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Campylobacter jejuni immune colloidal gold rapid detection test strip

A technology for detecting Campylobacter jejuni and test paper, which is applied in the field of bioengineering and can solve problems such as long cycle time and complicated operation for detecting Campylobacter jejuni

Active Publication Date: 2016-08-17
上海慧耘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a quick detection test strip of Campylobacter jejuni immune colloidal gold, which solves the problems in the prior art. Detection of Campylobacter jejuni complicated and long-term technical problems

Method used

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specific Embodiment

[0036] At first introduce the reagent and instrument used in the embodiment of the present invention:

[0037] main reagent

[0038] Freund's complete adjuvant and Freund's incomplete adjuvant, PEG, TWEEN-20Sigma; BSA was purchased from Shanghai Bright Biotechnology Co., Ltd.; colloidal gold solution was purchased from Shanghai Gold Standard Biotechnology Co., Ltd.; nitrocellulose membrane (NC membrane ) 135SMillipore; goat anti-mouse, HRP-goat anti-mouse, goat anti-rabbit were purchased from Shanghai Sangon Bioengineering Co., Ltd.; monoclonal antibody subtype identification kit was purchased from ZYMED Cat: 90-6550; Lot.60907259.

[0039] Standard strains such as Campylobacter jejuni were purchased from American Culture Collection Center (ATCC), China Medical Microbiology Collection Center (CMCC), China Industrial Microbiology Collection Center (CICC), and Inspection and Quarantine Microbiology Collection Center (IQCC) of AQSIQ.

[0040] main instrument

[0041] Microplate...

Embodiment 1

[0042] Embodiment 1 Campylobacter jejuni monoclonal antibody preparation

[0043] 1. Preparation of immunogen and positive standard

[0044] Campylobacter jejuni (CICC No. 22937) was inoculated on Brunner's broth, cultured with shaking at 37°C and 150 r / min for 17 hours under anaerobic conditions, counted, and inactivated by adding 0.3% formaldehyde solution at room temperature for 1 day. Use normal saline to adjust the concentration of Campylobacter jejuni (CICCNo.22937) to 5×109cfu / ml as an immunogen; use normal saline to adjust the concentration to 10 8 cfu / ml was used as a positive control standard, and Brooke's broth as a negative control standard.

[0045] 2. Preparation process of monoclonal antibody

[0046] (1) Experimental animals: 3 8-week-old female Balb / c mice weighing about 20 g were selected as experimental animals.

[0047] (2) Immunization method: each mouse was intraperitoneally injected with 0.2 ml of immunogen, and the same dose was boosted once every tw...

Embodiment 2

[0079] The determination of the optimal combination of colloidal gold test strip antibody of embodiment 2

[0080] 1. Determination of the optimal pH of each antibody coupled to colloidal gold

[0081] Take 100 μL of colloidal gold solution in 8 wells of a 96-well plate, adjust the pH to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, and add 10 μL of monoclonal antibody at a concentration of 1 mg / mL to each well. After reacting for 5 minutes, add 10 μL of 10% NaCl solution to each well, react for 5 minutes, observe the color change of the solution, and repeat three times.

[0082] figure 2 The pH values ​​in the wells of each group from left to right are 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0. Depend on figure 2 It is known that group A is the result of colloidal gold labeling of monoclonal antibody H3 at different pH values. When the pH is 7.5-8.0, the color of colloidal gold in the well is the reddest, and there is no coagulation, no fading or discoloration, that is, H3 si...

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Abstract

The invention discloses a campylobacter jejuni immune colloidal gold rapid detection test strip, comprising a sample pad, a gold mark pad tightly connected with the sample pad, a cellulose membrane tightly connected with the gold mark pad on the gold mark pad and a water absorption pad tightly connected with the other end of the cellulose membrane, a quality control line is arranged at one end, away from the gold mark pad, on the cellulose membrane, a detection line is arranged on the cellulose membrane between the quality control line and the gold mark pad, a monoclonal antibody coupled with colloidal gold is arranged on the gold mark pad, and is secreted by a hybridoma cell strain with a preservation number of CGMCC NO. 8457, and the detection is formed by a monoclonal antibody which is secreted by a hybridoma cell strain with a preservation number of CGMCC NO.8766. The gold mark pad and the detection line are respectively sprayed by adopting newly screened paired monoclonal antibodies, and an experiment shows that the detection specificity and flexibility on the campylobacter jejuni are higher.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a campylobacter jejuni, specifically a colloidal gold rapid detection test strip for the immune colloidal gold of the campylobacter jejuni. Background technique [0002] Campylobacter jejuni enteritis was isolated from the feces of patients with diarrhea by Butzler in 1973, and it has been recognized as one of the main pathogenic bacteria of human diarrhea. The incidence of Campylobacter jejuni enteritis exceeds bacillary dysentery in developed countries and is almost equal to bacillary dysentery in developing countries. As an important food-borne pathogen, this bacterium rapidly multiplies in an environment containing trace oxygen after food enters the intestinal tract, mainly invades the jejunum, ileum and colon, invades the intestinal mucosa, and causes congestion and hemorrhagic injury. To some strains can produce cholera-like enterotoxin, causing increased fluid se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56922
Inventor 方水琴万绍业杨标郭慧琴胡永辉刘箐
Owner 上海慧耘生物科技有限公司