60+ Years of In Vivo Bioassay at Los Alamos

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

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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

Journal of Endocrinology ◽

10.1677/joe.0.0910353 ◽

1981 ◽

Vol 91 (2) ◽

pp. 353-362 ◽

Cited By ~ 29

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.

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Quantification of follicle stimulating hormone (follitropin alfa): is in vivo bioassay still relevant in the recombinant age?

Current Medical Research and Opinion ◽

10.1185/030079902125001344 ◽

2003 ◽

Vol 19 (1) ◽

pp. 41-46 ◽

Cited By ~ 54

Author(s):

R. Driebergen ◽

G. Baer

Keyword(s):

Follicle Stimulating Hormone ◽

Follitropin Alfa ◽

In Vivo Bioassay ◽

Stimulating Hormone

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RhBMP-2 and -7 combined with absorbable collagen sponge carrier enhance ectopic bone formation: An in vivo bioassay

Asian Biomedicine ◽

10.5372/1905-7415.0501.010 ◽

2011 ◽

Vol 5 (1) ◽

pp. 85-92

Author(s):

Kanok Preativatanyou ◽

Sittisak Honsawek

Keyword(s):

Bone Formation ◽

Synergistic Effect ◽

Expression System ◽

Collagen Sponge ◽

In Vivo Studies ◽

Blood Vessel Invasion ◽

New Bone Formation ◽

Absorbable Collagen Sponge ◽

In Vivo Bioassay

Abstract Background: Recombinant human bone morphogenetic proteins (rhBMPs) have been characterized especially chondrogenic and osteogenic activity both in vitro and in vivo studies. However, delivery of more than one growth factor by sustained release carrier to orthopedic site has yet been questionable in terms of efficacy and synergism. Objective: Evaluate osteoinductivity and synergistic effect of rhBMP-2 and -7 using absorbable collagen sponge (ACS) carrier system in vivo. Methods: cDNA of BMP-2 and -7 active domains were cloned and expressed in Escherichia coli BL21 StarTM (DE3) using pRSETc expression system. Then, the purified rhBMPs were loaded onto ACS and evaluated by in vivo rat subcutaneous bioassay. Two and eight weeks postoperatively, all treated groups were histologically verified for evidence of new bone formation and neovascularization by hematoxylin-eosin staining and light microscopy. Results: The Wistar rat treated with rhBMP-2 or -7/ACS exhibited new bone formation, compared to ACS control. The group treated with ACS supplemented with both rhBMP-2 and -7 significantly showed the osteoid matrix very well-organized into trabeculae-like structure with significant blood vessel invasion. Conclusion: The osteogenic induction of rhBMPs was combined with ACS carrier in the in vivo bioassay. In addition, the combination of both two potent recombinant osteoinductive cytokines, rhBMP-2 and -7, with ACS carrier demonstrated synergistic effect and might be a more promising and effective choice for therapeutic applications.

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

Acta Endocrinologica ◽

10.1530/acta.0.1130017 ◽

1986 ◽

Vol 113 (1) ◽

pp. 17-22 ◽

Cited By ~ 30

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.

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