Immune colloidal gold test paper strip for fast detecting diarrhetic shellfish poison and method for making the same

A technology of diarrhea shellfish poisoning and colloidal gold test paper, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of expensive instruments, high technical requirements, and long detection time, and achieve simple and easy operation, high detection sensitivity, The effect of low detection cost

Inactive Publication Date: 2008-02-27
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the mouse method is simple and easy, and is suitable for the detection of large-scale and batch samples. The disadvantage of this method is that it cannot distinguish the type and structure of the toxin, has large interference, and is not accurate; it is now widely accepted and relatively complete HPLC analysis The method is used in many countries, and can accurately analyze and determine the content and type of toxins, and the detection limit can be as low as ng/g. The popularization and promotion of this kind of analytical method makes it impossible to realize on-site detection
The immunochemical method is an analytical method established by using the specific binding of antibodies to antigens. It has the advantages of high sensitivity, strong specificity, simple and easy operation of equipment, and relatively simple sample pretreatment. It is suitable for on-site monitoring and a large n...

Method used

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  • Immune colloidal gold test paper strip for fast detecting diarrhetic shellfish poison and method for making the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 (preparation embodiment)

[0031] Preparation method of immunocolloidal gold test strip for detecting diarrheal shellfish poisoning acid

[0032] 1. Preparation of halicic acid-carrier protein conjugates

[0033] Dissolve soft sponge acid (OA) in 1 mL of dimethyl sulfoxide, add water-soluble carbodiimide (EDC), N-hydroxysuccinimide (NHS), react at 25 °C for 1.5 hours, add dissolved Hemocyanin (KLH) or ovalbumin (OVA) in phosphate buffer (pH 7.0) was reacted at 25°C for 3 hours. The reaction mixture was transferred to a dialysis card and dialyzed for 3 days at 4°C, changing the medium every 12 hours. After aliquoting the dialyzate, store it at -20°C. The OA-KLH and OA-OVA conjugates were prepared for use.

[0034] 2. Preparation of monoclonal antibody against halicic acid

[0035] Using the prepared soft spongy acid-hemocyanin conjugate (OA-KLH) as an immunogen, immunize female BALB / c mice aged 6-8 weeks. For the first immunization, use 200 μL of OA-KL...

Embodiment 2

[0044] Embodiment 2 (application embodiment)

[0045] How to use the immunocolloidal gold test strip for detecting maic acid

[0046] 1. Pretreatment of shellfish and other samples

[0047] Wash the shellfish samples to remove the sediment, dig them open, remove the shells, take all the muscle tissue, homogenate and grind. Weigh 1 g of homogenate sample, add 4 mL of 80% methanol-water solution, stir and extract for 5 min, centrifuge, discard tissue residue, and supernatant is used for analysis.

[0048] 2. Detection

[0049] The above-mentioned shellfish methanol extract was diluted 4 times with 0.01M PBS, and 80 μL was taken into the sample hole with a micropipette gun, and left for 15 minutes to observe the color development. At the same time, take 80 μL 0.01M PBS and 80 μL 12.5ng / mL halicic acid standard solution in the sample holes of the other two test strips for negative blank and positive standard control experiments.

[0050] 3. Result judgment

[0051] As shown i...

Embodiment 3

[0052] Embodiment 3 (application embodiment)

[0053] Application of immunocolloidal gold test strips for detection of soft spongy acid

[0054] 1. Specificity experiment

[0055] The five components of diarrhea shellfish poisoning, Okadaic acid (Okadaic acid, OA), scallop toxin (pectenotoxin-2, PTX-2), scallop toxin (Yessotoxin, YTX), 13-Desmethyl spirolide C (decMeC, SPX1) and Gymnodimine (GYM) were configured into a sample of 0.1 μM (approximately equal to 80ng / mL), the main component of amnesic shellfish poisoning (Domoic acid, DA) was made into a sample of 1280ng / mL, and puffer fish The toxin (Tetrodotoxin, TTX) was prepared as a sample of 800ng / mL. The experiment was carried out according to the method described in Example 2. The experimental results showed that the detection line of Okadaic acid (OA) sample had no color, and the quality control line appeared red bands; ), scallop toxin (Yessotoxin, YTX), 13-Desmethyl spirolide C (decMeC, SPX1) and Gymnodimine (GYM),...

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Abstract

The invention relates to the detection device of detecting the red tide toxin. An immune colloidal gold test paper to detect the lienteric toxin includes the sample pad, colloidal gold pad, NC film, the suction pad and PVC backing. The PVC backing is adhibited with the sample pad, colloidal gold pad, NC film and the suction pad. The colloidal gold pad is covered with the monoclonal antibody against okadaic acid marked by colloidal gold. The NC membrane is encrusted with the detection line composed by the okadaic acid and ovalbumin and quality controlling line composed by the multi antibody of sheep against rat. The test paper has high specialty, detection sensitivity, quick detection and simple pretreatment character. It needs not any device and convenience to carry and has low detection cost; the test paper has good stability and can be stored for six months in room temperature; the detection result has high veracity and precision.

Description

Technical field: [0001] The invention relates to a detection device for detecting red tide toxin, in particular to a rapid detection test strip for Diarrhetic shellfish poisoning (DSP), and also to a preparation method thereof. Background technique: [0002] The red tide disaster is becoming more and more serious and has become a global marine ecological disaster. Diarrheal shellfish poisoning is a class of fat-soluble polycyclic ether natural compounds produced by toxic red tide algae Pynoflagellates and Prorocentrum. The main components are Okadaic acid (OA) and its derivatives. The food chain transmission of shellfish and other rate-eating animals can cause poisoning to human eaters, resulting in diarrhea, nausea, vomiting and gastrointestinal colic, etc. OA is a strong protein phosphatase inhibitor and a strong cancer-promoting agent. It is a marine biotoxin of great concern at present. The analysis methods of diarrheal shellfish poison mainly include biological mouse ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558
Inventor 梁玉波刘仁沿许道艳刘永健
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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