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Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide and its application

A monoclonal antibody and capsular polysaccharide technology, applied in the direction of antibodies, applications, antifungal agents, etc., can solve the problems of poor specificity and weak affinity, and achieve good stability, strong affinity, and good specificity

Active Publication Date: 2022-07-29
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN109879961A discloses an anti-cryptococcus capsular polysaccharide monoclonal antibody and the preparation and application of hybridoma cell lines. The antibody can specifically bind to the cryptococcal capsular polysaccharide and can be used for in vitro detection of cryptococcal infection. The price of the colloidal gold-labeled immunodiagnostic reagent developed with this antibody is as high as more than one million, with excellent affinity and good specific binding ability; However, the above-mentioned monoclonal antibodies are mouse-derived monoclonal antibodies. Although mouse-derived monoclonal antibodies are the most widely used antibodies, there are still problems of weak affinity and poor specificity.

Method used

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  • Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide and its application
  • Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide and its application
  • Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Animal immunization

[0054] (1) Preparation of Cryptococcus capsular polysaccharide (GXM)

[0055] Cryptococcus was inoculated into Sabouraud's liquid medium (1% peptone and 4% D-glucose per liter), cultured at 30°C for about 46 hours, and the shaker speed was 200 rpm / min. Stop the culture when it drops to 4.2~4.5; sterilize the culture solution at 121°C for 40 min with moist heat to kill the bacteria and possible spores, and collect the supernatant by centrifuging the obtained bacteria solution;

[0056] After washing the cells with physiological saline, slowly add calcium acetate powder to a final concentration of 5%, and then add glacial acetic acid to adjust the pH to 5.0; slowly add three times the volume of 95% ethanol to the mixture, precipitate out, and let stand at 4°C overnight; centrifuged to collect the precipitate, dried, and reconstituted with deionized water; added CTAB to treat the reconstituted solution, and centrifuged to collect the precip...

Embodiment 2

[0063] Example 2 Preparation and screening of hybridoma cells

[0064] The titer of the prepared rabbit antiserum was tested, and if qualified, the rabbit spleen was used for cell fusion to prepare a monoclonal hybridoma cell line. The method is as follows:

[0065] (1) Preparation

[0066] The immunized New Zealand big-eared rabbits were killed, and the spleen was taken out under aseptic conditions, washed once with cell culture medium, then ground, passed through a stainless steel mesh, and the obtained cells were centrifuged and washed twice with cell culture medium;

[0067] Take the SP2 / 0 myeloma cells in logarithmic growth phase and mix them with splenocytes, wash them once with cell culture medium without fetal bovine serum, centrifuge, discard the supernatant, add polyethylene glycol solution, and treat at 37°C for about 90 s ; Use cell culture medium without fetal bovine serum to terminate the reaction, centrifuge, resuspend cells with HAT selection medium containing...

Embodiment 3

[0071] Example 3 Isolation of antibody variable region genes from hybridoma cells by RT-PCR

[0072] After homogenizing the hybridoma cells, adding a cell lysate to extract RNA, using isopropanol to precipitate RNA from the aqueous layer, washing the precipitated RNA after centrifugation, removing impurities, resuspending and performing reverse transcription to obtain cDNA;

[0073] PCR was performed with specific primers from New Zealand rabbits, and the hybridoma cell cDNA was used as the template to amplify the variable region genes of the heavy and light chains of the antibody. A 50 μL system contained 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs and 0.5 μM Specific primers were used for PCR amplification under the following conditions: pre-denaturation at 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 50 s, 35 cycles; 72°C for 7 min; 1% agar for the obtained PCR product Glycogel electrophoresis was used to identify, recover the target fragments, send samples for seque...

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Abstract

The present invention provides a monoclonal antibody against Cryptococcus capsular polysaccharide and application thereof, wherein the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 7; The light chain variable region includes the amino acid sequence set forth in SEQ ID NO:8. The monoclonal antibody of the present invention is obtained from New Zealand rabbits, has strong binding activity and neutralization activity to Cryptococcus capsular polysaccharide, has good stability, and can be potentially applied to the detection and identification of clinical samples infected with Cryptococcus And the development of cryptococcal inhibitors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide and its application. Background technique [0002] Cryptococcus neoformans is an important opportunistic fungus that often invades the meninges, lungs and skin, causing fungal infections. Cryptococcus neoformans meningitis (CNM) is the most common type of fungal infection of the central nervous system. [0003] Monoclonal antibodies are highly homogeneous antibodies directed against specific antigenic epitopes produced by a single B cell clone, usually prepared by hybridoma cells. Based on cell fusion technology, sensitized B cells with the ability to secrete specific antibodies and unlimited The proliferative myeloma cells are fused into B cell hybridomas, and after culturing into a cell population, a specific antibody against an epitope, that is, a monoclonal antibody, can be prepared. [0004] Antibo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/14C12N5/20C12N15/13C12N15/85C12N5/10A61K39/395A61P31/10G01N33/569G01N33/577
CPCC07K16/14C12N15/85G01N33/56961G01N33/577A61P31/10C07K2317/92C07K2317/10C07K2317/33A61K2039/505G01N2333/37
Inventor 刘春龙付成华付彦凯翟栓柱盛长忠周泽奇粟艳
Owner DYNAMIKER BIOTECH TIANJIN
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