A comparison of preparations of highly purified human pituitary luteinizing hormone: differences in the luteinizing hormone potencies as determined by in vivo bioassays, in vitro bioassay and immunoassay

1982 ◽  
Vol 101 (3) ◽  
pp. 339-347 ◽  
Author(s):  
P. L. Storring ◽  
A. A. Zaidi ◽  
Y. G. Mistry ◽  
Monica Lindberg ◽  
Bridget E. Stenning ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
In Vitro Bioactivity ◽  
In Vitro Bioassay ◽  
Response Curves ◽  
Human Pituitary ◽  
International Reference ◽  
Reference Preparation ◽  
In Vivo Bioassay

Abstract. The LH potencies of 12 preparations of highly purified human pituitary LH, from 6 laboratories, were estimated by 2 in vivo bioassays and an in vitro bioassay in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (coded 69/104); and by immunoassay in terms of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay (IRP; coded 68/40). The LH potencies varied between preparations, including the IRP (68/40), from 864 to 5740 IU/mg by seminal vesicle weight gain (SVW) assay; from 1510 to 11500 IU/mg by ovarian ascorbate depletion (OAAD) assay; from 4490 to 14500 IU/mg by in vitro (testicular interstitial-cell testosterone production) bioassay; and from 2030 to 9180 IU/mg by immunoassay. Estimates of protein content were based on the assumption that the absorbance of LH at 280 nm (A 1% 1 cm) was 6.0. The LH potency of most preparations was highest by in vitro bioassay and lowest by SVW assay. The correlation between activities determined by SVW and OAAD assays was more marked than that between estimates by OAAD assay and in vitro bioassay; there was no correlation between estimates by SVW assay and in vitro bioassay. The slopes of the log dose-response curves of preparations in the OAAD assay were positively correlated with their potencies by OAAD assay and negatively correlated with the slopes of their log dose-response curves in the SVW assay. The qualitative differences between preparations are considered to be a reflection of the heterogeneity of LH and of its modification by different purification procedures. The present data, together with the different patterns of heterogeneity found in some of these preparations by isoelectric focusing in a separate study, suggest that the more basic molecular forms of LH, which are preferentially purified during the isolation of LH free from FSH and TSH, have shorter plasma survival times than the more acidic forms. The LH immunoreactivities of all preparations were significantly correlated with their potencies estimated by each of the in vivo bioassays but not with those estimated by in vitro bioassay. The ratios of in vitro bioactivity (in terms of IRP (68/40)): immunoreactivity varied between preparations from 0.53–1.5. The FSH content of each preparation was less than 2% (w/w) by bioassay and immunoassay. Most preparations were more potent by in vitro bioassay than by in vivo bioassay, which contrasted with, and complemented, findings for purified FSH preparations. This indicated that, as in the case of LH, the more basic molecular species of FSH are associated with lower ratios of in vivo: in vitro bioactivity than are the more acidic species. This study provides the most comprehensive comparison available of the activities of purified preparations of LH isolated from frozen and acetone-dried human pituitary glands in different experienced laboratories. These data are needed for selecting material for an international reference preparation of LH for immunoassay on the basis of high LH potency by in vivo bioassay, recommended by the WHO as a criterion for the identity of the hormone and for its freedom from contaminants. The consequences of the heterogeneity of LH are considered for the purification of the reference material and for the suitability of the latter for the various types of specimens which require LH assays.

A COMPARISON OF PREPARATIONS OF HIGHLY PURIFIED HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE: DIFFERENCES IN THE FOLLICLE-STIMULATING HORMONE POTENCIES AS DETERMINED BY IN-VIVO BIOASSAY, IN-VITRO BIOASSAY AND IMMUNOASSAY

1981 ◽  
Vol 91 (2) ◽  
pp. 353-362 ◽  
Author(s):  
P. L. STORRING ◽  
A. A. ZAIDI ◽  
Y. G. MISTRY ◽  
BERIT FRÖYSA ◽  
BRIDGET E. STENNING ◽  
...  
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
In Vitro Bioassay ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Potency ◽  
Reference Preparation ◽  
In Vivo Bioassay

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A1%1 cm 280 = 10. The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH. Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay. The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

THE INTERNATIONAL REFERENCE PREPARATION OF HUMAN PITUITARY LUTEINIZING HORMONE, FOR IMMUNOASSAY

1978 ◽  
Vol 88 (2) ◽  
pp. 250-259 ◽  
Author(s):  
P. L. Storring ◽  
D. R. Bangham ◽  
P. Mary Cotes ◽  
Rose E. Gaines ◽  
S. L. Jeffcoate
Keyword(s):  
Luteinizing Hormone ◽  
Reproductive Organ ◽  
Expert Committee ◽  
Confidence Limits ◽  
Binding Assays ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Standardization ◽  
Reference Preparation

ABSTRACT 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.

EFFECT OF STORAGE ON THE BIOACTIVITY OF TWO INTERNATIONAL REFERENCE PREPARATIONS OF HUMAN PITUITARY LUTEINIZING HORMONE IN VITRO

1979 ◽  
Vol 81 (1) ◽  
pp. 153-155 ◽  
Author(s):  
D. M. ROBERTSON ◽  
BERIT FRÖYSA
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Interstitial Cell ◽  
Saline Solution ◽  
Plasma Albumin ◽  
Human Pituitary ◽  
International Reference ◽  
Bovine Plasma ◽  
Reference Preparation

There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at −70 °C in a buffered 0·8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at −20 °C in either 0·1 or 1% BPA, or at −70 °C in the presence of 0·1% BPA showed a small but significant decrease (∼ 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at −70 °C in the presence of 1% BPA.

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

1989 ◽  
Vol 123 (2) ◽  
pp. 275-293 ◽  
Author(s):  
P. L. Storring ◽  
Gaines Das R. E.
Keyword(s):  
Geometric Mean ◽  
In Vitro Bioassay ◽  
International Standard ◽  
Receptor Assay ◽  
Specific Activities ◽  
In Vivo Bioassay ◽  
Ranking Order ◽  
Receptor Assays

ABSTRACT The International Standard for Pituitary FSH (IS; in ampoules coded 83/575) was assayed in terms of the Second International Reference Preparation of Human Pituitary FSH and LH for Bioassay (IRP 78/549) by 27 laboratories in 13 countries using bioassays, receptor assays and immunoassays. Estimates of the FSH content of the IS by in-vivo bioassay were homogeneous both within and between laboratories and gave a combined geometric mean (with 95% fiducial limits) of 79·9 (74·6–85·4) i.u./ampoule. Estimates by different in-vitro bioassays and receptor assays were also homogeneous between assays and laboratories, and gave a combined geometric mean (with 95% fiducial limits) of 31·2 (28·8–33·9) i.u./ampoule. However, estimates by the 19 different immunoassay systems were heterogeneous and varied between 5 and 31 i.u./ampoule. The material in ampoules coded 83/575 was established by the World Health Organization as the International Standard for Pituitary FSH. It was assigned a unitage of 80 i.u./ampoule on the basis of its calibration by in-vivo bioassay, because this assay best identifies and defines the hormone. However, the introduction of the new IS will necessitate the recalibration of immunoassay kits. FSH 84/530, prepared in the same way as the IS from the same FSH preparation, did not differ significantly from the IS in any of the assay systems studied and appeared to be equally suitable as a standard. Four highly purified preparations of human FSH (FSH A–D), differing in their isoform compositions and in their in-vivo: in-vitro bioactivity ratios, were also studied. The ranking order of the specific activities of FSH A–D by in-vitro bioassays paralleled their order by receptor assays and the order of their content of FSH isoforms with isoelectric points > 4·5. (Potency estimates of FSH B and C in terms of the IS were greater by receptor assay than by in-vitro bioassay.) The overall ranking order of the specific activities of FSH A–D by immunoassays was different. Contrary to expectation, estimates in terms of the IS of specific activities by immunoassay differed more between preparations than those by in-vitro bioassay or receptor assay. Differences in specificity between immunoassay systems were demonstrated not only in the calibration of the IS in terms of the crude FSH of IRP 78/549 but also in the comparisons of the highly purified FSH in the IS and FSH A–D. The differences in the immunoreactivities and bioactivities of FSH preparations differing in their isoform compositions greatly complicate the standardization of assays for FSH. Journal of Endocrinology (1989) 123, 275–293

INTERNATIONAL REFERENCE PREPARATION OF HUMAN CHORIONIC GONADOTROPHIN FOR IMMUNOASSAY: POTENCY ESTIMATES IN VARIOUS BIOASSAY AND PROTEIN BINDING ASSAY SYSTEMS; AND INTERNATIONAL REFERENCE PREPARATIONS OF THE α AND β SUBUNITS OF HUMAN CHORIONIC GONADOTROPHIN FOR IMMUNOASSAY

1980 ◽  
Vol 84 (2) ◽  
pp. 295-310 ◽  
Author(s):  
P. L. STORRING ◽  
ROSE E. GAINES-DAS ◽  
D. R. BANGHAM
Keyword(s):  
Geometric Mean ◽  
Collaborative Study ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Considerable Heterogeneity ◽  
International Reference ◽  
Reference Preparation ◽  
Α And Β Subunits

The preparation and nature of the International Reference Preparation of Human Chorionic Gonadotrophin (HCG) for Immunoassay (IRP), as well as that of a second batch of ampoules (HCG 75/589) prepared identically from the same HCG preparation, are described. A collaborative study of these materials was carried out by 11 laboratories in eight countries, using different bioassay and immunoassay methods. Using the various in-vivo and in-vitro bioassays and receptor assays, the mean log potency estimates for each method within each laboratory of the HCG content of ampoules of the IRP, in terms of the Second International Standard of Human Chorionic Gonadotrophin for Bioassay (IS), were homogeneous and gave an overall weighted geometric mean (95% confidence limits) of 650 (632–669) International Units (i.u.)/ampoule. There was considerable heterogeneity of potency estimates of the IRP in terms of the IS both within and between many of the immunoassay systems (reflecting the impurity of the IS), and hence attempts to calibrate the IRP with immunoassay systems of different specificities were invalid. Immunoassay estimates of the HCG content of preparations of serum and urine, in terms of the IRP, showed considerable heterogeneity between assay systems (although the degree of this heterogeneity was no greater than that observed using the IS as standard), but the ranking order between preparations was consistent. Confirmation was obtained that contamination of the IRP with HCG-α and HCG-β subunits was insignificant. Accelerated degradation studies of the IRP stored at increased temperatures suggested that its stability under normal storage conditions would be satisfactory. It was agreed that the IRP was suitable to serve as an international reference preparation for immunoassay, and it was assigned a unitage of 650 i.u./ampoule on the basis of bioassay calibration. Since the ampoules of HCG (75/589) did not differ significantly from the IRP in any of the assay systems studied, it appeared to be equally suitable as a reference preparation. The International Reference Preparations of the α and β Subunits of Human Chorionic Gonadotrophin for Immunoassay are also described.

Influence of the assay method used on the selection of the most active forms of FSH from the human pituitary

1986 ◽  
Vol 113 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Leif Wide ◽  
Bruce Hobson
Keyword(s):  
Biological Properties ◽  
Dominant Factor ◽  
Assay Method ◽  
Major Influence ◽  
Human Pituitary ◽  
Vitro Assay ◽  
Molecular Charge ◽  
In Vivo Bioassay

Abstract. The biological properties of different forms of human pituitary FSH, varying in their molecular charge, were investigated. FSH in two individual human pituitaries and a pool of 30 human pituitaries was extracted and subjected to electrophoresis. From each electrophoresis 14 consecutive fractions with the highest RIA activity were examined with in vitro and in vivo bioassays. The in vitro assay was based upon the estimation of oestradiol produced by cultured Sertoli cells from 10 day old rats. The in vivo bioassay was an hCG augmented test using immature female mice injected on 3 consecutive days. The increase in ovarian weight was the index of response. Both in the individual and in the pooled pituitary material the less negatively charged forms had the highest activity in the in vitro bioassay. In contrast, the more negatively charged forms had the highest activity in the in vivo bioassay. Forms of FSH from each of the two individual pituitary extracts were pooled according to their migration rate and injected iv into mice. The amount of FSH remaining in the circulation of the mouse after 1 h was related to the molecular charge. The highest value was obtained with the pool containing the more negatively charged forms of the hormone. The results indicate that the disappearance rate of the FSH molecule is a dominant factor in the in vivo bioassay. A consequence of these observations will be that the assay method chosen to monitor the purification of FSH will have a major influence on the biological properties of the final preparation.

Heterogeneity of rat FSH by chromatofocusing: studies on in-vitro bioactivity of pituitary FSH forms and effect of neuraminidase treatment

1985 ◽  
Vol 105 (1) ◽  
pp. 17-27 ◽  
Author(s):  
W. F. P. Blum ◽  
G. Riegelbauer ◽  
D. Gupta
Keyword(s):  
Apparent Molecular Weight ◽  
Maximum Response ◽  
Dependent Manner ◽  
In Vitro Bioactivity ◽  
Response Curves ◽  
Neuraminidase Treatment ◽  
Rat Pituitary ◽  
Exclusion Chromatography ◽  
Reference Preparation

ABSTRACT This study concerned the resolution of rat pituitary FSH utilizing chromatofocusing. Among the 11 components resolved and positively identified, ten had apparent isoelectric points (pI) between 3·1 and 5·1. Approximately 1% of pituitary FSH eluted at pH 9·4. Treatment with varying amounts of neuraminidase followed by refocusing generated FSH components of higher pI values. Treatment with other glycosidases did not alter the elution characteristics in chromato-focusing, while exclusion chromatography established an inverse relationship between apparent molecular weight and pi. Dose–response curves of various FSH components and of the reference preparation in the current radioimmunoassay system were parallel to each other. A study of their in-vitro bioactivity, utilizing granulosa cells which produce a plasminogen activator due to FSH in a dose-dependent manner, provided the following evidence: increased acidity of the components led to (1) an increase of maximum response and (2) an increase of the dose necessary for half-maximum response. Considering the observed alterations in the hetereogeneity of FSH with changing physiological states of the animal, it is concluded that qualitative changes of the FSH molecule are perhaps involved in a modulatory role in the biopotencies of the hormone. J. Endocr. (1985) 105, 17–27

IMMUNOLOGICAL CROSS REACTION BETWEEN HUMAN CHORIONIC GONADOTROPHIN AND HUMAN PITUITARY GONADOTROPHINS

1966 ◽  
Vol 53 (3) ◽  
pp. 420-428 ◽  
Author(s):  
C. Robyn ◽  
P. O. Hubinont ◽  
E. Diczfalusy
Keyword(s):  
Luteinizing Hormone ◽  
Follicle Stimulating Hormone ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Human Pituitary ◽  
Specific Antisera ◽  
Human Menopausal Gonadotrophin ◽  
Immunological Cross Reaction

ABSTRACT Immunologically mono-specific antisera prepared against human chorionic gonadotrophin (HCG) preparations completely neutralized in vitro as well as in vivo the luteinizing hormone (LH) and also the follicle-stimulating hormone (FSH) activity of both human hypophyseal gonadotrophin (HHG) and human menopausal gonadotrophin (HMG) preparations.

The International Reference Preparation of Tetracosactide for Bioassay: characterization and estimation of its (1–24)corticotrophin-tetracosapeptide content by physicochemical and biological methods

1984 ◽  
Vol 100 (1) ◽  
pp. 51-60 ◽  
Author(s):  
P. L. Storring ◽  
G. Witthaus ◽  
R. E. Gaines Das ◽  
W. Stamm
Keyword(s):  
High Performance ◽  
Storage Conditions ◽  
World Health ◽  
Expert Committee ◽  
Adrenocortical Cell ◽  
International Reference ◽  
Cell Assay ◽  
Reference Preparation

ABSTRACT The preparation and nature of the International Reference Preparation of Tetracosactide for Bioassay (IRP; in ampoules coded 80/590) are described. The IRP was studied by six laboratories in five countries using in-vivo and in-vitro bioassays and various physicochemical methods. The bulk (1–24)corticotrophin-tetracosapeptide (batch 000179) from which the IRP was prepared contained 10·4% (w/w) acetic acid and 8·3% (w/w) water; its (1–24)corticotrophin-tetracosapeptide content was estimated to be 71·7% (w/w) by amino acid analysis, 74·2% (w/w) by high performance liquid chromatography (HPLC) and 77·5% (w/w) by spectrophotometry. (1–24)Corticotrophin-tetracosapeptide accounted for more than 90% (w/w) of the total peptide in the IRP as judged by HPLC, thin-layer chromatography, carboxymethyl-cellulose chromatography, isoelectric focusing (IEF) and electrophoresis. The homogeneity of the peptide in the IRP was similar by all methods to that in batch 000179 from which it was prepared. The (1–24)corticotrophin-tetracosapeptide content of the IRP (with 95% confidence limits), in terms of batch 000179, was found to be 491 μg/ampoule by HPLC and spectrophotometry, 473 (433–513) μg/ ampoule by IEF and 505 (473–539) μg/ampoule by the in-vitro rat adrenocortical cell assay. A comparison in the same bioassay system of the IRP with a laboratory house standard of (1–24)corticotrophin-tetracosapeptide, which originated from a different manufacturer, gave similar results. Accelerated thermal degradation studies of the IRP by adrenocortical cell assay, HPLC and IEF suggested that more than 99·9% of its original content of (1–24)corticotrophin-tetracosapeptide would remain after 10 years under normal storage conditions of − 20 °C in the dark. Bioassay estimates of samples of the IRP which had undergone significant degradation were higher than estimates by HPLC, indicating that molecular species other than (1–24)corticotrophin-tetracosapeptide contributed to their corticotrophic activity. The corticotrophic activity of the IRP was demonstrated by cytochemical bioassay and by in-vivo bioassay as well as by the adrenocortical cell assay. After consideration of these data, the Expert Committee on Biological Standardization of the World Health Organization established the ampouled preparation, coded 80/590, as the International Reference Preparation of Tetracosactide for Bioassay and assigned to it a potency of 490 i.u./ampoule; thus the i.u. is represented by 1 μg (1–24)corticotrophin-tetracosapeptide. J. Endocr. (1984) 100, 51–60

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity

1997 ◽  
Vol 9 (5) ◽  
pp. 501 ◽  
Author(s):  
Patrick G. Burgon ◽  
Peter G. Stanton ◽  
Kim Pettersson ◽  
David M. Robertson
Keyword(s):  
Sialic Acid ◽  
Luteinizing Hormone ◽  
Acid Content ◽  
Specific Activity ◽  
Sialic Acid Residue ◽  
In Vitro Bioactivity ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Before And After

To establish whether sialic acid content is responsible for an observed 7–8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13–24 pituitary hLH isoform preparations of pI 7·03–8·98 in terms of sialic acid content (1–5 sialic acid residues per LH molecule), bioactivity in vitro (4030–30 000 I.U. mg-1), radioreceptor activity (6420–25 400 I.U. mg-1) and hLH immunoactivity (2900–4400 to 18 300–27 300 I.U. mg-1). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.

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