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Rapid detection test strip for detecting campylobacter jejuni specific antigen

A technology of Campylobacter jejuni and test strips, applied in applications, measuring devices, anti-bacterial immunoglobulins, etc., can solve problems such as limited significance of clinical diagnosis, achieve convenient storage and transportation, and have industrial effects

Inactive Publication Date: 2009-02-11
BEIJING ZHUANGDI HAOHE BIOMEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The IgA content in the serum and feces after infection increased rapidly for several weeks and then decreased rapidly, which has limited significance for clinical diagnosis, but it is of great significance for helping to determine the relationship between Campylobacter jejuni infection, GBS and arthritis

Method used

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  • Rapid detection test strip for detecting campylobacter jejuni specific antigen
  • Rapid detection test strip for detecting campylobacter jejuni specific antigen
  • Rapid detection test strip for detecting campylobacter jejuni specific antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of Genetic Engineering Antigen of Campylobacter jejuni Outer Membrane Protein PEB1

[0036] Extraction of CJ Genomic DNA:

[0037] Take 1ml of Campylobacter jejuni culture, centrifuge to collect cells, then suspend in 200ul TE solution, and extract DNA with Genomic DNA Extraction Kit.

[0038] Amplification of the peblA gene

[0039] According to the complete CDS of peblA reported by Genbank (No. AL139076), a pair of primers were designed by removing the signal peptide at the amino terminal, and BamHI and XhoI were selected as restriction sites to design the following primers:

[0040] Upstream is 5-GCGGATCCGCAGAAGGTAAACTTGAGTCTAT-3,

[0041] Downstream is 5-CCGCTCGAGTTATAAACCCCATTTTTTCGCT-3.

[0042] PCR amplification of the peblA gene. The amplification conditions were denaturation at 94°C for 4 minutes, denaturation at 94°C for 30 seconds, annealing at 53°C for 45 seconds, extension at 72°C for 1 minute, and extension at 72°C for 8 minutes ...

Embodiment 2

[0051] Rabbit anti-CJ whole bacteria antibody was used as the primary antibody; HRP-labeled goat anti-rabbit IgG was used as the secondary antibody, and Western blot was used to detect the immunoreactivity of rPEB1. The results showed that the recombinant protein Peb1 had good immunogenicity, and the ELISA titer of its rabbit serum reached 1:50000. Example 2: Preparation of monoclonal antibody against Campylobacter jejuni outer membrane protein PEB1

[0052] (1) Immunized mice

[0053] 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.

[0054] (2) Myeloma cells

[0055] SP2 / 0 myeloma cells: resuscitate SP2 / 0 cells stored in a liquid nitrogen tank, and cultu...

Embodiment 3

[0064] Example 3: Detection of anti-Campylobacter jejuni outer membrane protein PEB1 monoclonal antibody

[0065] (1) Monoclonal antibody ascites preparation:

[0066] 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.

[0067] Expanded culture of cell lines: take one tube of the production batch of cells to resuscitate, add nutrient solution to expand the culture, one production cell is only used once, and will not be frozen.

[0068] 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.

[0069] Ascites collection: 7-10 days after the injection of the cell line, or the ascites ...

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Abstract

The invention provides a quick test dipstick used for testing a specific antigen of Campylobacter jejuni, which comprises a reaction membrane and a binder release liner. The reaction membrane is provided with a testing strap coated with a monoclonal antibody mentioned in Claim 1 or a PEB1 polyclonal antibody and a quality control strap coated with anti-IgG; the binder release liner is coated with a PEB1 monoclonal antibody which is marked by colloidal gold and the binding site of which is linked with different antigen on the reaction membrane. A membrane chromatography double antibody sandwich method is applied to detecting the specific antigen of the Campylobacter jejuni in a specimen. The dipstick is applied to test, which has simple, convenient, quick and concise operation and does not require special equipment and facilities as well as professional training. Furthermore, the dipstick has clear and easy-identity results, simple operation and easy popularization, is applicable to matrixes, field tests of emergency on a large scale and the study of epidemiology and can aid in the infection diagnostics of the Campylobacter jejuni.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to the preparation of campylobacter jejuni outer membrane protein PEB1 genetic engineering antigen and the development of a monoclonal antibody, and a campylobacter jejuni colloidal gold rapid detection test strip containing the monoclonal antibody and its application. technical background [0002] Campylobacteriosis is an infectious disease caused by Campylobacter jejuni (Cjejuni). Enteritis caused by Campylobacter jejuni is a worldwide disease. In economically developed countries, it is the main foodborne infectious disease. Diarrhea caused by Campylobacter jejuni infection ranks first among bacterial diarrheal diseases. In developing countries, the incidence of diarrheal diseases is high, and the diarrhea caused by Campylobacter jejuni is second only to Escherichia coli and Shigella. Campylobacter jejuni infection is mostly sporadic, and outbreaks are rare. It is the main patho...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/31C12N15/63C07K14/205G01N33/577G01N33/558
Inventor 刘明
Owner BEIJING ZHUANGDI HAOHE BIOMEDICINE SCI & TECH
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