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BOR - Papers in Press, published online ahead of print September 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033654
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Submitted June 25, 2004
Returned for revision July 12, 2004
Accepted August 18, 2004

Pituitary


Validation of an Ultrasensitive and Specific Immunofluorometric Assay for Mouse Follicle-Stimulating Hormone

M. Jimenez , J. A. Spaliviero , A. J. Grootenhuis , J. Verhagen , C. M. Allan , and D. J. Handelsman *

* To whom correspondence should be addressed. E-mail: djh{at}anzac.edu.au.

Abstract
Sensitive and specific measurement of FSH is critical to reproductive biology research and the increasing availability of transgenic mouse models have created a need for a robust, sensitive and specific mouse FSH assay. This study has evaluated a time-resolved immunofluorometric assay (IFMA) for mouse FSH using monoclonal antibody to human FSH {beta} as a capture antibody and a biotinylated polyclonal antibody to rat {alpha} subunit as detection probe with signalling amplified by europium-labelled strepavidin. The mouse FSH IFMA lowered the detection limit 34 fold (5 vs 170 pg per sample) compared with standard mouse FSH radioimmunoassay (RIA). The mouse FSH IFMA demonstrated parallelism of response to dilutions of castrate mouse serum and rat FSH but no cross-reactivity with human FSH, mouse or human LH whereas the RIA demonstrated non-parallel cross-reactivity with human FSH. The IFMA has a wide analytical range with a good precision profile for within and between assay reproducibility. As the IFMA is a sandwich-type assay with strict dimer-specificity by design the lower readings and recovery compared with the RIA when both assays use a pituitary-purified mouse FSH assay standard which contains isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mouse FSH IFMA demonstrated expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mouse FSH in gonadotropin deficient mice. Furthermore, the IFMA measured mouse FSH with fidelity in intact and orchidectomized male mice without any interference from transgenic human FSH. The greatly enhanced sensitivity, specificity and technical convenience of this mouse FSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.

Key words: Testis • Follicle-stimulating hormone • Pituitary hormones • Sertoli cells • Spermatogenesis


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