A Sandwich Enzyme‐Linked Immunoabsorbent Assay for Measurement of Gonadotropin‐Releasing Hormone‐Toxin Conjugates

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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.