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Human mycoplasma pneumoniae monoclonal antibody and its application

A technology of mycoplasma pneumoniae and monoclonal antibody, which is applied in the field of immunology and vaccinology, can solve the problems of low positive separation rate, high nutritional requirements, complicated operation, etc., and achieve the effect of large application prospect, strong affinity and good specificity

Active Publication Date: 2020-03-17
SINOVAC RES & DEV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are many diagnostic and detection methods for human Mycoplasma pneumoniae. Isolation and culture is the gold standard for the diagnosis of Mycoplasma pneumoniae. However, this method is complicated to operate, takes a long time to cultivate, requires high nutrition, and has a low positive rate of isolation. It cannot be diagnosed quickly and early, and is not suitable for clinical practice. Has not been a routine practice for testing
Molecular biology diagnostic methods have high requirements on laboratory conditions and operator skills, and are not suitable for wide application

Method used

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  • Human mycoplasma pneumoniae monoclonal antibody and its application
  • Human mycoplasma pneumoniae monoclonal antibody and its application
  • Human mycoplasma pneumoniae monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 immunogen preparation and animal immunization

[0037] (1) Cultivate 20L of mycoplasma arginini (bacteria are purchased from ATCC, product number 15531) with the mycoplasma arginini broth culture medium that contains 20% calf serum, and formaldehyde in 1:2000 to the harvesting liquid of mycoplasma pneumoniae Inactivate at 37°C for 120h.

[0038] (2) Concentration of harvested solution of Mycoplasma pneumoniae: the inactivated solution of Mycoplasma pneumoniae was subjected to 300KD 0.1m 2 Concentrate by membrane bag ultrafiltration to 700ml.

[0039] (3) Centrifuge the human Mycoplasma pneumoniae concentrate with 30% and 55% sucrose at 30,000 r / min for 12 hours, and use 0.01mol / L PBS to wash and desugar the human Mycoplasma pneumoniae obtained by centrifugation.

[0040] (4) The protein content of Mycoplasma pneumoniae measured by the Lowry method was 2.7 mg / ml, and the protein concentration was diluted to 500 μg / ml.

[0041] (5) 1 mL of the purified Mycop...

Embodiment 2

[0043] Example 2 Cell Fusion and Strain Construction

[0044] (1) Preparation of myeloma cells: resuscitate and culture SP2 / 0 cell line two weeks before cell fusion, expand culture 3 days before fusion, and change medium of SP2 / 0 cells 1 day before fusion.

[0045] (2) Spleen cell preparation: sacrifice the mice for animal immunization, and prepare the mouse spleen cell suspension according to the conventional method.

[0046] (3) Add an appropriate amount of serum-free 1640 culture medium to splenocytes and myeloma cell SP2 / 0 according to the counting results, shake and mix SP2 / 0 cells, and pipette splenocytes evenly.

[0047] (4) Mix splenocytes and SP2 / 0 cells in a 50ml centrifuge tube at a ratio of 2:1 to 5:1, and mix well.

[0048] (5) Add serum-free 1640 culture medium to 50ml, centrifuge at 1500rpm for 5min, pour out the supernatant as much as possible, and use a pipette to suck up the liquid from the nozzle.

[0049] (6) Lightly tap the bottom of the fusion tube to l...

Embodiment 3

[0060] Example 3 Monoclonal Antibody Cell Line Ascites Preparation and Antibody Titer Detection

[0061] The frozen hybridoma cell MP-H8 obtained in Example 2 was revived according to conventional methods, cultured, and the cell growth status was observed under a microscope, 175cm 2 The cell culture flasks were inoculated when the cells covered 70%-80% of the bottom of the flasks; BALB / c female mice were inoculated intraperitoneally according to conventional methods, and ascites were collected regularly.

[0062] Ascites identification of human Mycoplasma pneumoniae monoclonal antibody: the titer of antibody was detected by indirect ELISA method. Antibody titer test results: the antibody titer of the ascites was detected after the human Mycoplasma pneumoniae 1 μg / ml was coated overnight, and the antibody titer was 10 7 .

[0063] Table 1 Antibody Titer Test Results

[0064]

[0065] Purification of human mycoplasma pneumoniae monoclonal antibody:

[0066] The ascites wa...

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Abstract

The invention provides a human mycoplasma pneumoniae monoclonal antibody and an application thereof and belongs to the field of immunology. According to the human mycoplasma pneumoniae monoclonal antibody, human mycoplasma pneumonia is cultivated, condensed and purified, and high-purity human mycoplasma pneumonia is obtained; after a small mouse is inoculated against the high-purity human mycoplasma pneumonia in an immune mode, mouse spleen cells are fused with mouse myeloma cells, and a hybridoma cell strain MP-H8 capable of steadily secreting the human mycoplasma pneumoniae monoclonal antibody is screened. The preservation number of the hybridoma cell strain MP-H8 is CGMCC No.11287; the titer of the monoclonal antibody secreted by the hybridoma cell strain MP-H8 is high, and can reach 107; specificity is good; affinity is strong; and the affinity constant reaches to 3.92x10<8> L / mol. The human mycoplasma pneumoniae monoclonal antibody can be used for preparing a content detecting reagent box of the human mycoplasma pneumonia and an antibody detecting reagent box, and also used for the differential diagnosis of the human mycoplasma pneumonia, so that the human mycoplasma pneumoniae monoclonal antibody has wide application prospects and market value.

Description

technical field [0001] The present invention relates to the fields of immunology and vaccinology, and in particular relates to an anti-human mycoplasma pneumoniae monoclonal antibody, a hybridoma cell line producing the antibody and the application of the antibody. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae) is a common pathogen with a simple structure, no cell wall, and a high degree of pleomorphism. It is transmitted through the respiratory tract and has a high infection rate in children. In addition to causing pneumonia, Mycoplasma pneumoniae infection can also cause extrapulmonary infections, such as bronchitis, tracheitis, pharyngitis, tonsillitis, etc., and can also induce asthma. Mycoplasma pneumoniae has no cell wall, so antibacterial drugs that act on the cell wall, such as penicillins and cephalosporins, are not sensitive. Children are in the growth and development stage, and there are fewer drug options for treating children with My...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569A61K39/40A61P31/04
CPCA61K39/00C07K16/1253C07K2317/92
Inventor 童钦张博金亚明陈慧芬张星星高强尹卫东
Owner SINOVAC RES & DEV
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