Heterologous Radioimmunoassay for Llama and Alpaca Luteinizing Hormone with a Monoclonal Antibody, an Equine Standard and a Human Tracer

Abstract

A radioimmunoassay for llama and alpaca LH was developed using a human I¹²⁵LH tracer from a commercial kit, equine LH diluted in human LH free serum as standard, and a monoclonal antibody (518B7) specific for LH but with low species specificity. A 60-min delay in the addition of the tracer and overnight incubation gave a sensitivity of 0.8 μg L⁻¹. The intra-assay coefficient of variation was 37% at 1 μg L⁻¹, declined to 15% at 4 pg L⁻¹ and was below 6% for concentrations up to 32 μg L⁻¹. The inter-assay coefficients of variation for 3 control samples were 20% (2.8 μg L⁻¹), 16% (7.1 μg L⁻¹) and 9.8% (19 μg L⁻¹). In an attempt to increase sensitivity, all tubes were preincubated for 4 h at room temperature before adding the tracer, and the sample volume was increased from 50 μL to 100 μL· (in the standard curve the increased volume was compensated for by human LH free serum). With this protocol, the assay sensitivity was 0.5 μg L⁻¹. The assay was validated clinically and demonstrated increased concentrations of LH after mating in llamas and alpacas. Furthermore, the assay was used to monitor LH responses to a single dose of GnRH in llamas (adult males and females at different ages).