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Rabies virus nucleoprotein monoclonal antibody and application thereof

A technology of monoclonal antibody and rabies virus, which is applied in the field of viral protein immune analysis, can solve problems that plague human beings, and achieve high sensitivity and high specificity

Active Publication Date: 2016-04-20
ABMAX BIOPHARMACEUTICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a monoclonal antibody that can specifically bind to the nucleoprotein in rabies virus and produce the antibody against the current situation that rabies still plagues human health and there are still various difficulties in the prevention and treatment process. hybridoma cells

Method used

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  • Rabies virus nucleoprotein monoclonal antibody and application thereof
  • Rabies virus nucleoprotein monoclonal antibody and application thereof
  • Rabies virus nucleoprotein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The establishment of embodiment 1 hybridoma cell line

[0027] 1. Experimental materials

[0028] 1. Immunogen: The rabies virus SRV9 strain was used as the immunogen.

[0029] 2. Culture medium: DMEM medium was purchased from Hyclone Company; HAT, HT selection medium and pristane were purchased from Sigma Company.

[0030] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0031] 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 Company; other reagents were domestic analytically pure products.

[0032] 2. Establishment of hybridoma cell lines

[0033] 1. Animal immunity

[0034] 1) Basic immunization: The immunogen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, and injected subcutaneously in points, each injection volume of...

Embodiment 2

[0050] The preparation of the monoclonal antibody of embodiment 2 anti-rabies virus nucleoproteins

[0051] 1. Antibody preparation

[0052] Adult BALB / c mice were selected, and pristane was inoculated intraperitoneally, 0.5ml per mouse. After 7-10 days, the 16th generation cells of the two strains of hybridoma cells in Example 1 were inoculated into the peritoneal cavity respectively, and each mouse was 1×10 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.

[0053] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Purify with ProteinG~SepharoseCL-4B, the upper column liquid is 20mM PBS buffer, and the column chromatography eluent is: pH 2.7, 20mM glycine buffer, respectively, to obtain monoclonal antibodies against rabies virus, which specifically recog...

Embodiment 3

[0063] Example 3 Application of Purified Antibody to Prepare Anti-rabies Virus Detection Reagent

[0064] ELISA double-antibody sandwich method: 5H7 and 1C7 antibodies were used for pairing experiments, and 5H7 was determined as the coating antibody, and HRP-labeled 1C7 was used as the detection antibody. The ELISA detection method was determined. The detection sensitivity of the kit can reach 0.003125IU / ml. The results are shown in Table 3, and the fitting curve formulated according to the results is as follows Figure 4 shown. Antibodies were labeled with the modified sodium periodate method.

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Abstract

The invention provides a rabies virus nucleoprotein monoclonal antibody and application thereof. The amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.2; or the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.4. The monoclonal antibody has no cross reaction with other kinds of protein of rabies viruses, other antigens and pathogens, has the advantages of high specificity and high sensitivity, can accurately detect the content of each effective component in a rabies combined vaccine in a sample and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of virus protein immune analysis, and in particular relates to a rabies virus nucleoprotein monoclonal antibody and an application thereof. Background technique [0002] Rabies is a Class B infectious disease under the statutory reporting management in my country. It is one of the acute infectious diseases with the highest human mortality rate so far. Its pathogen is Rabies virus, and humans and all warm-blooded animals are susceptible to this virus. According to the statistics of my country Center for Disease Control and Prevention, in 2014, there were 969 rabies cases and 873 deaths in China. The lethal rate of rabies is close to 100%. Although human anti-rabies virus antibody drugs have been greatly developed in recent years, there is still no very effective therapeutic drug developed. Therefore, rabies vaccine is still the most effective way to prevent and treat rabies. s method. [0003] The post-expo...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20C12N15/13G01N33/68G01N33/577G01N33/569A61K39/42A61P31/14C12R1/91
Inventor 孙乐李茂华王慕旸李英姿
Owner ABMAX BIOPHARMACEUTICALS
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