Enzyme-linked immunosorbent assay kit for rapid detection of gonylin gtx2/3 and its preparation method

A genus algae toxin and kit technology, applied in the direction of anti-animal/human immunoglobulin, anti-fungal/algae/lichen immunoglobulin, microorganism-based methods, etc., can solve the problem of expensive instruments and analytical reagents, early sample preparation Deal with problems such as troublesome and lack of reference materials, and achieve the effect of rapid detection, low detection cost and low cost

Inactive Publication Date: 2011-12-28
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its disadvantages are expensive instruments and analytical reagents, lack of standard subst...

Method used

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Embodiment 1

[0021] Embodiment 1 (preparation embodiment)

[0022] Preparation method of competitive inhibition enzyme-linked immunosorbent assay kit for detecting paralytic shellfish poison gonylin GTX2 / 3

[0023] 1. Preparation of saxitoxin-carrier protein conjugates

[0024]Dissolve saxitoxin (STX) in 0.002M sodium acetate buffer, add hemocyanin (KLH) or ovalbumin (OVA) dissolved in phosphate buffer (pH 7.0), and react at 25°C for 72 hours, and 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-OVA conjugates were prepared for use.

[0025] 2. Preparation of anti-paralytic shellfish poison monoclonal antibody

[0026] 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 inject...

Embodiment 2

[0030] Embodiment 2 (application embodiment)

[0031] Method for using the competitive inhibition enzyme-linked immunosorbent assay kit for detection of gonylin GTX2 / 3

[0032] 1. Shellfish sample processing

[0033] Weigh 3 g of shellfish homogenate, add 3 ml (dilute 16 times with 0.00125M HCL, pH=4.16), stir well, boil for 5 min, centrifuge at 3000 r / min for 10 min, and take the supernatant for analysis.

[0034] 2. Detection

[0035] Use the STX-OVA conjugate as the coating antigen, dilute it with carbonate buffer (pH 9.6) and add it to the 96-well ELISA plate (100 μL / well), overnight at 4°C in a wet box; discard the residual solution and wash (PBS-Tween20) was washed 3 times, and after being patted dry, 300 μL of PBS solution containing 1% PVA was added to each well and incubated at 37°C for 3 hours. ). When measuring, discard the blocking solution, wash the plate 3 times, pat dry, add 50 μL serially diluted GTX2 / 3 standard substance solution or shellfish sample extrac...

Embodiment 3

[0036] Embodiment 3 (application embodiment)

[0037] Application of Indirect Competitive ELISA for the Detection of Paralytic Shellfish Poison GTX2 / 3

[0038] 1. GTX2 / 3 specific antibody titer and optimum working concentration experiment

[0039] After the start of immunization, the tail blood of the immunized mice was collected at the fifth and seventh weeks respectively, and the serum titer (titer) was determined by direct ELISA method. After 5 times of immunization, the titer can reach more than 11,000. The ascites titer of the monoclonal antibody produced after cell fusion is more than 18,000. Through the square array titration test, in the direct ELISA method, according to the absorbance value of monoclonal antibody ascites diluted in different times, the absorbance value ranges from 1.2 to 1.7, and the optimal dilution ratio of the coated antigen is 1:800; goat anti-rabbit enzyme The optimal dilution ratio of the labeled secondary antibody is 1:20000, the optimal dil...

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PUM

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Abstract

The invention relates to a detection technology for detecting paralytic shellfish poisoning, in particular to a method for preparing an anti-paralytic shellfish poisoning monoclonal antibody by using protein coupling to synthesize an antigen, and relates to the use of monoclonal antibody and gonylin GTX2/3 antigen Combination, and the preparation method of the quick detection kit that analyzes the content of gonylin GTX2/3 in the biological sample. An enzyme-linked immunoassay kit for rapid detection of the main component of paralytic shellfish poisoning, gonyellin GTX2/3, including a 96-well microtiter plate with detachable strips, enzyme-labeled secondary antibody, and buffer salt wash containing Tween-20 solution, substrate solution and stop solution, using the prepared monoclonal antibody containing specific anti-gonitoxin GTX2/3, on the 96-well ELISA plate coated with the coated antigen of the synthetic protein conjugate. The kit of the invention has the advantages of simple pretreatment, easy operation, and is suitable for on-site, fast, and large-volume sample detection. The kit has low cost and low price, and the detection limit of the kit is less than 15ng/ml.

Description

Technical field: [0001] The invention relates to a detection device for detecting red tide toxin, a rapid detection kit for paralytic shellfish poisoning (PSP). In particular, it relates to a method of using protein coupling to synthesize antigens to prepare monoclonal antibodies against the paralytic shellfish poison GTX2 / 3, and involves the use of monoclonal antibodies combined with GTX2 / 3 antigens to analyze and detect the GTX2 / 3 in biological samples. Preparation method of rapid and convenient detection kit for paralytic toxin content. Background technique: [0002] The red tide disaster is becoming more and more serious and has become a global marine ecological disaster. Red tide is one of the most serious ecological disasters in my country's marine environment. The sea areas of red tides in our country are expanding year by year, and the disasters are increasing year by year. The economic losses caused by red tides are very huge every year. Red tide organisms secret...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18C07K16/14G01N33/543G01N33/577C12R1/91
Inventor 梁玉波刘仁沿许道艳刘磊
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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