form; those two were in ascitic fluid and purified by Protein A chromatography. Polyclonal sheep antiserum against human LH (LH 9) was kindly provided by Prof. Leif Wide (Academic Hospital, Uppsala, Sweden). The immunoglobulin fraction was purified by ion-exchange chromatography with diethylaminoethyl-Trisacryl M (Reactifs IBF, Pointet Girard, France) as described previously(9). The International Reference Preparation of pituitary LH (ICSH) Human for Immunoassay (code 68/40) was obtained from the National Institute for Biological Standards and Control (London, U.K.). Highly purified standards of the glycoprotein hormones follitropin (FSH), thyrotropin (TSH), and choriogonadotropin (hCG) were purchasedfrom Diagnostika(Falkenberg, Sweden). From the same source we obtained ultrapure preparations of TSH and FSH (LH contamination
(_)
17 >67
1.3 1.9
“34 >80 ‘67
03 10 >67
29 1.1 1.8
Fig. 1. Results of comparing two-site immunometric
assays of 11
Mabs against LH CA. = capture antibody, TA. = tracer antibody,B = betaspecific,A = alpha specific, I = intact specific. (-) and (+) indicatesdetection limit
