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Colloidal gold chromatographic rapid diagnosis test paper strip used for detecting acetylcholine esterase and preparation method thereof

An acetylcholinesterase and rapid diagnosis technology, applied in the field of immunodiagnostic detection, can solve the problems of limited application, affecting the interpretation of results, reducing the sensitivity of sensors, etc., achieving good sensitivity and specificity, small amount of reagents and samples, and not easy to fake The effect of a negative result

Pending Publication Date: 2017-10-24
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The poor stability of the chip directly limits its clinical application
[0007] 2. The convenience of using the chip is poor: in the existing scheme, before starting the detection, the chip is first activated, then glutaraldehyde is combined with the chip, and then the antibody is injected into the reaction pool of the sensor
The operation is cumbersome, and the antibody needs to be refrigerated or frozen. Improper storage of the antibody will reduce the titer and reduce the sensitivity of the sensor
[0008] 3. The equipment used in the scheme is expensive, and each equipment costs about RMB 500,000. Even if the configuration is simplified, it needs more than RMB 250,000
In addition, this scheme is cumbersome to operate, and operators need more skilled technology
[0009] 4. There are many interference factors, which affect the interpretation of the results
It is difficult to rule out the influence of solution viscoelastic factors

Method used

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  • Colloidal gold chromatographic rapid diagnosis test paper strip used for detecting acetylcholine esterase and preparation method thereof
  • Colloidal gold chromatographic rapid diagnosis test paper strip used for detecting acetylcholine esterase and preparation method thereof
  • Colloidal gold chromatographic rapid diagnosis test paper strip used for detecting acetylcholine esterase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of monoclonal antibody against acetylcholinesterase

[0042] 1. Acquisition of immunogen

[0043] 1) Using the monoclonal antibodies previously obtained by our research group (invention authorization number: ZL2012 1 0516 218.3; monoclonal antibodies with monoclonal antibody clone numbers AP0691, AP0962, 53E11c9, 62D12H4, 1H1H4 were prepared according to the method in this patent), Add 20 μl monoclonal antibody (clone number: AP0961, 53E11c9, 62D12H4, 1H1H4, AP0962) and 50 μl Anti-mouse IgG ( H+L), F(ab')2Fragment( Bead Conjugate) (Cell Signaling Technology company, product number: 5946), 20 μl self-made rabbit anti-acetylcholinesterase polyclonal antibody with a concentration of 1 mg / ml, 50 μl Protein GAgarose Beads (Cell Signaling Technology company, product number 37478), 50 μl Mouse IgG ( Bead Conjugate) (Cell Signaling Technology Company, Cat. No. 3420), shaker overnight at 4°C. Centrifuge at 12,000 g at 4°C for 15 minutes, discard th...

Embodiment 2

[0059] Example 2: Preparation of colloidal gold chromatography rapid diagnostic test strips for detection of acetylcholinesterase

[0060] A colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase, said diagnostic test strip comprises a water-absorbing filter paper 1, a nitrocellulose membrane ( Millipore company, 135S, pore diameter 8 μ m) detection pad 2 and colloidal gold pad (absorbent glass fiber) 3 and sample pad ( Water-absorbing glass fiber) 4, the quality control area 5 on the detection pad is fixed with commercially available rabbit anti-mouse IgG (Abcam company ab46540), and the detection area 6 on the detection pad is immobilized with acetylcholinesterase monoclonal antibody (clone number: 1F4-C4-F2 -D5).

[0061] Assembling method of the test strip: Assemble the absorbent filter paper 1, the test pad 2 and the sample pad 4 on the plastic bottom plate according to the figure, and the accessories and the plastic bottom plate can...

Embodiment 3

[0068] The specific method is the same as in Example 1 and Example 2, except that the diameter of the colloidal gold pad used is 20nm, and the concentration of the monoclonal antibody (clone number: 2H12-F-7) bound to the colloidal gold is 58 μg / ml ; The detection zone 6 on the nitrocellulose membrane is a paired monoclonal antibody (clone number: 1F4-C4-F2-D5). Apply different concentrations of acetylcholinesterase PBST solution (concentrations are 1ng / ml, 10ng / ml, 100ng / ml, 1μg / ml, 10μg / ml) onto the sample pad, and run the sample for 10 minutes. If there are two obvious red lines in area 6 and quality control area 5, it is a positive result, and if there is only one red line in quality control area 5, it is negative.

[0069] In the present embodiment, when the concentration of acetylcholinesterase was 1 μg / ml, no typical positive results occurred (see Image 6 ).

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Abstract

The invention discloses a colloidal gold chromatographic rapid diagnosis test paper strip used for detecting acetylcholine esterase and a preparation method thereof. The diagnosis test paper strip comprises, in a successively connected manner on a hard plastic bottom board, water-absorbing filter paper, a cellulose acetate membrane detection pad on which an anti-acetylcholine esterase monoclonal antibody (detection zone) and a rabbit-anti-mouse IgG (quality control zone) are immobilized, a colloidal gold pad on which the anti-acetylcholine esterase monoclonal antibody is labeled, and a sample pad for placing a sample. The colloidal gold test paper strip is used for detecting the acetylcholine esterase in a solution by naked eyes and has the following advantages: 1) usage amounts of reagents and sample are very low, wherein the sample size is low to 10-20 [mu]l, so that the concentration of the acetylcholine esterase in a specimen eluant can be increased without less possibility of false negative results; 2) the test paper strip is free of expensive instruments and adapts field application better; 3) detection time is greatly reduced and detection speed is increased; and 4) the test paper strip can be preserved at room temperature and only needs to keep dry.

Description

technical field [0001] The invention relates to the field of immunodiagnostic detection, in particular to a colloidal gold chromatography rapid diagnostic test strip for detecting acetylcholinesterase and a preparation method thereof. Background technique [0002] With the development of social industry, the incidence of peripheral nerve injury is increasing year by year. At present, the main treatment method is peripheral nerve suture reconstruction technique. The biggest challenge in nerve suturing is how to quickly and accurately identify the motor bundle and sensory bundle in the nerve trunk during surgery. At present, there is still a lack of clinical methods that can accurately identify the nature of peripheral nerve bundles in a short period of time. Once the wrong anastomosis of different types of nerve bundles occurs, it will seriously affect the recovery rate of postoperative nerve function. Therefore, how to quickly and accurately identify the nature of peripher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N33/577G01N33/531
CPCG01N33/531G01N33/558G01N33/577G01N33/54387
Inventor 眭涛戈应滨曹晓建唐健葛大伟
Owner JIANGSU PROVINCE HOSPITAL
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