A Sandwich Enzyme-Linked Immunoabsorbent Assay for Measurement of Gonadotropin-Releasing Hormone-Toxin Conjugates
Authors: Yang, Wei-Hsiung1; Allen, Matt C.1; Wieczorek, Maciej2; Michael Glode, L.2; Nett, Terry M.1
Source: American Journal of Reproductive Immunology, Volume 55, Number 3, March 2006 , pp. 208-216(9)
Publisher: Blackwell Publishing
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Abstract:
Problem Biological effectiveness of targeted cytotoxins is dependent on their stability, circulating half-life, receptor binding ability, and cytotoxicity. The objective of this study was to compare stability of gonadotropin-releasing hormone (GnRH)-toxin conjugates made with disulfide linkers to those using a maleimidodibutyryl (mb) linkage. Method of study We developed a sandwich enzyme-linked immunoabsorbent assay recognizing both GnRH analog and cytotoxin to ensure the conjugate measured was intact. Anti-D-Leu6-GnRH was used for capture and anti-pokeweed antiviral protein (anti-PAP) or anti-RNase for quantification. Specificity was verified by lack of reactivity with ovine FSH and LH, PAP, RNase, and D-Lys6-GnRH. Results Conjugates prepared using disulfide linkages were not stable in serum in vitro (half-lives <10 min), whereas mb conjugates had half-lives >2 hr. Clearance of mbGnRH-PAP from the circulation of sheep was rapid (t1/2 <20 min). Conclusion The assays were found to be specific, sensitive and accurate for measurement of GnRH-toxin conjugates in vitro and in vivo.Keywords: Enzyme-linked immunoabsorbent assay; GnRH; pokeweed antiviral protein; RNase
Document Type: Research article
DOI: 10.1111/j.1600-0897.2005.00350.x
Affiliations: 1: Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA 2: Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA
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