276. The technical and biological validation of an LH assay for use with the Western Grey Kangaroo (Macropus fuliginosis) and Black-flanked Rock Wallaby (Petrogale lateralis lateralis)

Abstract

Methods for the measurement of marsupial LH invariably rely upon the similarity of the LH molecule between different species and usually use anti-ovine or anti-bovine LH antibody and an ovine or bovine labelled LH preparation. Initial attempts to measure plasma LH in the Western Grey Kangaroo with assays using antibodies to 4 different isoforms of ovine LH raised in 7 different rabbits were unsuccessful. An enzymeimmunoassay (EIA) developed for the Asian elephant (Zoo Biology 23:45–63) was then applied to the Western Grey Kangaroo and the Black-flanked Rock Wallaby. This EIA has an anti-bovine-LH monoclonal antibody (518B7 provided by Dr Jan Roser, University of California, Davis, USA), biotinylated ovine LH label and bovine LH standard (NIADDK-oLH-26 and NIH-bLH-B10, both provided by Dr Janine Brown and Nicole Abbondanza, Smithsonian Institute, Front Royal, Virginia USA). Technical validation showed that serial dilution down to 1:8 of plasma from 7 individuals of each species showed parallelism to the assay standard curve, and control samples (1.24–5.30 ng/mL) had between-assay coefficients of variation <9%. Biological validation was achieved by challenging animals with intramuscular GnRH (Fertagyl®, 2.5 µg/kg) and measuring LH before and 25 min after the injection. Significant increases in plasma concentrations of LH (mean ± sem; all P > 0.0005) were seen after GnRH for both the Western Grey Kangaroo (from 5.0 ± 0.8 ng/mL to 9.4 ± 1.2 ng/mL; n = 19) and the Black-flanked Rock Wallaby (from 6.0 ± 0.7 ng/mL to 10.6 ± 0.6 ng/mL; n = 28). In conclusion, this assay can be successfully used to measure LH in these two species.