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A kind of anti-Aspergillus galactomannan monoclonal antibody and its application

A monoclonal antibody and antigen technology, applied in the direction of antibodies, applications, antifungal agents, etc., can solve problems such as weak affinity, and achieve the effect of strong affinity, strong affinity, and high homology

Active Publication Date: 2021-01-01
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN109609466A discloses a mouse anti-Aspergillus polysaccharide hybridoma cell line, monoclonal antibody and application; CN109628410A discloses a mouse anti-Aspergillus polysaccharide hybridoma cell line, monoclonal antibody and application, obtained mouse-derived Aspergillus polysaccharide monoclonal Antibodies and Aspergillus polysaccharides have high titer and strong binding specificity, and can be used for the detection of Aspergillus polysaccharides, but both monoclonal antibodies are mouse monoclonal antibodies, although mouse monoclonal antibodies are the most widely used antibodies, But there is still the problem of weak affinity

Method used

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  • A kind of anti-Aspergillus galactomannan monoclonal antibody and its application
  • A kind of anti-Aspergillus galactomannan monoclonal antibody and its application
  • A kind of anti-Aspergillus galactomannan monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Embodiment 1 animal immunization

[0055] (1) Preparation of Aspergillus galactomannan (GM)

[0056] Inoculate Aspergillus fumigatus in Sabouraud liquid medium (each liter of medium contains 1% peptone and 4% D-glucose), culture at 30°C for about 46h, and the shaker speed is 200rpm / min, when the pH value of the culture solution drops to Stop culturing at 4.2-4.5; sterilize with damp heat at 121°C for 40 minutes to kill bacteria and possible spores, filter the obtained bacteria solution through qualitative filter paper at room temperature, and then filter to remove bacteria with a 0.22 μm filter membrane;

[0057] Transfer the filtrate to a new centrifuge tube, add 4 times the volume of absolute ethanol, and let stand overnight at 4°C; centrifuge at 16,000g for 15 minutes at 4°C, dissolve the precipitate in deionized water, add 4 times the volume of absolute ethanol, and let stand 1h;

[0058] Centrifuge, wash the precipitate three times with absolute ethanol, centrifu...

Embodiment 2

[0064] Preparation and screening of embodiment 2 hybridoma cells

[0065] 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:

[0066] (1) Preparation

[0067] 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;

[0068] 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;

[0069] Terminate the reaction with cell culture medium without fetal bovine serum and centrifuge, resuspend the cells in HAT selection medium contain...

Embodiment 3

[0074] Example 3 Isolation of Antibody Variable Region Genes from Hybridoma Cells Using RT-PCR

[0075] 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;

[0076] 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 for resisting aspergillus galactomannan and an application of the monoclonal antibody. The variable region of heavy chain of the monoclonal antibody comprises an amino acid sequence as shown in SEQID NO:7, and the variable region of light chain of the monoclonal antibody comprises an amino acid sequence as shown in SEQID NO:8. The monoclonal antibody isobtained from New Zealand large-ear rabbits, has high combination activity and neutralization activity for aspergillus galactomannan, is good in stability, and can be potentially applied to detectionand identification of clinical samples infected by aspergillus, research and development of an aspergillus inhibitor and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a monoclonal antibody against Aspergillus galactomannan and application thereof. Background technique [0002] In recent years, due to the extensive use of broad-spectrum antimicrobials and the increase in cancer patients and organ transplant patients, the morbidity and mortality caused by deep fungal infections have increased year by year. Aspergillus fumigatus is an opportunistic pathogen that often infects immunocompromised or immunocompromised patients, and is gradually becoming an important pathogenic fungus. The gold standard for the detection of invasive Aspergillus infection is tissue biopsy and sterile body fluid culture. However, when infected by Aspergillus, the disease develops rapidly. The traditional culture method has a long cycle and a low positive detection rate, which often leads to missed diagnosis and misdiagnosis. , It is easy to delay the condition. Therefore, n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/14C12N15/13C12N5/20C12N15/85A61K39/395A61P31/10G01N33/569G01N33/577
CPCA61K2039/505A61P31/10C07K16/14C07K2317/10C07K2317/14C07K2317/33C07K2317/92C07K2317/94C12N15/85G01N33/56961G01N33/577G01N2333/38
Inventor 刘春龙翟栓柱付成华张舟盛长忠周泽奇粟艳
Owner DYNAMIKER BIOTECH TIANJIN
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