Colloidal gold rapid detection test paper for enterovirus 71 (EV71)

A technology for detecting enteroviruses and test strips, applied to measuring devices, instruments, scientific instruments, etc., to achieve clear and easy results, save manpower and material resources, and simple operation

Inactive Publication Date: 2012-01-18
BEIJING ZHUANGDI HAOHE BIOMEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, so far, there is no report on a detection scheme that can detect enterovirus 71 conveniently and quickly.

Method used

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  • Colloidal gold rapid detection test paper for enterovirus 71 (EV71)
  • Colloidal gold rapid detection test paper for enterovirus 71 (EV71)
  • Colloidal gold rapid detection test paper for enterovirus 71 (EV71)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of anti-enterovirus 71 type VP1 protein monoclonal antibody

[0030] (1) Immunized mice

[0031] After the prepared genetically engineered antigen was taken out from the -20 low-temperature refrigerator and dissolved, it was subcutaneously injected into the back of BALB / C mice (0.2ml / only) with an interval of 10 days. Three days before the fusion, mice were challenged with 0.15 ml of antigen in the peritoneal cavity. The immune effect was detected by ELISA method.

[0032] (2) Myeloma cells

[0033] SP2 / 0 myeloma cells: purchased from the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences. Resuscitate the SP2 / 0 cells stored in the liquid nitrogen tank, and culture them in DMEM medium containing 10% calf serum for 48-72 hours. Number split, ready to fuse.

[0034] (3) cell fusion

[0035] Prepared SP2 / 0 cells and splenic lymphocytes of immunized BALB / C mice were prepared in 2×10 7 / ml and 1×10 8 / ml. Take 1ml ...

Embodiment 2

[0042] Embodiment 2: Assay of anti-enterovirus 71 type VP1 protein monoclonal antibody

[0043] (1) Monoclonal antibody ascites preparation:

[0044] Mice: SPF grade mice, no rodent virus contamination after inspection, if the animals are found to be unhealthy, bitten or infected during the ascites production process, they should be discarded.

[0045] Expanded culture of cell lines: Take one tube of the production batch of cells to resuscitate, add nutrient solution to expand the culture, and one production cell is only used once, and will not be frozen.

[0046]Inoculation of hybridoma cell lines: preparation of ascites should be carried out under sterile conditions, before injection of hybridoma cells, each mouse was intraperitoneally injected with liquid paraffin 0.5ml. One week later, each mouse was intraperitoneally injected with hybridoma cells 1-3×10 6 / 0.2ml.

[0047] Ascites collection: 7-10 days after the injection of the cell line, or the ascites was collected o...

Embodiment 3

[0056] Embodiment 3: enterovirus type 71 (EV71) detection test strip (colloidal gold) (see Figure 1)

[0057] (1) Preparation of colloidal gold-VP1 monoclonal antibody conjugate:

[0058] It was determined by experiments that the optimum binding pH value of the anti-VP1 monoclonal antibody colloidal label was 8.0, and the ratio of colloidal gold and antibody was 40 μg / ml colloidal gold. After the labeled colloidal gold is treated with a stabilizer, the colloidal gold-antibody conjugate solution is taken in an amount of 65 μl per square centimeter, evenly adsorbed on glass fibers, freeze-dried, and stored in a dry environment.

[0059] (2) Coating antigen on nitrocellulose membrane:

[0060] The monoclonal antibody of another strain of VP1 recognizing different antigenic sites was diluted to 1.5mg / ml with 0.01MPBS. Anti-mouse IgG polyclonal antibody was diluted to 2mg / ml with 0.01MPBS. Spray the two on the nitrocellulose membrane at a speed of 1 μl / cm with a film spraying ma...

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Abstract

The invention provides colloidal gold rapid detection test paper for enterovirus 71 (EV71). The rapid detection test paper is characterized by coating the monoclonal antibody or polyclonal antibody of a specific antigen VP1 of EV71 and quality control anti-mouse IgG (immunoglobulin G) on a nitrocellulose membrane (NC membrane), and combining the monoclonal antibody of the specific antigen VP1 of EV71 marked by colloidal gold; and adopting a membrane chromatography double antibody sandwich method to detect the EV71 in a sample. The test paper provided by the invention can be used for detecting in a simple, convenient, rapid and direct mode without special apparatus and professional training; the test paper has a quite distinct result, is simple in operation and easy for popularization, and is suitable for basic level, suitable for the large scale field test for emergency, and suitable for early diagnosis, and plays an additional role for diagnosing the infection of the EV71.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to an enterovirus 71 (EV71) antigen detection test strip and an application thereof. Background technique [0002] Enterovirus 71 (EV71) belongs to Enterovirus type A and is a close relative of Coxsackie virus A16 (CA16). EV71 infection can cause hand, foot and mouth disease (HFMD), encephalitis (encephalitis) , aseptic meningitis (asepicmeningitis) and poliomyelitis-like paralysis (poliomyelitis-like paralysis) and other diseases related to the nervous system. At present, humans are the only known natural host of EV71 virus, and the virus is mainly spread through close contact between people. According to the differences in nucleotide sequences, EV71 can be divided into three genotypes: A, B, and C. Types B and C can be further divided into subtypes B1, B2, B3, B4, and C1 and C2. The 7 strains of EV71 virus (SHZH03, SHZH98, SH2F1, SH2F2, SH2H25, SH2H26 and CHN287) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
Inventor 刘明
Owner BEIJING ZHUANGDI HAOHE BIOMEDICINE SCI & TECH
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