Method for detecting aeromonas hydrophila B11 through lutetium-europium co-luminescence time-resolved fluoroimmunoassay

An Aeromonas hydrophila, luminescence time technology, used in fluorescence/phosphorescence, material excitation analysis, material analysis by optical means, etc.

Active Publication Date: 2019-04-05
JIMEI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Co-luminescent TRFIA method for detection of pathogenic bacteria has never been reported

Method used

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  • Method for detecting aeromonas hydrophila B11 through lutetium-europium co-luminescence time-resolved fluoroimmunoassay
  • Method for detecting aeromonas hydrophila B11 through lutetium-europium co-luminescence time-resolved fluoroimmunoassay
  • Method for detecting aeromonas hydrophila B11 through lutetium-europium co-luminescence time-resolved fluoroimmunoassay

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Embodiment Construction

[0022] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

[0023] 1. Experimental part

[0024] 1. Instrument

[0025] PerkinElmer VICTOR X4 multi-label analyzer, AKTA Purifier 100 protein purification system, MerckMilliporeDirect-Q3 ultrapure water integrated system, GE Healthcare HiTrap TM rProtein AFF protein A column, LGJ-10 freeze dryer.

[0026] 2. Reagents and materials

[0027] Perkin-Elmer Eu 3+ Labeling kit (1244-302), Sigma-Aldrich Triton X-100, 2-thienoyltrifluoroacetone (TTA), 1,10-phenanthroline (phen), europium trioxide (Eu 2 o 3 , 99.99%), Alfa-Aesar lutetium oxide (Lu 2 o 3 , 99.9%).

[0028] After Aeromonas hydrophila (Aeromonas hydrophila) B11 was inoculated on the solid medium for culture, the concentration was determined by the plate colony counting method, and the concentration was adjusted to 1×10 8 cfu / mL spare.

[0029] 3. Preparation of main reagents

[0030] PBS solution (...

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Abstract

The invention provides a method for detecting aeromonas hydrophila B11 through lutetium-europium co-luminescence time-resolved fluoroimmunoassay, and relates to co-luminescence time-resolved fluoroimmunoassay. The method comprises the steps that an aeromonas hydrophila antiserum is prepared; a B11 antibody is purified; a co-luminescence system is established; and lutetium-europium co-luminescenceTRFIA of the B11 is detected. In the presence of excessive Lu<3+>, the fluorescence intensity of a Eu<3+>-TTA-phen-Tritonx-100 system is enhanced. Condition optimization is conducted on the co-luminescence system, and a result shows that when the concentration of Lu3+, the concentration of TTA, the concentration of phen and the concentration of Triton X-100 is 7.5*10<-6> mol/L, 5*10<-5> mol/L, 4*10<-4> mol/L and 0.075% respectively, the fluorescence intensity of the co-luminescence system is the highest. By applying the direct co-luminescence TRFIA method to detect the B11, the bacterium withthe concentration of 1*10<3> cfu/mL is detected and is two magnitude order lower than a detection result obtained by using direct TRFIA.

Description

technical field [0001] The invention relates to co-luminescence time-resolved fluorescence immunoassay, in particular to a method for detecting Aeromonas hydrophila B11 by co-luminescence time-resolved fluorescence immunoassay of lutetium and europium. Background technique [0002] Aeromonas hydrophila (Aeromonas hydrophila) can cause human sepsis, meningitis, diarrhea and other diseases (Xin Zhiming, et al. Development of colloidal gold rapid detection test strips for Aeromonas hydrophila. Chinese Veterinary Science. 2012,42 :708-712). Highly sensitive detection of Aeromonas hydrophila can effectively reduce the losses caused by diseases and play a role in disease prevention and control. [0003] Immunoassay is easy to operate and has strong specificity, and is commonly used in the detection of pathogenic bacteria. However, the sensitivity of enzyme-linked immunosorbent assay (ELISA) and fluorescent antibody method is not high. Time-resolved fluorescent immunoassay (TRFIA)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N21/64
CPCG01N21/6408G01N33/56911
Inventor 林鹏冯建军郭松林王艺磊陈锦民王淑红谢东琴
Owner JIMEI UNIV
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