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A kind of monoclonal antibody and its application

A monoclonal antibody and polyclonal antibody technology, applied in the biological field, can solve problems such as prone to false positives, short detection time, expensive equipment, etc., and achieve the effects of low cost, convenient detection, and easy operation

Active Publication Date: 2021-10-29
宁波博肽生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological detection has high accuracy and short detection time, but the equipment is expensive and prone to false positives
Serological detection is simple and fast, and the detection rate is high after 7 days of onset. Zika virus IgM antibodies have strong cross-reactions with flaviviruses such as dengue virus, yellow fever virus, and West Nile virus, and are prone to false positives; in short, A simple, rapid and sensitive detection method for Zika virus infection is still lacking in the current market of diagnostic reagents

Method used

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  • A kind of monoclonal antibody and its application
  • A kind of monoclonal antibody and its application
  • A kind of monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: the preparation of anti-ZIKV-NS1 specific polyclonal antibody and monoclonal antibody

[0041] 1. Antigen preparation

[0042] 1.1, Construction of pET-32a (+)-NS1 prokaryotic expression vector

[0043]Take out the preserved Zika virus (ZIKV, SZ-WIV01) from the -80°C refrigerator, inoculate Vero cells in RMPI-1640 medium containing 1% serum, and culture in a cell culture incubator for 72 hours; collect the supernatant, 12,000×g Centrifuge for 10 minutes, discard the pellet, use the viral DNA / RNA Extraction Kit to extract the supernatant RNA, and use HiScript® II 1st StrandcDNA Synthesis Kit to reverse transcribe into cDNA. Use the cDNA of Zika virus as a template for PCR amplification, verify the PCR products with 2% agarose gel, and recover the target bands that meet the expected results with agarose gel electrophoresis DNA gel; connect the products recovered from the gel To the pMD-19T vector, spread the bacteria on an ampicillin plate and culture over...

Embodiment 2

[0059] Example 2: Establishment of double-antibody sandwich ELISA detection method

[0060] 1. Preparation of HRP-2A7, 4G6, 3F1, 1F4, 3G12, 2C4, 1G2, 2C9, 1F12 probes

[0061] 1) Dissolve 5mg HRP in 0.5mL 0.1mol / L NaHCO 3 solution; add 0.5mL 10mmol / L NaIO 4 Solution, mix well, tightly cap the bottle stopper, and protect from light at room temperature for 2 hours;

[0062] 2) Add 0.75mL 0.1mol / L Na 2 CO 3 mix;

[0063] 3) Add 0.75mL of mouse ascites that has been treated, or purified monoclonal antibody (15mg / mL), and mix well;

[0064] 4) Weigh 0.3g of Sephadex G25 dry powder and add it into the outer cylinder of a 5mL syringe with a glass wool pad on the lower mouth; then transfer the above-mentioned cross-linked product into the outer casing of the syringe; cover it tightly and let it work at room temperature (protect from light) for 3 hours or overnight at 4°C ;

[0065] 5) Use a little PBS to wash out all the cross-linked products, collect the eluate, add 1 / 20 volum...

Embodiment 3

[0110] Example 3: Evaluation of specificity and sensitivity of double-antibody sandwich ELISA

[0111] 1. Coating

[0112] Dilute the rabbit polyclonal antibody with CBS to a final concentration of 10 μg / mL, then add 100 μL of antibody to each well and coat at 37°C for 2 hours;

[0113] 2. Closed

[0114] After the coating is completed, discard the liquid in the well, wash with PBS-T for 3 times, add 200 μL of blocking solution to each well, and block at 37°C for 2 hours;

[0115] 3. Add antigen

[0116] The recombinant NS1 protein or inactivated virus culture supernatant was incubated at 37°C for 2h.

[0117] 3. Add enzyme-labeled antibody

[0118] After the reaction, discard the liquid in the well, wash with PBS-T for 3 times, add 100 μL of HRP-1F12 monoclonal antibody (1:1000) diluted with blocking solution to each well, and react at 37°C for 1 hour;

[0119] 4. Color rendering

[0120] After incubation, discard the liquid in the well, wash with PBS-T for 3 times, add...

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Abstract

The invention discloses a monoclonal antibody, which comprises a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; the present invention will recognize Zika The polyclonal antibody R1 of the virus NS1 protein and the mouse monoclonal antibody 1F12 were used in the preparation of the Zika virus NS1 protein double-antibody sandwich ELISA detection reagent, and the rapid detection method of the Zika virus NS1 protein double-antibody sandwich ELISA was established, which can be detected quickly and accurately Zika virus: This technology integrates the specificity of antibodies and the high sensitivity of ELISA. It is accurate, fast, efficient, specific, sensitive and does not require virus isolation and RNA extraction. It is suitable for import and export quarantine and patient infection. On-site rapid detection of Zika virus is of great significance for the control of the epidemic, ensuring the safety of the country, and guiding the use of clinical drugs in a timely and accurate manner.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double-antibody sandwich ELISA for detecting Zika virus NS1 protein. Background technique [0002] Zika virus (ZIKV) belongs to the Flaviviridae family, the genus Flavivirus, a single-stranded positive-sense RNA virus with a diameter of 20nm, mainly transmitted by Aedes aegypti mosquitoes, and can also be transmitted through blood and sex. The clinical features are mainly fever, rash, arthralgia or conjunctivitis, which can cause death in severe cases. The World Health Organization (WHO) believes that neonatal microcephaly and Guillain-Barré syndrome may be related to Zika virus infection. New evidence suggests Zika virus has potentially adverse effects on the heart, according to findings presented at the American College of Cardiology's 66th Annual Academic Meeting. In recent years, ZIKV has exploded on a large scale in countries around the world, including 1.5 milli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
CPCG01N33/558G01N33/56983G01N33/577G01N2333/08
Inventor 张金阳张立定陈志鑫宋玉竹夏雪山杜雪薇
Owner 宁波博肽生物技术有限公司
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