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Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof

An immunochromatographic test strip, Escherichia coli technology, applied in biological testing, measuring devices, analytical materials, etc., can solve the problems of difficulty in promotion, easy contamination, time-consuming and labor-intensive, etc., and achieves low single-use cost, intuitive test results, and The effect of high antibody titers

Inactive Publication Date: 2010-11-17
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional F5 + The separation and identification method of ETEC has strong specificity, but it is time-consuming and laborious, and does not meet the needs of clinical testing; the agglutination test is easy to operate, fast and suitable for on-site testing, but single-factor serum is not easy to purchase, and it is easy to be contaminated after repeated use; small intestinal epithelial cell adhesion test The required small intestine fragments are not easy to obtain and preserve; MRHA and HI used for agglutination of red blood cells are not suitable for long-term storage; nucleic acid detection and ELISA methods are highly sensitive, but because the use of specific instruments is limited to laboratory operations, it is difficult to promote

Method used

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  • Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof

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specific Embodiment approach 1

[0016] Specific implementation mode one: combine figure 1 Describe this embodiment, the colloidal gold immunochromatography test strip used for detecting Escherichia coli F5 pili in this embodiment, consists of PVC backing 1, sample pad 2, binding pad 3, nitrocellulose membrane 4 and water-absorbing pad 5 , on the PVC backing 1, a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 5 are continuously adhered in sequence, and there is an overlap between the binding pad 3 and the nitrocellulose membrane 4, so There is an overlap between the nitrocellulose membrane 4 and the water-absorbing pad 5; the binding pad 3 is coated with an anti-F5 pili monoclonal antibody-colloidal gold marker, and the nitrocellulose membrane 4 is coated with The detection line is composed of anti-F5 pili polyclonal antibody and the quality control line is composed of goat anti-mouse IgG.

[0017] The PVC backing 1 , sample pad 2 , binding pad 3 , nitrocellulose membra...

specific Embodiment approach 2

[0019] Specific embodiment two: the preparation method of the colloidal gold immunochromatography test strip that this embodiment is used to detect Escherichia coli F5 pilus is realized by the following steps: one, induce prokaryotic expression F5 pilus protein, purify this fusion protein, i.e. immunization The original; two, the preparation of anti-F5 pili polyclonal antibody, then coated on the nitrocellulose membrane to form a detection line, and then goat anti-mouse IgG coated on the nitrocellulose membrane 4 to form a quality control line; three, the preparation of anti- F5 pilus monoclonal antibody; 4. Prepare colloidal gold solution by trisodium citrate reduction method, then add anti-F5 pilus monoclonal antibody to the colloidal gold solution to prepare anti-F5 pilus monoclonal antibody-colloidal gold marker , and then coated on the binding pad 3; five, on the PVC backing 1, continuously adhere to the sample pad 2, the binding pad 3 obtained in step 4, the nitrocellulos...

specific Embodiment approach 3

[0037] Specific embodiment three: the difference between this embodiment and specific embodiment two is the specific steps of preparing anti-F5 pilus polyclonal antibody in step two: a, the immunogen whose immune concentration is 1mg / mL, by volume ratio 1:1 Add Freund's complete adjuvant to mix and emulsify, and then inject subcutaneously at multiple points on the back of rabbits, the injection volume is 0.2mL / rat; b. Two weeks later, use Freund's incomplete adjuvant to emulsify the immunogen, and carry out the second step in the same way. Secondary immunization, and booster immunization every three weeks, blood collection from ear veins every week, and indirect ELISA method to detect antibody titer until the titer reaches 6.4×10 4 Afterwards, blood was collected from the heart to separate the serum, and then purified by crude extraction with ammonium caprylate sulfate and ProteinG affinity chromatography to obtain polyclonal antibodies against F5 pili. Others are the same as ...

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Abstract

The invention discloses a colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and a preparation method thereof, which relate to a test paper strip for detecting escherichia coil pilus and a preparation method thereof. The invention provides the colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and the preparation method thereof. The test paper strip consists of a PVC back lining, a sample pad, a combination pad, a cellulose nitrate membrane and a water absorbent pad. The preparation method comprises the following steps of: preparing an immunogen; coating an anti-F5 pilus polyclonal antibody and a sheep anti-mouse IgG on the cellulose nitrate membrane; preparing an anti-F5 pilus monoclonal antibody; coating the anti-F5 pilus monoclonal antibody and a colloidal gold marker on the combination pad; and finally, continuously adhering the sample pad, the combination pad, the cellulose nitrate membrane and the water absorbent pad on the PVC back lining in turn, and cutting the PVC back lining into the test paper strip. The colloidal gold immuno-chromatography test paper strip has high specificity, stability and sensibility, can be used for identifying enterotoxigenic escherichia coil F5 pilus, has an intuitive result, and is convenient to be conserved.

Description

Technical field [0001] The present invention involves a test note and its preparation methods for detecting E. coli. Background technique [0002] Statistics show that the incidence and mortality of animal diarrhea caused by E. coli in my country accounted for 56.2%and 24.7%of the incidence and mortality of the entire diarrhea, respectively, which caused huge economic losses to the production of pig industry.E. E. E. E. E. ETEC) is one of the important pathogens that cause diarrhea caused by piglets, but other pathogens, such as pig infectious gastroenteritis virus, pig flow diarrhea virus, pig wheat virus, etc.Diarrhea, therefore, sensitive and accurately identifying the pathogen is an important prerequisite for diagnosis and prevention of pig diarrhea. [0003] E. coliic bacteria plays a leading role in diarrhea caused by ETEC. F5 (K99) positive bacteria are the most common in ETEC that causes pigment diarrhea. At presentSmall intestine epithelial cell adhesion tests, as well a...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/52G01N33/531G01N33/558
Inventor 师东方杨旭东于迪
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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