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Pertussis PRN protein monoclonal antibody and application thereof

A monoclonal antibody and pertussis technology, applied in the direction of antibodies, anti-bacterial immunoglobulins, anti-bacterial drugs, etc., can solve the problems such as the loss of efficacy of pertussis vaccines, and achieve high specificity, high sensitivity and accurate detection effect

Active Publication Date: 2015-09-09
ABMAX BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Potential progressive loss of potency of current pertussis vaccines due to antigenic drift and continued selection of circulating strains less susceptible to vaccine strains has thus far not been demonstrated

Method used

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  • Pertussis PRN protein monoclonal antibody and application thereof
  • Pertussis PRN protein monoclonal antibody and application thereof
  • Pertussis PRN protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, establishment of hybridoma cell line

[0029] 1. Experimental materials

[0030] 1. Immunogen: The PRN protein in pertussis vaccine (purchased from Hualan Bioengineering Co., Ltd.) was used as the immunogen.

[0031] 2. Medium: DMEM medium was purchased from Hyclone Company; HAT, HT selective medium, and pristane were purchased from Sigma Company.

[0032] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture.

[0033] 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.

[0034] 2. Establishment of hybridoma cell lines

[0035] 1. Animal immunity

[0036] 1) Basic immunization: The immunogen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, and injected sub...

Embodiment 2

[0050] The preparation of the monoclonal antibody of embodiment 2 anti-PRN protein

[0051] Primary 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 Protein G~Sepharose CL-4B, the upper column solution is 20mM PBS buffer solution, and the column chromatography eluent is: pH2.7, 20mM glycine buffer solution to obtain monoclonal antibodies against PRN protein respectively, wherein The mono...

Embodiment 3

[0059] Example 3 Preparation of Anti-Pertussis PRN Protein Detection Reagent Using Purified Antibody

[0060] 1. Anti-PRN protein ELISA double antibody sandwich method

[0061] Using 4C8 and 2B9 antibodies for pairing experiments, it was determined that 4C8 was used as the coating antibody, and HRP-labeled 2B9 was used as the detection antibody. The ELISA detection method was determined, and the detection sensitivity of the kit could reach 7.8125ng / ml ( figure 2 ). Antibodies were labeled with the modified sodium periodate method.

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Abstract

The invention provides a pertussis PRN protein monoclonal antibody and an application thereof, which belong to the biological product field. The monoclonal antibody is prepared by taking PRN protein in bordetella pertussis as immunogen immune mice, the obtained two monoclonal antibodies are respectively as, an amino acid sequence of a light-chain variable region is shown in a SEQ ID No.1, the amino acid sequence of a heavy-chain variable region is shown in a SEQ ID No.2, the amino acid sequence of the light-chain variable region is shown in a SEQ ID No. 3, and the amino acid sequence of the heavy-chain variable region is shown in a SEQ ID No.4. The provided monoclonal antibody enables no cross reaction with bordetella pertussis other protein and other antigen and pathogen, has the advantages of high specificity and high sensitivity for detecting the pertussis PRN components, can accurately detect PRN content of pertussis vaccine in a sample, and can be widely used in vaccine and clinical detection.

Description

technical field [0001] The present invention relates to a hybridoma cell line and the monoclonal antibody produced by it and its application, in particular to the monoclonal antibody of PRN protein in pertussis bacteria, the hybridoma cell line secreting the antibody and the application of the antibody. Background technique [0002] Pertussis is an acute respiratory infectious disease that seriously threatens the health of infants and young children. It is highly contagious and the population is generally susceptible. Even in countries with high vaccination coverage, whooping cough remains a relatively high public health concern. According to WHO estimates, there were about 17.6 million whooping cough cases in the world in 2003, 90% of which occurred in developing countries, and about 279,000 people died of it. It is also estimated that global vaccination against pertussis in 2003 averted approximately 38.3 million infections and 607 000 deaths. [0003] The causative agen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N5/20A61K39/395A61P31/04G01N33/577G01N33/569C12R1/91
Inventor 孙乐李茂华张翠娟张小刚李英姿陈兴张萍萍
Owner ABMAX BIOTECHNOLOGY CO LTD
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