Development of Monoclonal Antibodies Against Phenobarbital and Monitor of Phenobarbital Concentration in Serum by Competitive ELISA
WANG Zi-liang1，WANG Jian-na2，ZHANG Hai-tang1，WANG Xuan-nian1，YANG Yan-yan3，ZHANG Gai-ping3
Author information+
1.College of Animal Science，Henan Institute of Science and Technology，Xinxiang 453003，China； 2.Northwest Agriculture and Forestry University Hospital，Yangling 712100，China； 3.Henan Provincial Key Laboratory for Animal Immunology，Zhengzhou 450002，China
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History+
Received
Published
2005-12-29
2006-12-05
Issue Date
2006-12-05
Abstract
OBJECTIVE To generate monoclonal antibody against phenobarbital (PB mAb) and to establish competitive ELISA for monitoring PB concentration in serum. METHODS PB was modified chemically to form p-amidophenobarbital (pAPB), diazotization was used to conjugate pAPB to BSA and immunogen BSA-pAPB was obtained. Balb/c mice immunized with BSA-pAPB were chosen for cell fusion by indirect ELISA and blocking ELISA. The hybridoma lines secreting PB mAb were filtered with cell fusion and the immunological traits of PB mAb were characterized. A competitive ELISA kit for monitoring PB (PB-Kit) was developed with PB mAb and its traits were tested. RESULTS PB immunogen was synthesized successfully. The best one among four hybridoma lines was 4E6, which secreted PB mAb with indirect ELISA titers of 1∶6.4×105 in ascites, a high affinity constant (Ka) of 2.08×1010 L·moL-1, a good sensitivity with 50% inhibitive concentration (IC50) of 9.84 μg·L-1.The PB mAb had 7.9% cross-reactivity to Barbital and little or no cross-reactivity to other compounds. PB-Kit had the linear detection of 1.0 to 128 μg·L-1, the sensitivity of 0.89 μg·L-1 and the detection limit of 1.0 μg·L-1. The recoveries of PB spiked in serum were 94.7%. The precision and accuracy of the assay determined by inter-assay and intra-assay coefficient variation were both below 10%. Its coincidence rate was 100% compared with differential spectrophotometry (DS). CONCLUSION PB mAb with high-titer, sensitivity and specificity has been generated successfully, a competitive ELISA has been established with PB mAb, which can be used to monitor PB concentration in serum.
WNG Zi-ling;WNG Jin-n;ZHNG Hi-tng;WNG Xun-nin;YNG Yn-yn;ZHNG Gi-ping.
Development of Monoclonal Antibodies Against Phenobarbital and Monitor of Phenobarbital Concentration in Serum by Competitive ELISA [J]. Chinese Pharmaceutical Journal, 2006, 41(23): 1826-1830
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