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Immune colloidal gold test strip for rapidly detecting paralytic shellfish poison and preparation method thereof

A colloidal gold test paper and paralytic technology, which is applied in the field of rapid detection test strips of paralytic shellfish poisoning, can solve the problems of expensive instruments, large variability of analysis results, long detection time, etc., and achieve simple and easy operation and high detection sensitivity The effect of high and low detection cost

Inactive Publication Date: 2012-02-08
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 toxins, has large interference, and is not accurate.
The now widely accepted and relatively complete high-performance liquid chromatography-derived fluorescence analysis method is used in many countries. It can accurately analyze and determine the content and structure of toxins, and the detection limit can be as low as ng / g. The disadvantage is that the sample pretreatment is cumbersome and time-consuming, and the instrument is expensive. , the technical requirements for operators are high, which seriously hinders the popularization and promotion of this analysis method, and it is impossible to realize on-site detection.
The immunochemical method is an analysis 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 number of samples under certain conditions. Rapid screening; at present, the ELISA detection kits for PSP produced by companies such as Germany and Japan are based on the principle of competitive enzyme-linked immunosorbent reaction. The whole detection process takes 2-4 hours, and multiple samples can be detected at one time, which can be qualitative It can also be quantified, but there is a problem of false positives; because ELISA is a very sensitive technique, the variability of the analysis results is large, and the results of different analysts are different, and the result judgment often requires experienced operators, which is prone to false detection At the same time, there are many chemical structures of paralytic shellfish poisoning components, and there are certain antigenic differences among the isomers, and the detection time is relatively long, so it is difficult to realize the real field operation

Method used

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  • Immune colloidal gold test strip for rapidly detecting paralytic shellfish poison and preparation method thereof
  • Immune colloidal gold test strip for rapidly detecting paralytic shellfish poison and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0030] Embodiment 1 (preparation embodiment)

[0031] Preparation method of immune colloidal gold test strip for detecting paralytic shellfish poisoning

[0032] 1. Preparation of saxitoxin-carrier protein conjugates

[0033] Dissolve saxitoxin (STX) in 0.002M sodium acetate buffer, add hemocyanin (KLH) or bovine serum albumin (BSA) dissolved in phosphate buffer (pH7.0) and a small amount of formaldehyde, 25 °C for 72 hours, then overnight at 4 °C. The reaction mixture was dialyzed for 3 days at 4°C, and the medium was changed every 12 hours. The STX-KLH and STX-BSA conjugates were prepared for use.

[0034] 2. Preparation of anti-paralytic shellfish poison monoclonal antibody

[0035]The prepared saxitoxin-hemocyanin conjugate (STX-KLH) was used as an immunogen to immunize female BALB / c mice aged 6-8 weeks. For the first immunization, 150 μg of STX-KLH and an equal volume of complete Freund's adjuvant were used, and after being fully emulsified, the mice were injected in...

Embodiment 2

[0043] Embodiment 2 (application embodiment)

[0044] Method of using immunocolloidal gold test strip for detecting paralytic shellfish poisoning

[0045] 1. Pretreatment of shellfish and other samples

[0046] Weigh 3 g of shellfish homogenate, add 3 ml (dilute HCL, pH=4), stir well, boil for 5 min, centrifuge at 3000 r / min for 10 min, and take the supernatant for analysis.

[0047] 2. Detection

[0048] Take 80 μL of the above-mentioned shellfish extract into the sample hole with a micropipette, and let it stand for 15 minutes to observe the color development. At the same time, take 80μl 0.01M PBS and 80μL 110ng / mL saxitoxin STX standard solution in the sample wells of the other two test strips, and do negative blank and positive standard control experiments.

[0049] 3. Result judgment

[0050] like figure 2 As shown, if the color of the detection line of the test strip of the sample to be tested is lighter than that of the negative blank test strip, and a red band ap...

Embodiment 3

[0051] Embodiment 3 (application embodiment)

[0052] Application of immunocolloidal gold test strips for detecting paralytic shellfish poisoning

[0053] Simulated detection experiment of paralytic shellfish poison in samples

[0054] Add paralytic shellfish poison saxitoxin standard substance respectively in shellfish meat, make the sample test solution of 0ng / ml, 200ng / ml and 110ng / ml by embodiment 2, and 3 negative samples and 12 positive samples (mouse biological method), and according to the method described in embodiment 2, with test strip of the present invention to the sample detection of 0ng / ml, 200ng / ml and 110ng / ml, repeat experiment 3 times, the result shows that the 0ng / ml sample The color of the test strip test line is the same as that of the negative blank test line, and at the same time, a red band appears on the quality control line, which is judged as a negative sample; while the test strip test line of the 200ng / ml and 110ng / ml samples has no color or is v...

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Abstract

The invention relates to a detection device for detecting red tide virus. An immune colloidal gold test strip for rapidly detecting paralytic shellfish poison disclosed herein comprises a sample pad, a gold jed pad, a Sartorius NC film, an absorbent pad and a PVC backing, wherein, the sample pad, the gold jed pad, the Sartorius NC film, and the absorbent pad are attached to the PVC backing orderly, the gold jed pad is coated with a colloidal gold-labeled paralytic shellfish poison monoclonal antibody, and the Sartorius NC film is respectively coated with a detection line consisting of Saxitoxin-bovine serum albumin conjugate and a quality control line consisting of goat-anti-mouse antibodies. The test strip has the advantages of strong specificity, high detection sensitivity, rapid detection, simple pre-treatment, no need of any device, convenience in carrying, low cost of the detection, simple operation, no need of professional personnel to operate, convenient storage, good stability, and high accuracy and high precision of the detection result, and the test strip can be stored for at least 6 months at room temperature.

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 Paralytic Shellfish Poisoning (PSP), and also relates 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. Paralytic Shellfish Poisoning (PSP) is one of the most common red tide biotoxins or shellfish poisons in coastal waters of my country, and is often detected in shellfish in coastal waters. Various algae, such as Alexandrium tamarense, can produce paralytic shellfish poisoning. Paralytic shellfish poisoning (PSP) is a class of compounds with a tetrahydropurine structure, which is alkaline, easily soluble in water, relatively stable under weak acid and low temperature conditions, and oxidized under alkaline conditions. More than 20 kinds of such compounds have been found, which can be divided into four categ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
Inventor 刘仁沿梁玉波刘磊许道艳
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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