Abstract
A novel, direct noncompetitive flow injection enzyme immunoassay for α-fetoprotein (AFP) was developed by enhanced chemiluminescence detection. The method was based on off-line incubation of AFP and horseradish peroxidase (HRP)-labeled anti-AFP, and then trapping of the unbound enzyme conjugate by an immunoaffinity column filled with AFP-modified Sepharose. The immunocomplex formed in incubation passed through the column and then was directly detected by a postcolumn chemiluminescence technique. The optimal conditions for the immunoassay procedure and chemiluminescence detection were established. At a 1:10 dilution of enzyme conjugate solution, the linear range for chemiluminescence detection of AFP was from 2.0 to 75 ng/mL with a correlation coefficient of 0.993 and a coefficient of variation of 2.67% at 30 ng/mL. The detection limit was 0.5 ng/mL. This method was flexible, sensitive, and rapid. The immunoaffinity column of 200 µL could be repeatedly used 100 times without a single decrease. The whole assay time including the preincubation step was only 30 min for one sample.
Article PDF
Similar content being viewed by others
References
Yamagata, Y., Katoh, H., Nakamura, K., Tanaka, T., Satomura, S., and Matsuura, S. (1998), J. Immunol. Methods 212, 161–168.
Yusof, Y. A. M., Salleh, M. N., and Abdullah, N. A. (1997), J. Biochem. Mol. Biol. 2, 18–22.
Webb, A., Scott-Mackie, P., Cunningham, D., Norman, A., Andreyev, J., O’Brien, M., and Bensted, J. (1996), Eur. J. Cancer 32, 63–68.
Kubota, M., Tsubota, N., Yoshimura, M., Murotani, A., and Sakamoto, T. (1995), Lung Cancer 12, 290.
Kishida, T., Yamada, H., Negishi, H., Sagawa, T., Makinoda, S., and Fujimoto, S. (1996), Eur. J. Obstet. Gynecol. R. B. 69, 77–82.
Wang, C. C., Hong, J. H., Hsu, K. H., Tsang, N. M., Leung, W. M., and Simon, G. T. (1996), Int. J. Radia. Oncol. Biol. Phys. 36, 163.
Wang, X. W. and Xu, B. (1998), Int. J. Cancer 75, 596–599.
Kirkpatrick, A. and Nakamura, H. T. (1977), Clin. Chem. 23, 50–59.
Xue, M., Haruyama, T., Kobatake, E., and Aizawa, M. (1996), Sens. Actuators B 36, 458–462.
Matsumoto, K., Yuan, J. L., Wang, G. L., and Kimura, H. (1999), Anal. Biochem. 276, 81–87.
Ito, K., Oda, M., Tsuji, A., and Maeda, M. (1999), J. Pharm. Biomed. 20, 169–178.
Ci, Y. X., Qin, Y., Chang, W. B., and Li, Y. Z. (1995), Anal. Chim. Acta 300, 273–276.
Nakamura, K., Imajo, N., Yamagata, Y., Katoh, H., Fujio, K., and Tanaka, T. (1998), Anal. Chem. 70, 954–957.
Wang, R. Y., Lu, X. M., and Ma, W. Y. (2002), J. Chromatogr. B 779, 157–162.
Della Ciana, L., Bernacca, G., De Nitti, C., and Massaglia, A. (1996), J. Immunol. Methods 193, 51–62.
Pollema, C. H., Ruzicka, J., and Christian, G. D. (1992), Anal. Chem. 64, 1356–1361.
Gunaratna, P. C. and Wilson, G. S. (1993), Anal. Chem. 65, 1152–1157.
Nandakumar, R., Nandakumar, M. P., and Mattiasson, B. (2000), Biosens. Bioelectron. 15, 241–247.
Bereczki, A. and Horváth, V. (1999), Anal. Chim. Acta 391, 9–17.
Bjarnason, B., Bousios, N., Eremin, S., and Johansson, G. (1997), Anal. Chim. Acta 347, 111–120.
Burestedt, E., Kjellström, S., Emnéus, J., and Marko-Varga, G. (2000), Anal. Biochem. 279, 46–54.
Lövgren, U., Kronkvist, K., Bäckström, B., Edholm, L. E., and Johansson, G. (1997), J. Immunol. Methods 200, 159–168.
önnerfjord, P., Eremin, S. A., Emnéus, J., and Marko-Varga, G. (1998), J. Chromatogr. A 800, 219–230.
Piran, U., Riordan, W. J., and Livshin, L. A. (1996), Biotechnol. Adv. 14, 519.
Lim, T. K., Nakamura, N., and Matsunaga, T. (1998), Anal. Chim. Acta 370, 207–214.
Zhu, Q. Z., Liu, F. H., Li, D. H., Xu, J. G., Su, W. J., and Huang, J. W. (1998), Anal. Chim. Acta 375, 177–185.
Dodeigne, C., Thunus, L., and Lejeune, R. (2000), Talanta 51, 415–439.
Thorpe, G. H. G., Kricka, L. J., Gillespie, E., Moseley, S., Amess, R., Bagget, N., and Whitehead, T. P. (1985), Anal. Biochem. 145, 96–100.
Zhang, H. L. and Horacio, A. M. (1996), Analyst 121, 211–218.
Navas Diaz, A., Gonaalez Garcia, J. A., and Lovillo, J. (1997), J. Biolumin. Chemilumin. 12, 199–205.
Kricka, L. J. and Ji, X. Y. (1997), Talanta 44, 1073–1079.
Osipov, A. P., Zaitseva, N. V., and Egorov, A. M. (1996), Biosens. Bioelectron. 11, 881–887.
Díaz, A. N., Sánchez, F. G., Lovillo, J., and García, J. A. G. (1996), Anal. Chim. Acta 321, 219–224.
Botchkareva, A. E., Fini, F., Eremin, S., Mercader, J. V., Montoya, A., and Girotti, S. (2002), Anal. Chim. Acta 453, 43–52.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lin, J., Yan, F. & Ju, H. Noncompetitive enzyme immunoassay for α-fetroprotein using flow injection chemiluminescence. Appl Biochem Biotechnol 117, 93–102 (2004). https://doi.org/10.1385/ABAB:117:2:093
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1385/ABAB:117:2:093