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Important heating pathogen fast screening system

A pathogenic and important technology, applied in the field of immune technology and clinical detection, can solve the problems of difficult to meet the needs of rapid screening, difficult to achieve simultaneous screening of multiple etiologies, etc., and achieve the effect of cost saving

Inactive Publication Date: 2009-08-12
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rapid detection technologies such as PCR for pathogenic nucleic acids rely on the gene amplification and electrophoresis process of the instrument, as well as the technical level of the operator, which is difficult to meet the needs of simple and rapid screening at the port
The general method mainly detects a single pathogen or infectious disease. Facing a febrile patient, it is difficult to screen multiple causes at the same time

Method used

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  • Important heating pathogen fast screening system
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Coupling of Coated Antigens to Microspheres with Known Numbers:

[0035] 1. Take coded microspheres No. 27, 31, 32, 33, 43, and 44 respectively, oscillate the microsphere suspension with a vortex oscillator for 30 seconds, and ultrasonicate for 30 seconds to mix the microspheres evenly;

[0036] 2. Take 1.25×106 microspheres of each of the above codes into a 1.5mL centrifuge tube, centrifuge at 10000g-14000g for 4min, carefully suck out the supernatant and discard it;

[0037] 3. Add 100 μL of bead washing buffer (PBS, pH 7.4, 0.05% TWEEN-20) to suspend the microspheres, shake for 30 seconds, sonicate for 30 seconds, centrifuge at 10000g-14000g for 4min, carefully suck out the supernatant and discard it;

[0038] 4. Add 80 μL of bead activation buffer (3g NaH 2 PO 4 , 5N NaOH40drops / 250mL H 2 O), use the vortex shaker to vibrate the microsphere suspension;

[0039] 5. Add 10 μL of freshly prepared EDC (50 mg / mL), followed by 10 μL of freshly prepared Sulf...

Embodiment 2

[0051] Embodiment 2: the suspension chip detection of sample:

[0052] The immunological detection mode of the indirect method is adopted, and all reactions are carried out on a 96-well filter plate during the detection process.

[0053] 1. Add 150 μL detection buffer to each well to pre-wet the wells, and filter with a vacuum pump;

[0054] 2. Add 50 μL of the working solution containing the corresponding coded microspheres to each well, wash the lotion and filter twice with a vacuum pump;

[0055] 3. Add 50 μL of the diluted test sample, mix well, shake at room temperature in the dark for 30 minutes, wash the lotion and filter three times;

[0056] 4. Add 50 μL of appropriate concentration of biotinylated secondary antibody diluted with antibody diluent, mix well, shake at room temperature in the dark for 30 minutes, wash with 100 μL of lotion for 3 times, and filter with vacuum pump;

[0057] 5. Add 50 μL of SA-PE, mix well and shake at room temperature for 10 minutes in ...

Embodiment 3

[0060] Example 3: Detection of different concentrations of rabbit anti-TB IgG by multiple microsphere system

[0061] 1. Each antigen is coupled with different encoded microspheres, the specific operation method is as in Example 1, and the conjugates of different antigens and encoded microspheres are obtained;

[0062] 2. Take the No. 27 carboxyl microsphere conjugate coated with the 38KD protein and 16KD protein of the antigen Mycobacterium tuberculosis, the No. 32 carboxyl microsphere conjugate of the avian influenza H5 antigen, and the No. 43 carboxyl microsphere conjugate of the Yersinia pestis F1 antigen. Ball coupling, No. 44 carboxyl microsphere coupling of SARS-CoV N protein, 3500 pieces each, add to the same centrifuge tube, add microsphere dilution to a total volume of 50 μL as the detection working solution of the multiple microsphere system;

[0063] 3. Make 4-fold gradient dilution of rabbit anti-TB IgG with the sample diluent as the test sample. The concentration...

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Abstract

The invention discloses a method for detecting a system for quickly screening a fever pathogen, and mainly relates to detection of a tuberculosis antibody, a flu antibody, a bird flu antibody, a plague antibody and an SARS antibody in blood serum. The chip mainly comprises coding microspheres, a coating antigen, a detection antibody, a biotinylated antibody and a streptavidin-phycoerythrin. The method comprises the following steps that: the coating antigen and the coding microshperes are coupled; the detection antibody is respectively coupled with the corresponding microspheres in separate specificity; the red laser excites the classified fluorescence on the spherical substrate; the type is determined according to different colors of the spherical substrates, wherein the biotinylated antibody is combined with the detection antibody; and the streptavidin-phycoerythrin is combined with the biotin of the detection antibody captured by the microshperes; the green laser excites the phycoerythrin, the number of report fluorescence molecules combined on the spherical substrate is measured, and the number is used for indirectly determining the content of the detection antibody combined onthe spherical substrate, so that whether the pathogen infection exists is determined, and the aim of quickly screening the fever pathogen is achieved.

Description

technical field [0001] The present invention relates to a protein suspension chip and its preparation method and application, especially to the detection of several infectious pathogenic antibodies that cause fever at the same time, such as the protein suspension of tuberculosis antibody, influenza antibody, bird flu antibody, plague antibody, SARS antibody, etc. The chip and its preparation method and application belong to the field of immune technology and clinical detection technology. Background technique [0002] In 2003, the global SARS epidemic, frequent outbreaks of bird flu in Asian countries, dengue fever, falciparum malaria on the China-Myanmar border, encephalitis, influenza, and plague that entered an active stage again, West Nile fever in the United States, and Ebola hemorrhage in Africa Fever, etc., aroused widespread fear of infectious diseases among the public. According to the World Health Organization's analysis report on infectious diseases in 2002, 1,50...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/546G01N33/538
Inventor 王静杨永莉孙肖红杨宇胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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