Development of a Sensitive Enzyme-linked Immunosorbent Assay (ELISA) for the Determination of Pulsatile Luteinizing Hormone Secretion in Mice (#225)
The stimulation of luteinizing hormone (LH) from the anterior pituitary gland is regulated through the activation of hypothalamic gonadotrophin releasing hormone (GnRH) neurons. Assessment of pulsatile LH secretion in transgenic mouse models will significantly advance efforts to assess key factors involved in the initiation of pubertal development. Due to blood volume constraints, repeated measurement of circulating levels of LH in mice remains highly challenging. We developed a simple technique for the detection of pulsatile levels of LH in freely moving mice. This was achieved through the development of an Enzyme-linked Immunosorbent Assay (ELISA) for detection of mouse LH in small quantities (2 μl) of whole blood. The specificity and accuracy of this assay was validated following guidelines established by the International Union of Pure and Applied Chemistry (IUPAC). We incorporated an established method for tail-clip blood sample collection to determine circulating levels of LH secretion in 36 whole blood samples collected consecutively over a period of 6 hours. A standard curve was generated following serial dilution of a known amount of mouse LH, and demonstrates accurate detection of LH in the range of 0.0019 to 1.00 ng/ml. The accuracy of LH detection across this range was assessed by spike recovery analysis in whole blood and plasma samples. We observed, on average, 83.75±4.32% and 81.96±3.19% recovery for whole blood and plasma samples, respectively. The upper and lower detection limits of this assay in 50 μl of whole blood (diluted at 1:30) were 30 and 0.057 ng/ml, respectively. Assessment of repeated blood samples collected from postpubertal male C57/Bl6J mice demonstrate peak secretion periods of LH and inter-pulse stable baseline secretion periods. This method provides a sensitive and accurate tool for the assessment of pulsatile LH secretion in mice, and will allow for the reliable assessment of pulsatile LH secretion in transgenic mouse lines.
This work was supported by the Australian National Health and Medical Research Council (NHMRC),collaborative project funding between the University of Queensland and the University of Otago. Ying Wan receives a University of Queensland International Scholarship (UQI) from the University of Queensland.