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14 type pneumococcal neutralizing monoclonal antibody and application

A monoclonal antibody and pneumococcal technology, applied in the field of immunology and vaccinology, can solve the problems of low antibody level, high purchase cost, large dosage, etc., and achieve good specificity, good type specificity and potency, and high efficiency price effect

Inactive Publication Date: 2014-12-31
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the widely used antibody is the pneumonia serum for pneumococcus identification from the Danish Serum Institute (SSI). This serum is a rabbit polyantiserum. The purchase cost is high and the purchase cycle is relatively long. There are certain differences between different batches. , the low level of antibodies and the large dosage restrict the research and development of pneumonia vaccines by domestic enterprises

Method used

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  • 14 type pneumococcal neutralizing monoclonal antibody and application
  • 14 type pneumococcal neutralizing monoclonal antibody and application
  • 14 type pneumococcal neutralizing monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 immunogen preparation and animal immunization

[0037] (1) Soybean peptone comprehensive medium was used to ferment the bacterium of type 14 pneumococcus strain 31602 (sourced from China Medical Bacteria Collection and Management Center).

[0038] (2) After fermentation, add formalin to the fermented cells to a final concentration of 2%, and sterilize overnight at 2-8°C.

[0039](3) Centrifuge at 8000rpm for 10 minutes, discard the supernatant, collect the bacteria, and blow and suspend the bacteria with 0.01M PBS / 0.5% formalin.

[0040] (4) Grinding the bacteria, resuspending and diluting with 0.01M PBS / 0.5% Forin Malin after grinding.

[0041] (5) Mix the above-mentioned resuspended bacteria with Freund's complete adjuvant in equal volumes, and immunize BALB / c mice with the emulsion formed on day 0, day 14, and day 28, 0.2ml / mouse.

[0042] (6) One week later, the blood was collected to detect the antibody titer. If it was higher than 1:1000, the emulsi...

Embodiment 2

[0043] Example 2 cell fusion

[0044] (1) Preparation of myeloma cells: two weeks before cell fusion, the SP2 / 0 cell line was revived and cultured, expanded and cultured 3 days before fusion, RPMI 1640 cell culture medium (Gibco) was removed one day before fusion, and culture medium was added again.

[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 appropriate amount of incomplete IMDM medium (Gibco) to splenocytes and myeloma cell SP2 / 0 according to the counting results, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly.

[0047] (4) Mix splenocytes and SP2 / 0 cells in a ratio of 1:2 to 10:1 in a 50ml centrifuge tube, and mix well.

[0048] (5) Add incomplete IMDM culture medium to 50 ml, centrifuge for 5 minutes, pour off the supernatant as much as possible, and use a pipette to suck up the liquid from the nozzle.

[0049] (6) L...

Embodiment 3

[0059] Cloning of embodiment 3 hybridoma cells (limiting dilution method)

[0060] (1) Hybridoma cells were counted, and the hybridoma cells were diluted with HT medium containing 20% ​​serum.

[0061] (2) Diluted hybridoma cells were added to a 96-well plate for culture, 100 μl per well.

[0062] (3) 37°C, 5% CO 2 Wet culture for 7-10 days, when clones visible to the naked eye appear, detect antibody activity in time. Observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection.

[0063] (4) Move the cells in the positive wells to a 24-well plate for expanded culture, and then transfer to a 25 or 175 cell bottle for expansion.

[0064] (5) Freeze the cell lines as soon as possible after amplification, number them, and store them in liquid nitrogen.

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Abstract

The invention relates to an anti 14 type pneumococcal capsular polysaccharide neutralizing monoclonal antibody and a hybridoma cell strain for producing the anti 14 type pneumococcal capsular polysaccharide neutralizing monoclonal antibody, and the accession number of the hybridoma cell strain is CGMCC No.9455. The monoclonal antibody is high in titer and good in specificity, can be effective in vitro neutralization of 14 type pneumococcal infection, can effectively remove bacteria, has wide application prospect in the aspects of pneumonia diagnosis and treatment, can be applied to the pneumonia vaccine polysaccharide content detection, can be used for the polysaccharide content detection by enzyme linked immunosorbent assay, and breaks through the current detection methods.

Description

technical field [0001] The invention relates to the field of immunology and vaccinology, in particular to a neutralizing monoclonal antibody against type 14 pneumococcal capsular polysaccharide, a hybridoma cell line producing the antibody and the application of the antibody. Background technique [0002] Streptococcus pneumoniae, also known as Streptococcus pneumoniae or Diplococcus pneumoniae, has 91 serotypes and is a common Gram-positive diplococcus pathogen. In 1881, Pasteur in France and G.M.Sternberg in the United States were the first to isolate the Gram-positive diplococcus, Streptococcus pneumoniae, from patient sputum in France and the United States, and named it Pasteur. Micrococcus. In 1886, Weichselbaum discovered that pneumococcus is the main pathogenic bacteria of lobar pneumonia in humans, which can cause pneumonia and other diseases. Before the advent of sulfa drugs and antibiotics, pneumococcal morbidity and mortality were extremely high. In the 1930s a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577C12R1/91
Inventor 蔡芳王欣陈磊童钦高强尹卫东
Owner SINOVAC RES & DEV
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