Quick detection method and kit for trace-amount endotoxin

A technology of detection kits and detection methods, which are applied in measurement devices, preparation of test samples, instruments, etc., can solve the problems of killing a large number of protected animals, the color and turbidity of the test result samples themselves, and improving the detection The effect of sensitivity

Inactive Publication Date: 2014-09-17
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this method still has following two main disadvantages: (1) the detection result of limulus test method is easily affected by the color and the turbidity of sample itself;
At present, there is no report on a method that integrates sample pretreatment and ELISA detection. Therefore, the MIP-ELISA rapid detection method that integrates specific sample pretreatment and ELISA detection can not only be used for rapid and accurate detection of endotoxin, but also is expected to be used in other The rapid detection of pollutants will have greater application value in the fields of medicine, biology and the environment

Method used

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  • Quick detection method and kit for trace-amount endotoxin
  • Quick detection method and kit for trace-amount endotoxin
  • Quick detection method and kit for trace-amount endotoxin

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Sensitivity analysis of MIP-ELISA method

[0037] Add a series of concentration gradient endotoxin standards (10, 50, 100, 1000, 10000, 100000ng / mL) into the wells of the enzyme-labeled plate containing artificial antibodies, 100μL per well, 20-40℃ after 1-5h Wash 2-8 times with phosphate buffer. Then add 100μL of rabbit anti-E. coli endotoxin polyclonal antibody to each well (diluted with antibody diluent at 1:100~1:2000), incubate at 20-40℃ for 1-5h and wash 2-8 times with phosphate buffer . Add 100μL of horseradish peroxidase-labeled goat anti-rabbit antibody to each well (diluted with antibody diluent at 1:500~1:5000), incubate at 20-40℃ for 1-5h and wash with phosphate buffer 2 -8 times. Finally, add 200μL of enhanced luminescence working solution to each well (molar concentration is 5*10 -4 mol / L Luminol, 4*10 -3 mol / L of H 2 O 2 And 4*10 -4 mol / L PIP), immediately use the multifunctional microplate reader to detect the luminous intensity.

[0038] From ...

Embodiment 2

[0039] Example 2: Specificity analysis of MIP-ELISA

[0040] The MIP-ELISA method was used to determine the endotoxin content produced by various bacteria, and compared with the standard method limulus reagent method and icELISA method. Cultivate 3 kinds of Gram-negative bacteria (E. coli, Photobacterium luminescens, Pseudomonas aeruginosa), and 1 kind of Gram-positive bacteria (Staphylococcus aureus). Combine 4 kinds of bacteria liquid (all 10 5 cfu / mL) boiled in boiling water for 2.5h to inactivate, inactivate the endotoxin in the bacteria and release it. The endotoxin content in various bacterial lysates was determined according to the MIP-ELISA method established in Example 1. The endotoxin concentrations in Gram-negative bacteria-Escherichia coli, Photobacterium luminescens and Pseudomonas aeruginosa were 99.15±2.79 ng / mL, 112.63±3.68ng / mL, 113.74±3.58ng / mL, the test result of Gram-positive bacteria-Staphylococcus aureus was negative. Simultaneously use the standard limulu...

Embodiment 3

[0041] Example 3: MIP-ELISA method and stability analysis of the corresponding kit

[0042] (1) Method stability. Place the collected river water (Wuhan Yangtze River) in a container and let it settle naturally for 24 hours to remove large particles and suspended solids. Different concentrations of endotoxin standards (10, 50, 100 ng / mL) are added to the post-settlement In the river water samples, the endotoxin content in the unspiked and spiked river water samples was determined according to the MIP-ELISA method established in Example 1. The recovery rate of endotoxin addition was 83.6%-101.7%, and the intra-day deviation was 2.1~ 4.0%, the day-to-day deviation is 1.6-4.6%, all less than 5% (n=6). At the same time, icELISA was used to detect the same sample. The recovery rate of endotoxin was 79.3%-92.5%, the intra-day deviation was 4.2-7.1%, and the inter-day deviation was 3.9-8.6% (n=6). It shows that MIP-ELISA has good stability, while the icELISA which uses natural antibod...

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Abstract

The invention method provides a quick detection method for trace-amount endotoxin. The method can detect the trace-amount endotoxin in various samples directly and sensitively in a high throughput. The invention also provided a corresponding kit which is composed of a 96-hole enzyme label plate, an endotoxin standard substance solution, a rabbit-anti-endotoxin polyclonal antibody (first antibody), a goat-anti-rabbit antibody which is labeled by horseradish peroxidase (second antibody) and a chemiluminescence reagent. According to the method, a specific pretreatment of a sample and ELISA detection are integrated together. An artificial antibody is high-temperature-resistant, acid-resistant, alkali-resistant and organic-solvent-resistant and can be used for high-efficient specifically separating and enriching the trace-amount endotoxin in the sample. The ELISA detection can specifically and quickly detect the enriched endotoxin. By means of combination with a high-sensitivity chemiluminescence method, the ELISA detection is effectively improved in sensitivity, accuracy and stability when endotoxin is detected so that the trace-amount endotoxin in various samples can be detected sensitively, quickly and in a high throughput.

Description

Technical field [0001] The present invention relates to the field of molecular imprinting and immunoanalysis, in particular to the preparation of a molecularly imprinted artificial antibody (MIP) capable of specifically adsorbing endotoxin, and the combined use of the MIP and ELISA method to establish a set of specific sample pretreatment and ELISA detection is integrated, a new type of enzyme-linked immunoassay method for the direct, sensitive and high-throughput detection of trace endotoxins in a variety of samples: MIP-ELISA method. And provide the corresponding kit. technical background [0002] Endotoxin is the main component of the cell wall of gram-negative bacteria. It is located in the outermost layer of the cell wall. It will be released when the bacteria are lysed or the structure of the bacteria is artificially destroyed. The human body is extremely sensitive to endotoxins. A very small amount of endotoxin (1-5ng / kg body weight) can increase the body temperature, pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/535G01N1/34
Inventor 吕斌黄正钟剑张梦石云
Owner HUAZHONG UNIV OF SCI & TECH
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