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A Sandwich Enzyme‐Linked Immunoabsorbent Assay for Measurement of Gonadotropin‐Releasing Hormone‐Toxin Conjugates
Author(s) -
Yang WeiHsiung,
Allen Matt C.,
Wieczorek Maciej,
Michael Glode L.,
Nett Terry M.
Publication year - 2006
Publication title -
american journal of reproductive immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.071
H-Index - 97
eISSN - 1600-0897
pISSN - 1046-7408
DOI - 10.1111/j.1600-0897.2005.00350.x
Subject(s) - conjugate , disulfide linkage , in vivo , in vitro , toxin , rnase p , chemistry , cytotoxicity , enzyme , gonadotropin releasing hormone receptor , receptor , gonadotropin , biochemistry , gonadotropin releasing hormone , hormone , biology , luteinizing hormone , rna , microbiology and biotechnology , cysteine , gene , mathematical analysis , mathematics
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‐Leu 6 ‐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‐Lys 6 ‐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 ( t 1/2 <20 min). Conclusion The assays were found to be specific, sensitive and accurate for measurement of GnRH‐toxin conjugates in vitro and in vivo .