A sandwich enzyme-linked immunoabsorbent assay for measurement of gonadotropin-releasing hormone-toxin conjugates.
OBJECTIVE
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.


METHODS
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).


CONCLUSIONS
The assays were found to be specific, sensitive and accurate for measurement of GnRH-toxin conjugates in vitro and in vivo.
Yang
Wei-Hsiung
WH
Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Allen
Matt C
MC
Wieczorek
Maciej
M
Michael Glode
L
L
Nett
Terry M
TM
D000818:Animals; D003603:Cytotoxins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000097:blood / Q000276:immunology*; D011817:Rabbits; D012680:Sensitivity and Specificity; D012756:Sheep; D013997:Time Factors
D003603:Cytotoxins; D007987:Gonadotropin-Releasing Hormone