Immunological and physiological validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of growth hormone in goat (Capra hircus) plasma

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Abstract

The current study describes the optimization and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of growth hormone (GH) in blood plasma of goats, Capra hircus. A microtiter plate based competitive assay was developed using a capture ELISA for anti-ovine GH and biotinylated ovine GH. Ovine GH standards were prepared in charcoal–dextran stripped goat plasma. The assay was optimized in terms of sensitivity, specificity and precision. The sensitivity of the assay was 50 pg GH/100 μl, which corresponds to 0.5 ng/ml plasma. A dose–response inhibition curve resulting from serially diluted goat plasma was parallel to the standard curve using ovine GH and thus it confirmed the similar specificity of ovine GH standard and endogenous GH in goat plasma for ovine GH antibody. The precision of the assay, i.e., the intra- and inter-assay coefficients of variations (for repeatability and reproducibility, respectively) for the pooled plasma samples containing two GH concentrations (32 and 2 ng/ml) were 6.72%, 11.2%, 7.1% and 12.8%, respectively. To physiologically validate the assay, goats were treated with human growth hormone-releasing factor (hGRF) and there was an immediate rise in plasma GH levels in hGRF-treated goats. This ELISA is economic, safe, quick (requires only 48 h), convenient and the first competitive enzyme immunoassay described for caprine GH.

Introduction

Growth hormone (GH), also named somatotropin, is obligatory for growth and development, but is also involved in the process of sexual differentiation and pubertal maturation and it participates in gonadal steroidogenesis, gametogenesis and ovulation. It also has additional roles in pregnancy and lactation (Hull and Harvey, 2001). GH is necessary for fetal and postnatal somatic growth (Etherton and Kensinger, 1984). GH is thus an important regulatory hormone in several physiological processes in animals. Commonly, GH is estimated in blood plasma by employing radioimmunoassay (RIA) which requires 125I as the trace isotope. Although this method is reliable and accurate, radioisotope use restricts assay use to specialized laboratories. The availability of 125I for radio iodination is also a limitation for GH RIA. Moreover, the short half-life of 125I isotope limits its use in the RIA. Recently, a simple and sensitive enzyme immunoassay (EIA) procedure was validated to estimate luteinizing hormone (LH) in goat plasma (Haldar et al., 2009). While EIA procedures have been developed for the estimation of GH in cattle (Hennies and Holtz, 1993, Roh et al., 1997), buffalo (Prakash et al., 2003, Mishra et al., 2007), fish (Fukada et al., 1997, Lal and Singh, 2005) and human (Strasburger et al., 1996, Moura et al., 2004), no immunoassay has thus far been optimized for caprine plasma GH. The objective of this study was to optimize an EIA technique for estimation of GH in blood plasma of goats. The physiological validation of the method was tested by estimating GH in blood plasma of goats after a challenge by human growth hormone releasing factor (GRF).

Section snippets

Chemicals

Ovine GH (NIDDK-oGH-15, Lot # AFP 9220A), rabbit antiserum to ovine GH (NIDDK-anti-oGH-IC-1, Lot # AFP 2861453P) and human growth hormone- releasing factor (hGRF 1-29 amide, BACHEM, Lot # FGRF 1299701) procured from Dr. A.F. Parlow, National Hormone Peptide Program (NHPP), Harbor-UCLA Medical Centre, Carson, CA, USA were used in the present study. Goat IgG anti-rabbit IgG (second antibody), prepared by Anandlaxmi and Prakash (2001), was obtained from Dr. B.S. Prakash, National Dairy Research

Optimization of anti-oGH and biotinylated oGH concentrations

A two-dimensional titer determination test for the optimum dilution of biotinylated-oGH and the anti-oGH antiserum was performed. Initially, antibody dilutions ranging from 1:10,000 to 1:6.4 million and a biotinylated-oGH dilution of 1:500 to 1:16,000 were tested. However, the optical density (OD) for most of the combinations was low. A total of 7 titer tests were then run repeatedly, changing the antibody dilutions (ranging between 1:2500 and 1:1.6 million) with the biotinylated-oGH dilutions

Discussion

The heterogeneity of molecular isomers of circulating GH, the epitope specificity of the antibodies and the interference from matrix components such as GH binding protein in plasma are among the reasons why standardizing GH immunoassay is difficult (Popii and Baumann, 2004, Bidlingmaier and Strasburger, 2007). To the best of our knowledge, this is the first report describing the optimization of an ELISA technique for the estimation of goat plasma GH levels using a competitive capture ELISA with

Conclusion

A simple and highly sensitive ELISA technique has been optimized and validated on second antibody coated 96-well microtiterplate using biotin–streptavidin peroxidase amplification system in a competitive assay. The ELISA technique for GH estimation described here offers a reliable alternative to RIA. It is safe, quick and convenient. The greatest advantage in ELISA technique is that biotinylated GH has a much longer shelf life (several years) as compared to iodinated-GH (60 days). Thus, the

Acknowledgements

The authors gratefully acknowledge the financial supports of ICAR, New Delhi, India for providing National Fund for Basic, Strategic and Frontier Application Research in Agriculture (Project no. NFBSRA/PCN/AP-06/2006-07). The authors wish to thank to Dr. A.F. Parlow, National Hormone Peptide Program (NHPP), Harbor-UCLA Medical Centre, Carson, CA, USA for providing ovine GH, ovine GH antiserum and hGRF. The necessary help and cooperation extended by the Director, ICAR Research Complex for North

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