Immunity test strip based on fluorescent microsphere for detecting infectious pleural pneumonia antibody of goat as well as preparation and detection method

A fluorescent microsphere, pleuropneumonia technology, applied in the field of immunological detection, can solve the problems of low sensitivity, poor repeatability or specificity, and need laboratory instruments, and achieves the effect of high sensitivity and shortened time.

Inactive Publication Date: 2018-03-02
LUOYANG LAIPSON INFORMATION TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Rapid and accurate diagnosis is beneficial to the prevention and control of Mccp. At present, there are many methods for the detection of Mccp antibodies that have been reported at home and abroad. The main techniques include indirect hemagglutination test, direct and indirect fluorescent antibody test, complement fixation test, and enzyme-linked immunosorbent immunoassay. Adsorption test (ELISA) and PCR technology recommended by OIE, but there are many disadvantages such as complex operation, poor repeatability or specificity, and the need for laboratory equipment, etc., especially not suitable for rapid, easy-to-operate and grass-roots use in the actual diagnosis o

Method used

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  • Immunity test strip based on fluorescent microsphere for detecting infectious pleural pneumonia antibody of goat as well as preparation and detection method
  • Immunity test strip based on fluorescent microsphere for detecting infectious pleural pneumonia antibody of goat as well as preparation and detection method
  • Immunity test strip based on fluorescent microsphere for detecting infectious pleural pneumonia antibody of goat as well as preparation and detection method

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0025] Example 1. Preparation of test paper card

[0026] 1. Preparation of bonding pad

[0027] (1) Washing: Take 50μL of fluorescent microspheres into a 1.5mL centrifuge tube, add 1mL 0.01M MES buffer, shake and mix, centrifuge at 15000r / min for 15min, discard the supernatant, add 1mL 0.01M MES buffer, and ultrasound Disperse the microspheres; repeat this step three times to achieve the purpose of cleaning the microspheres, and finally get the cleaned microsphere solution;

[0028] (2) Activation: Add 250μL of EDC solution to the cleaned 1mL microsphere solution, activate for 2h in the dark, centrifuge at 15000r / min for 15min, discard the supernatant, add 1mL 0.01M MES buffer solution, ultrasonically disperse the microspheres, 15000r Centrifuge for 15 min at / min, discard the supernatant;

[0029] (3) Labeling: add 1mL of 0.05M borate buffer, ultrasonically disperse, add 20μL of Mycoplasma capricolum recombinant protein and mix well, and react for 2h in the dark;

[0030] (4) Block ...

Example Embodiment

[0048] Example 2. Detection method and comparison test of goat infectious pleuropneumonia antibody in goat serum

[0049] 1. Tear off the aluminum foil packaging bag of the test card, take out the test card, and place it on a flat and clean table;

[0050] 2. Use a matching pipette to suck freshly collected goat serum, and add 2-3 drops (about 60μl) vertically and slowly into the sample hole;

[0051] 3. Read the results in 5-10 minutes, insert the test paper card into the card slot of the fluorescence immunoassay analyzer according to the arrow direction shown on the card cover, and read the T / C value;

[0052] 4. Judgment criteria: T / C value<0.1 is negative; T / C value≧0.1 is positive; C value is 0, invalid;

[0053] 5. Comparison with colloidal gold test strips: select 20 weakly positive samples detected by the Goat Infectious Pleuropneumonia Hemagglutination Diagnosis Kit, test the results with colloidal gold test strips, 5 positive and 15 negative; use Fluorescent microsphere test ...

Example Embodiment

[0055] Example 3. Comparison of the stability of the colloidal gold-labeled Mycoplasma amnioticum recombinant protein and the fluorescent microsphere-labeled anti-Mycoplasma amnioticum recombinant protein

[0056] 1. Place the prepared binding pads of colloidal gold-antigen complexes and fluorescent microspheres-antigen complex binding pads in a blast drying oven at 37°C;

[0057] 2. On the 3rd, 5th, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 20 weeks, 36 weeks, and 48 weeks, test the negative serum and the positive serum to observe their stability;

[0058] 3. The results are as follows

[0059]

[0060] As shown in the above table, the detection effect of the colloidal gold-labeled antigen complex decreases with the extension of the destruction experiment time, and the detection situation at the 20th week has a significant change; while the test strip using the fluorescent microsphere-antigen complex binding pad The positive serum of the test showed a downward trend at the 36th we...

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Abstract

The invention relates to an immunity test strip based on a fluorescent microsphere for detecting an infectious pleural pneumonia antibody of a goat as well as a preparation and detection method, whichbelong to the detection field of immunity. The test strip comprises a shell and a test strip, wherein the test strip comprises a bottom plate as well as a water absorption pad, a detection pad, a combination pad and a sample pad which are successively lapped and adhered onto the bottom plate; a nitrocellulose membrane is provided with a quality control line C and a detection line T, the quality control line C covers a His tag monoclonal antibody, and the detection line T covers a goat mycoplasma pneumonia recombinant protein; the combination pad is a glass cellulose membrane coating with thegoat mycoplasma pneumonia recombinant protein marked by time resolution fluorescent microspheres; and the sample pad is a dry glass cellulose membrane after being soaked in sample treatment liquid. The test strip prepared by the invention has better stability and higher sensitivity, and can achieve a purpose of rapid semi-quantitative detection by virtue of the fluorescent signal analysis.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to an immune test strip for detecting goat infectious pleuropneumonia antibody based on fluorescent microspheres, a preparation method and a detection method. Background technique [0002] Goat contagious pleuropneumonia (Mccp), also known as Mycoplasma capricum pneumonia, is the pathogen of Mycoplasma capricosum pneumonia, which belongs to the subspecies of Mycoplasma capricosum, and is a unique acute or chronic highly contagious infectious disease of goats. The disease is clinically characterized by high fever, cough, serous and fibrinous inflammation in the lungs and pleura, and has a high mortality rate. In recent years, the pathogens of Mccp have been isolated or detected in Gansu, Shandong, Qinghai and other places in China, and its morbidity and mortality are on the rise, which has caused great economic losses to the goat breeding industry in my country. ...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/558G01N33/543G01N33/577
CPCG01N33/533G01N33/54313G01N33/558G01N33/577
Inventor 李秀梅陈小云任雪建张志鹏王俊峰张小辉王蓝天
Owner LUOYANG LAIPSON INFORMATION TECH
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