Citation: LU Jie, GUO Song-Lin, FENG Jian-Jun, WANG Yi-Lei, ZHU Hong-Quan, LIN Peng. Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica[J]. Chinese Journal of Analytical Chemistry, ;2012, 40(11): 1709-1714. doi: 10.3724/SP.J.1096.2012.20139 shu

Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica

1.
Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen 361021, China

Corresponding author: LIN Peng,
Received Date: 25 February 2012
Available Online: 5 June 2012
Fund Project: 本文系国家自然科学基金(Nos. 30972281, 31001136) (Nos. 30972281, 31001136)福建省高等学校新世纪优秀人才支持计划(闽教科[2007]20号) (闽教科[2007]20号)集美大学创新团队基金项目(No. 2010A001)资助 (No. 2010A001)

Klebsiella Oxytoca B12, isolated from diseased Anguilla janponica, was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria. The rabbit immunoglobulin (IgG) was purified by SPA affinity column. A microplate sandwich immunoassay for the detection of K. Oxytoca was developed utilizing time-resolved fluoroimmunoassay (TRFIA). Antibodies immobilized on microtiter plates were used to capture the bacteria. The bound antigen-antibody complexes were then detected using the same antibodies labeled with Eu³⁺ chelates. The effects of antibody concentration, immunoreaction time and dissociation time on assay were studied during assay development. The limit of detection was 5.0×10³ cfu/mL and the linear range was 5.0×10³-1.0×10⁷ cfu/mL with the correlation coefficients more than 0.99. Cross reaction showed that it had high specificity. The intra-assay and inter-assay standard deviation were 5.6% and 2.3%, respectively. The method was optimized to detect the tissues including gill, kidney, intestine, liver and muscle from infected K. Oxytoca with satisfactory results.
- Time-resolved fluoroimmunoassay,
- Klebsiella Oxytoca,
- Anguilla janponica
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