Development of a simple, rapid sandwich enzyme immunoassay for the measurement of serum rat LH
Article Sidebar
22)How to Cite
87
Abstract
The present study describes the development and validation of a rapid, sensitive, specific and precise enzyme immunoassay (EIA) sandwich suitable for measuring luteinizing hormone (LH) in rat serum. Ninety-six well polystyrene microtiter plates were coated with 100 pl (250 ng/ml) of a well-characterized monoclonal antibody (518B7, Roser, UC Davis) generated against bovine LH. A polyclonal antiserum raised in rabbits against ovine FSH (G4-215B, Papkoff) was conjugated to sodium periodate-activated horseradish peroxidase (HRP), and used as the second antibody of the sandwich assay. This anti-ovine FSH antiserum cross-reacted more than 200 % with rat LH. Standards (r-LH-RP-3, NIADDK, range 0 pg/well to 2.5 ng/well or 100 p.1) diluted in a 3(N-Morpholino) propane sulfonic acid (MOPS) buffer, or serum, were incubated with the solid phase antibody for 2 hours. Plates were washed and the anti-oFSH:HRP (100 pl) in MOPS buffer was added and incubated a further 2 hours before a second wash and the addition of the substrate (TMB, 3,3’,5,5’-tetramethylbenzidine dihydrochloride and H2O2). The least detectable concentration of LH was 16.1 ± 1.42 pg/ml. The recovery of known concentrations of LH added to several samples was 93.5 ± 1.70 %. Mean intra-assay and inter-assay coefficients of variation (%) were less than 10 % (n = 20). The anti-FSH:HRP showed less than 8.0 % cross reactivity with rFSH in this LH EIA system. The correlation coefficient (r) of samples analyzed by EIA in parallel with RIA was r = 0.90 (p < 0.001, n = 26). Results showed levels between 105.21 and 633.87 pg/ml. This new LH EIA sandwich offers a stable, rapid, and improved EIA system for the measurement of serum LH concentrations of this species over previously reported methods.The present study describes the development and validation of a rapid, sensitive, specific and precise enzyme immunoassay (EIA) sandwich suitable for measuring luteinizing hormone (LH) in rat serum. Ninety-six well polystyrene microtiter plates were coated with 100 pl (250 ng/ml) of a well-characterized monoclonal antibody (518B7, Roser, UC Davis) generated against bovine LH. A polyclonal antiserum raised in rabbits against ovine FSH (G4-215B, Papkoff) was conjugated to sodium periodate-activated horseradish peroxidase (HRP), and used as the second antibody of the sandwich assay. This anti-ovine FSH antiserum cross-reacted more than 200 % with rat LH. Standards (r-LH-RP-3, NIADDK, range 0 pg/well to 2.5 ng/well or 100 p.1) diluted in a 3(N-Morpholino) propane sulfonic acid (MOPS) buffer, or serum, were incubated with the solid phase antibody for 2 hours. Plates were washed and the anti-oFSH:HRP (100 pl) in MOPS buffer was added and incubated a further 2 hours before a second wash and the addition of the substrate (TMB, 3,3’,5,5’-tetramethylbenzidine dihydrochloride and H2O2). The least detectable concentration of LH was 16.1 ± 1.42 pg/ml. The recovery of known concentrations of LH added to several samples was 93.5 ± 1.70 %. Mean intra-assay and inter-assay coefficients of variation (%) were less than 10 % (n = 20). The anti-FSH:HRP showed less than 8.0 % cross reactivity with rFSH in this LH EIA system. The correlation coefficient (r) of samples analyzed by EIA in parallel with RIA was r = 0.90 (p < 0.001, n = 26). Results showed levels between 105.21 and 633.87 pg/ml. This new LH EIA sandwich offers a stable, rapid, and improved EIA system for the measurement of serum LH concentrations of this species over previously reported methods.
Authors
References
Abdul-Ahad, W. G. and Gosling, J. P. (1985): Ir. J. Med. Sci., 154, 284.
Abdul-Ahad, W. G. and Gosling, J. P. (1987): J. Reprod. Fert., 80, 653-661. https://doi.org/10.1530/jrf.0.0800653
Abraham, G. E. (1975): In "Steroid Immunoassay" (Cameron, E. H.D., Hillier, S.G., Griffiths, K., eds). Alpha-Omega Publishers Ltd., Cardiff, Wales, pp 67-78.
Anouassi, A. and Combarnous, Y. (1991): J. Reprod. Fert., 93, 409-414. https://doi.org/10.1530/jrf.0.0930409
Bieglmayer, Ch. and Fischl, F. (1987): J. Clin. Chem. Clin. Biochem., 25, 747-750.
Bieglmayer, Ch., Fischl, F and Janisch, H. (1990): FertiL SteriL, 53, 842-846. https://doi.org/10.1016/S0015-0282(16)53519-5
Clough, K. M., Cole, F. X., Seaver, S. S., Vesprini, A., Kuo, A. Y. and Lasley, B. L. (1992): Fertil. SteriL, 57, 1241-1246. https://doi.org/10.1016/S0015-0282(16)55081-X
Feldkamp, C. S. and Smith, S. W. (1992): In "Immunoassays: a practical guide" (Chan, D.W., ed.). Academic Press Inc., New York, pp 46-96.
Fernandez-Galaz, C. (1989): Medical Thesis, Facultad de Medicina, UCM, Spain.
Haavisto, A. M., Pettersson K., Bergendahl, M., Perheentupa, A., Roser, J. F. and Huhtaniemi, I. (1993): Endocrinology, 132, 1687-1691. https://doi.org/10.1210/endo.132.4.8462469
Khatkhatay, M. L, Desai, M. P., Sankolli, G. M. and Joshi, U. M. (1992): Eur. J. Clin. Chem. Clin. Biochem., 30, 297-300. https://doi.org/10.1515/cclm.1992.30.5.297
Matteri, R. L., Roser, J. F., Baldwin, D. M., Lipovetsky, V. and Papkoff, H. (1987): Domestic Anim. Endocrinol., 4, 157-165. https://doi.org/10.1016/0739-7240(87)90011-7
Munro, C. J. and Lasley, B. L. (1988): In "Nonradiometric assays: Technology and Application in Polipeptide and Steroid Hormone Detection" (Albertson B.D., Haseltine F.P., eds) Alan R. Liss Inc., New York, pp 289-329.
Mutayoba, B. M., Meyer, H. H. D., Schams, D. and Schallenberger, E. (1990): Acta Endocrinol., 122, 227-232. https://doi.org/10.1530/acta.0.1220227
Niswender, G. D., Reichert Jr., L. E., Midgley, A. B. and Nalbandov, A. V. (1969): Endocrinology, 84, 1166-1173. https://doi.org/10.1210/endo-84-5-1166
Rahe, C. H., Owens, R. E., Fleeger, J. L., Newton, H. J. and Harms, P. G. (1980): Endocrinology, 107, 498-503. https://doi.org/10.1210/endo-107-2-498
Rodbard, D. (1974): Clin. Chem., 20, 1255-1270. https://doi.org/10.1093/clinchem/20.10.1255
Snook, R. B., Saatman, R. R. and Hansel, W. (1971): Endocrinology, 88, 678-686. https://doi.org/10.1210/endo-88-3-678
Spearow, J. L. and Trost, B. A. (1987): Biol Reprod., 37, 595-605. https://doi.org/10.1095/biolreprod37.3.595
Stupnicki, R. and Madej, A. (1976): Endocrinology, 68, 6-13. https://doi.org/10.1677/joe.0.0680013
Vilja, P. (1990): Clin. Chem., 36, 1897-1901. https://doi.org/10.1093/clinchem/36.11.1897
Wilson, M. B. and Nakane, P. K. (1978): In "Immunofluorescence and related staining techniques" (Holubar, K. and Wick, G., eds). Elsevier/ North-Holiand Biomed. Press, Amsterdam.pp. 215-224.
Metrics
|
|