Development and validation of a simple, sensitive enzyme immunoassay for quantification of androstenedione in bull plasma

  • Mallick, Smrutirekha (Dairy Cattle Physiology Division, National Dairy Research Institute) ;
  • Kumar, BS Bharath (Dairy Cattle Physiology Division, National Dairy Research Institute) ;
  • Prakash, BS (Division of Animal Sciences, Indian Council of Agriculture Research) ;
  • Aggrawal, Anjali (Dairy Cattle Physiology Division, National Dairy Research Institute) ;
  • Pandita, Sujata (Dairy Cattle Physiology Division, National Dairy Research Institute)
  • Received : 2014.09.03
  • Accepted : 2014.12.11
  • Published : 2015.04.30


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 $40{\mu}l$ of bull plasma. The androstenedione standards ranged from 0.20 to 200 pg/$40{\mu}l$/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.


Androstenedione;Enzyme immunoassay;Bull;Plasma;Standardisation


  1. Mongkonpunya K, Hafs HD, Convey EM, Tucker HA, Oxender WD. Serum luteinizing hormone, testosterone and androstenedione in pubertal and prepubertal bulls after gonadotropin releasing hormone. J Anim Sci. 1975;40:682-6.
  2. Mondain-Monval M, Bonnin M, Scholler R, Canivenc R. Androgens in peripheral blood of the red fox (Vulpes Vulpes L.) during the reproductive season and the anestrous. J Steroid Biochem. 1979;11:1315-22.
  3. Cook SJ, Rawlings NC, Kennedy RI. Quantitation of six androgens by combined high performance liquid chromatography and radioimmunoassay. Steroids. 1982;40:369-80.
  4. Thorneycroft IH, Riberio WO, Stone SC, Tillson SA. A radioimmunoassay of androstenedione. Steroids. 1973;21:111-22.
  5. Palme R, Mostl E. Biotin-Streptavidin Enzyme Immunoassay for the Determination of Oestrogens and Androgens in Boar Faeces. In: Gorog S, editor. Advances in Steroid Analysis '93. Budapest: Akademiai Kiado; 1994. p. 111-7.
  6. Illera JC, Silvan G, Munro CJ, Lorenzo PL, Illera MJ, Liu IKM, et al. Amplified androstenedione enzyme immunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulfate methods. J Steroid Biochem Mol Biol. 2003;84:377-82.
  7. Ananadlaxmi N, Prakash BS. Production and purification of goat antirabbit IgG. Indian J Dairy Sci. 2001;54:332-4.
  8. Liebermann S, Erlanger BF, Beiesr SM, Agate FJ. Steroids-Protein conjugates: their chemical, immunological and endocrinological properties. Prog Horm Res. 1959;15:165-96.
  9. Meyer HHD. Possibilities to Improve Enzyme Immunoassay (EIA) Techniques and their Application in Animal Production. In: IAEA and FAO Proceedings of an International Symposium On the use of Nuclear Techniques in Studies of Animal Production and Health in Different Environment. 1986. p. 256-62.
  10. Rawlings NC, Hafs HD, Swanson LV. Testicular and blood plasma androgens in Holstein bulls from birth through puberty. J Anim Sci. 1972;34:435-40.
  11. Kiser TE, Milvae RA, Hafs HD, Oxender WD, Louis TM. Comparison of testosterone and androstenedione secretion in bulls given prostaglandin $F2{\alpha}$ or luteinizing hormone. J Anim Sci. 1978;46:436-42.
  12. Moura AA, Erickson BH. Age-related changes in peripheral hormone concentrations and their relationships with testis size and number of Sertoli and germ cells in beef bulls. J Reprod Fertil. 1997;111:183-90.
  13. Abdel Malak Bedair G, Thibier M. Peripheral plasma androstenedione and testosterone concentrations in bulls before and during puberty. J Reprod Fertil. 1979;56:7-10.