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Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide, and application thereof

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

Active Publication Date: 2020-03-20
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 application thereof
  • Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide, and application thereof
  • Rabbit-derived monoclonal antibody against Cryptococcus capsular polysaccharide, and application thereof

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Experimental program
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Effect test

Embodiment 1

[0053] Embodiment 1 animal immunization

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

[0055] Inoculate Cryptococcus in Sabouraud liquid medium (each liter of medium contains 1% peptone and 4% D-glucose), culture at 30°C for about 46 hours, and the shaker speed is 200rpm / min, when the pH value of the culture solution drops to Stop the culture at 4.2-4.5; sterilize the culture solution at 121°C for 40 minutes with damp heat to kill the bacteria and possible spores, and centrifuge the obtained bacterial solution to collect the supernatant;

[0056] After washing the bacteria with normal saline, slowly add calcium acetate powder to a final concentration of 5%, 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 stand at 4°C Overnight; collect the precipitate by centrifugation, dry, and redissolve with deionized water; add CTAB to treat the reconstituted solution, and...

Embodiment 2

[0063] Preparation and screening of embodiment 2 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, ground, passed through a stainless steel screen, centrifuged and washed twice with cell culture medium;

[0067] Take SP2 / 0 myeloma cells in the logarithmic growth phase and mix them with splenocytes, wash once with cell culture medium without fetal bovine serum, centrifuge, discard the supernatant, add polyethylene glycol solution, and treat at a constant temperature of 37°C for about 90s; Terminate the reaction with cell culture medium without fetal bovine serum and centrifuge, resuspend the cells in HAT selection medium containing 20% ​...

Embodiment 3

[0071] Example 3 Isolation of Antibody Variable Region Genes from Hybridoma Cells Using RT-PCR

[0072] After the hybridoma cells were homogenized, the cell lysate was added for RNA extraction, the RNA was precipitated from the aqueous layer with isopropanol, the precipitated RNA was washed after centrifugation, impurities were removed, and cDNA was obtained by reverse transcription after resuspension;

[0073] Use the specific primers of New Zealand big-eared rabbits for PCR, and use the hybridoma cell cDNA as a template to amplify the heavy and light chain variable region genes of the antibody. The 50 μL system contains 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs and 0.5 μM specificity Primers, PCR amplification was carried out according to the following conditions: pre-denaturation at 94°C for 5min; 35 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 50s; 72°C for 7min; the obtained PCR products were identified by 1% agarose gel electrophoresis, The target fragments were rec...

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Abstract

The invention provides a monoclonal antibody against Cryptococcus capsular polysaccharide, and an application thereof. A heavy chain variable region of the monoclonal antibody comprises an amino acidsequence as shown in SEQ ID NO:7; and a light chain variable region of the monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO:8. The monoclonal antibody of the invention is obtained from New Zealand big-ear rabbits, has relatively strong binding activity and neutralizing activity to the Cryptococcus capsular polysaccharide, has good stability, and can be potentially appliedto detection and identification of Cryptococcus infected clinical samples, development of Cryptococcus inhibitors, and the like.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a rabbit-derived monoclonal antibody against cryptococcal capsular polysaccharide and application thereof. Background technique [0002] Cryptococcus neoformans is an important conditional pathogenic fungus, which often invades the meninges, lungs and skin, causing fungal infections. Cryptococcal neoformans meningitis (CNM) is the most common type of fungal infection of the central nervous system. In recent years, the morbidity and mortality of CNM have shown an obvious upward trend. According to statistics, about 5% to 10% of AIDS patients in the United States suffer from cryptocephaly, and in some developing countries, the incidence of AIDS patients suffering from cryptocephaly is higher. At the same time, pulmonary cryptococcosis caused by Cryptococcus neoformans infection of the respiratory system is also increasing year by year. [0003] Monoclonal antibody is a highly u...

Claims

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Application Information

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Patent Type & Authority Applications(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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