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The preparation and nature of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay are described. A collaborative assay of this material (coded 68/40) in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH (ICSH)), for Bioassay (coded 69/104) was carried out by twelve laboratories in seven different countries, using different bioassay and immunoassay methods. The weighted combined potency estimate (with 95 % confidence limits) was 52.1 (46.3–58.7) IU/ampoule with male accessory reproductive organ weight gain assays; 80.2 (73.2–87.9) IU/ampoule with ovarian ascorbate depletion assays; 127 (124–129) IU/ampoule with in vitro Leydig cell testosterone production assays; and 124 (121–126) IU/ampoule with testis receptor binding assays. Immunoassay estimates in terms of the same standard were heterogeneous and gave an unweighted mean potency estimate of 33.2 IU with 95% confidence limits of 14.8– 74.4 IU/ampoule. Estimates from different methods gave significantly different results, and the reasons for this are discussed in terms of the differences between the materials being compared and the methods used in the comparison. These data illustrate the conceptual difficulties involved in comparing hetero geneous reference preparations, especially by both bioassay and immunoassay, and some of the causes of inevitable discontinuity of assay results, as described in the 26th Report of the WHO Expert Committee on Biological Standardization. On the basis of these results, and in the interest of maintaining continuity of its unitage, the International Reference Preparation has been allocated a potency of 77 IU/ampoule.

Dear Sir,We read with concern the article by Parte and Juneja (Assessment of Bioactivity of WHO/NIH Research Standard for Inhibin [code 86/690] using two separate pituitary cell culture systems. Parte, P. P. and Juneja, H. H, Horm. Metab. Res. 25: 356-359 [1993]). As distributors of this standard, we wish to make the following comments:The preparation of porcine inhibin ampouled in 1986 under code number 86/690, was intended to serve as an internationally available interim research standard for in vitro bioassays pending the availability of more suitable highly purified material, ideally recombinant human inhibin, in sufficient quantity for ampouling. This research standard was initially assessed for bioactivity in a number of independent expert laboratories involved in the characterisation of inhibin at that time. In the interests of making a research standard for in vitro bioassay available on a world wide basis as quickly as possible, the results of that study were published in 1987. It is significant that Parte and Juneja do not cite that reference (Waites, Bialy, Gordon, Findlay, de Jong, Robertson, Schwartz and Storring 1987).A formal international collaborative study under the auspices of the World Health Organization was initiated in 1988, this time with ten independent expert laboratories in eight countries and again assessing the biological activity of 86/690 with a number of other ampouled or local preparations of different inhibins in in vitro bioassays. A detailed report of that study was drafted in 1990 and circulated to all the participants (who included scientists from the same address as Parte and Juneja) to invite their comments. The assigned potency of 2000 International Units (to maintain continuity with the biological potency originally adopted for its research use) was agreed by the participants who recommended to the WHO Expert Committee on Biological Standardisation that ampoule code 86/690 appeared suitable to serve as a biological standard for in vitro bioassay. The publication of the report of that international collaborative study appeared in Journal of Reproduction and Fertility in 1992 (Gaines-Das, Rose and Zanelli 1992) -again, Parte and Juneja do not cite that reference.It is clear that the experimental data presented by Parte and Juneja do not include a positive control such as a test of the responsiveness of their two pituitary cell systems to inhibins other than 86/690 (eg recombinant human inhibin which is now available). The preparation of porcine inhibin in 86/690 was well described in 1987 as being of 1 -3 % purity and of course the possibility of other active components cannot be excluded as discussed further in reference (Gaines-Das, Rose and Zanelli 1992). Nevertheless, this preparation appears to have served the scientific research community well since 1987; some 300 ampoules have been distributed and, to our knowledge, the report by Parte and Juneja is the first to suggest that the preparation does not show a dose dependent inhibition of basal FSH release in a pituitar...

SUMMARY.Prolactin has been reported to be present in cervical mucus at concentrations higher than those found in blood. Our initial findings appeared to confirm this and the material fulfilled criteria of validity generally applied when an immunoassay is employed on a new biological matrix, i.e. parallelism and chromatographic identity. Further experiments demonstrated that prolactin concentrations in cervical mucus were less than 40 mUlL and the prolactin-like immunoreactivity originally detected was due to the action of the enzyme bromelin which was used to liquefy the mucus. Bromelin has a similar molecular weight to prolactin and appeared to digest prolactin tracer and reduce its ability to bind antiserum in a manner paralleling the effect of adding pituitary prolactin.Prolactin has been reported to be present in relatively high concentrations in a number of body fluids including amniotic fluid;' rnilk.i semen" 4 and cervical mucus-where it has been suggested that it may alter the receptivity of the mucus to spermatozoa at mid-cycle by altering the concentration of electrolytes."Many studies have shown that in plasma, prolactin, as identified by radioimmunoassay, is present in three different molecular forms:": 7 the smallest (mol. wt. 22 0(0) corresponding to monomeric prolactin, and the larger forms (mol. wt. 50 000 and greater than 100 000 daltons) to dimer and aggregate or precursor respectively. Size heterogeneity of immunoreactive prolactin has been reported in amniotic fluid," cerebrospinal fluid" and seminal plasma. III This study was originally undertaken to confirm that prolactin was present in cervical mucus and to investigate whether it was present in different molecular weight forms.Samples of cervical mucus were obtained from a seminology laboratory attached to an inferility out-patient clinic. These samples had been collected for sperm antibody investigations and had already been treated with bromelin, a peptidase obtained from plants of the pineapple family, 11 in order to liquefy the mucus and make it easier to pipette.Correspondence: S B Sufi. 316During the course of the investigation it became apparent that this enzyme was interferring in the radioimmunoassay for prolactin. This paper is a report of this effect. Materials and methods SAMPLESSamples of mucus were collected, during the periovulatory period, from subjects having regular menstrual cycles who were attending an infertility clinic. They were instructed to abstain from sexual intercourse for 3 days prior to collection. TREATMENT OF SAMPLESThree methods of pretreatment of samples before assay were investigated. All samples were stored at -20°C after liquefaction. Bromelin treatmentOne vial of bromelin (Blood Transfusion Service, Central Laboratory, Amsterdam, Netherlands) was diluted with 2 mL of 0·88% sodium chloride solution to give a stock solution containing ()·5% bromelin. An equal volume of bromelin solution was added to each sample of cervical mucus and the sample was left to liquefy at ambient temperature. Freezing and thaw...