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Monoclonal antibody for hand-foot-mouth EV71 virus and application thereof

A monoclonal antibody and virus technology, applied in the direction of antiviral immunoglobulin, instruments, biochemical equipment and methods, etc., can solve the problems of discomfort and rapid detection, inability to distinguish EV71 and CA16, etc., and achieve good application prospects

Active Publication Date: 2012-07-18
ABMAX BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the effective detection of EV71 virus monoclonal antibody in China. There are related antibodies abroad. However, this antibody has cross-reaction with CA16, and EV71 and CA16 cannot be distinguished, and this antibody is not suitable for direct and rapid detection of patient samples.

Method used

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  • Monoclonal antibody for hand-foot-mouth EV71 virus and application thereof
  • Monoclonal antibody for hand-foot-mouth EV71 virus and application thereof
  • Monoclonal antibody for hand-foot-mouth EV71 virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, establishment of hybridoma cell line

[0025] 1. Experimental materials

[0026] 1. Immunogen: In this example, the VP1 protein of EV71 virus is used as the target protein, and two polypeptide sequences are selected from it. The sequences are as follows: SP55: PESRESLAWQTATNPC, SP70: YPTFGEHKQEKDLEYC. The two polypeptides are prepared by chemical synthesis, and the purity requirement is greater than 90 %. The two polypeptides are respectively coupled with KLH to prepare an immunogen.

[0027] 2. Culture medium: DMEM medium was purchased from Hyclone Company; HAT, HT selection medium and pristane were purchased from Sigma Company.

[0028] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0029] 4. Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma Company; PEG4000 was purchased from Fluka Company; HRP-goat anti-mouse IgG antibody was purchased from JacksonImmune ...

Embodiment 2

[0058] The preparation of the monoclonal antibody of embodiment 2 anti-EV71

[0059] Primary antibody preparation

[0060] Adult BALB / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation hybridoma cells were inoculated intraperitoneally, 1×10 per mouse 6 -2×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.

[0061] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Carry out purification with Protein G~Sepharose CL-4B, the PBS damping fluid of upper column liquid is 20mM, and the eluent of column chromatography is: the glycine buffer fluid of pH2.7, 20mM, obtain the monoclonal antibody against EV71 virus (will be obtained by SP55 The monoclonal antibody produced by the sequence is called clo...

Embodiment 3

[0071] Example 3 Application of Purified Antibody to Prepare EV71 Virus Detection Reagent

[0072] 1. ELISA double antibody sandwich method

[0073] Using clone 20D12 and Clone 22A12 antibodies for pairing experiments, it was confirmed that clone22A12 was used as the coating antibody, and HRP-labeled Clone 20D12 was used as the detection antibody. The ELISA detection method was determined, and the detection sensitivity of the kit could reach 1ng / mL ( Figure 4 ). Antibodies were labeled with the modified sodium periodate method.

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Abstract

The invention provides a monoclonal antibody for hand-foot-mouth EV71 virus, which is obtained through immunogen preparation by using VP1 protein of the EV71 virus as target protein to design and synthesize a polypeptide sequence and coupling the polypeptide sequence with vector protein serving as immunogen. The monoclonal antibody has no cross reaction with other proteins of the EV71 virus, CA16virus, other antigens or pathogens, has the advantages of high specificity and high sensitivity in detection, can accurately detect the level of the EV71 virus in a detected sample, and is expected to be widely applied in clinical detection.

Description

technical field [0001] The present invention relates to a hybridoma cell line and the monoclonal antibody produced therefrom and applications thereof, in particular to a monoclonal antibody against enterovirus 71, one of the pathogens of hand, foot and mouth disease, a hybridoma cell line secreting the antibody and the application of the antibody. technical background [0002] Hand, foot, and mouth disease (HFMD) is an infectious disease caused by enteroviruses, which mostly occurs in infants and young children under the age of 5, and can cause fever, rashes, and ulcers on hands, feet, and mouth. Individual patients can cause myocarditis, pulmonary edema, aseptic meningoencephalitis and other complications. There are more than 20 kinds of enteroviruses that cause HFMD, among which Coxsackieviruses 16 (CA16) and enterovirus 71 (Enterovirus71, EV71) are the most common. Enterovirus 71 is referred to as EV71. In 1957, the first large-scale outbreak broke out in New Zealand and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/577
Inventor 孙乐毛春明马树君王晓庆付非孙哲
Owner ABMAX BIOTECHNOLOGY CO LTD
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