Mallick et al. Journal of Animal Science and Technology (2015) 57:13 DOI 10.1186/s40781-014-0035-z
Development and validation of a simple, sensitive enzyme immunoassay for quantification of androstenedione in bull plasma Smrutirekha Mallick1*, BS Bharath Kumar1, BS Prakash2, Anjali Aggrawal1 and Sujata Pandita1
Abstract As an alternative to radioimmunoassay a simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for androstenedione quantification in plasma of Karan Fries bulls using second antibody coating technique. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was performed to analyze androstenedione directly in 40 μl of bull plasma. The androstenedione standards ranged from 0.20 to 200 pg/40 μl /well and the sensitivity of the assay was 5 pg/ml plasma. Serially diluted bull plasma containing high endogenous androstenedione showed good parallelism with bovine androstenedione standard curve. Intra- and inter-assay coefficients of variation (CV) were found to be 8 and 9%, respectively. Peripheral plasma androstenedione concentrations determined in young and adult bull samples ranged between 104–990 pg/ml and 184–2040 pg/ml, respectively. Keywords: Androstenedione, Enzyme immunoassay, Bull, Plasma, Standardisation
Background Androstenedione is the common precursor of male and female sex hormones. Being the predominant steroid in case of young animals, measurement of androstenedione provides a useful marker of androgen biosynthesis. Several radioimmunoassay (RIA) procedures [1-4] for estimation of androstenedione were developed and standardised, however these procedures require an extraction step which entails the use of hazardous solvents. Additionally the use of a radioisotope limits the application to labs which possess license; other disadvantages include proper disposal of radioactive waste, use of expensive instrumentation and requirement of adequate lab space. Although a few enzyme immunoassay (EIA) procedures have been developed [5,6] in recent years, the direct coating of hormone specific antibody on microtitre plate employed by these procedures resulted in extensive usage of the expensive antiserum. Advantages of second antibody coating EIA technique over direct coating technique are reduced time dependent drift, less
expensive, high precision and sensitivity. Hence a study was designed to develop a highly sensitive and specific EIA using second antibody coating technique for androstenedione determination in bovine plasma.
Methods Enzyme immunoassay for androstenedione
The development of a heterologous competitive ELISA for androstenedione determination was done using 96well flat bottomed polystyrene microtitre plates (Greiner Labotrechnik, Germany) using the second antibody coating technique and androstenedione–HRP as a label in unextracted bull plasma. The affinity-purified goat IgG (antirabbit IgG) was developed following the procedure of Anandlaxmi and Prakash . The antiserum used in these assays was highly specific for androstenedione. Percentage cross-reaction of androstenedione antiserum with related steroids at 50% binding was determined. Preparation of hormone-free plasma and enzyme label
* Correspondence: [email protected]
1 Dairy Cattle Physiology Division, National Dairy Research Institute, Karnal 132001, India Full list of author information is available at the end of the article
For preparation of androstenedione-free plasma, blood samples were collected from aged Karan Fries cows. Plasma was separated after centrifugation of blood and
© 2015 Mallick et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Mallick et al. Journal of Animal Science and Technology (2015) 57:13
in-order to remove traces of androstenedione, it was treated with charcoal and dextran mixture as follows: Mixture of activated charcoal (14 g) and dextran T-70 (1.4 g) per 100 ml plasma were taken and the mixture was washed with distilled water by thorough mixing, using a magnetic stirrer for overnight at room temperature. After allowing the mixture to stand for 5 min, the supernatant was discarded and the mixture was again subjected to the washing process for three more times at 8 h intervals to remove all traces of suspended fine charcoal particles. Cow plasma was then added to the washed charcoal–dextran mixture and mixed thoroughly for 2 h at 4°C. Then the mixture was centrifuged at 1500 × g for 1 h at 4°C and the supernatant was filtered to remove suspended particles. The filtrate was again centrifuged at 5000 × g for 1 h at 4°C and the supernatant was re-filtered. The plasma thus obtained was tested for androstenedione concentration in an assay. The concentration of androstenedione in this charcoal dextran stripped plasma was below detectable limit of the assay (