Anti-cryptococcal capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof

A hybridoma cell line, monoclonal antibody technology, applied in application, antifungal/algae/lichen immunoglobulin, botanical equipment and methods, etc., can solve the problem of high price and achieve good affinity and good specificity The effect of binding capacity

Active Publication Date: 2019-06-14
GENOBIO PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is only one rapid immunoassay product on the market for the clinical detection of cryptococcus, namely, the American Immuno-Mycologics company’s cryptococcal antigen detection kit (colloidal gold immunochromatography), which is expensive

Method used

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  • Anti-cryptococcal capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof
  • Anti-cryptococcal capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof
  • Anti-cryptococcal capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Preparation method of monoclonal antibody against cryptococcal capsular polysaccharide

[0085] 1. Polysaccharide extraction

[0086]Cultivate cryptococcus (the strain number is ATCC208821) with liquid Sabouraud medium, culture at 20-25°C for 3-4 days, centrifuge the supernatant after the bacteria solution is inactivated, add sodium acetate to the supernatant to a final concentration of 5 %, adjust the pH=5.0; add 3 times the volume of absolute ethanol, overnight at 4°C, a creamy precipitate at the bottom can be seen; after discarding the supernatant, the precipitate is cryptococcal capsular polysaccharide, air-dry the precipitate, and weigh (or calculate the mass by concentration) The precipitate was dissolved in 0.2M NaCl, dialyzed in a 50KD dialysis bag for 24 hours, and concentrated by suspension at 4°C; slowly added 0.3% CTAB which was three times the weight of the polysaccharide precipitate, and centrifuged after standing, the obtained precipitate was w...

Embodiment 2

[0103] Embodiment 2 colloidal gold immune test strip preparation

[0104] The anti-cryptococcus capsular polysaccharide monoclonal antibody was labeled with colloidal gold to prepare a double-antibody sandwich method immune colloidal gold test strip, which was named as the cryptococcal capsular polysaccharide detection card (colloidal gold method). The specific process is as follows:

[0105] Step 1 Gold-labeled antibody preparation

[0106] Measure 50mL colloidal gold solution, add it to a sterilized and dried beaker, place it on an electric heating magnetic stirrer, 500-600rpm, add 0.1M K while stirring 2 CO 3 solution, adjust the pH to 9.5; labeling: add 0.3 mL (2 mg / mL) of anti-cryptococcal capsular polysaccharide monoclonal antibody, and stir for 1 h at the same speed as above; sealing: add 5 mL of 10% bovine serum albumin (BSA) solution, 2 %PEG20000 2.5ml, the same speed as above, stir for 1h; low-speed centrifugation: 1500 rpm, 4 ° C for 30 min, discard the precipitate,...

Embodiment 3

[0113] Embodiment 3 colloidal gold immune test strip clinical sample comparison

[0114] The cryptococcal capsular polysaccharide detection card (colloidal gold method) prepared by Example 2 is used as an assessment reagent to carry out clinical trials, and the cryptococcal antigen detection kit (colloidal gold immunochromatography method) of the U.S. Immuno-Mycologics company is selected as a comparison reagent , Blind detection of serum and cerebrospinal fluid samples into the group, statistical analysis of the detection results, and investigation of the consistency of the detection results of the above test strips and contrast reagents. Among the cerebrospinal fluid samples, 321 cases had the same positive test results of the assessment reagent and the comparison reagent, 22 cases had positive test results of only the test reagent, 10 cases had positive test results of only the comparison reagent, and 10 cases had negative test results. The number of cases with consistent r...

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Abstract

The invention relates to an anti-cryptococcal capsular polysaccharide hybridoma cell strain and an anti-cryptococcal capsular polysaccharide monoclonal antibody secreted thereof. The antibody can be specifically bound to cryptococcal capsular polysaccharide and can be used for in vitro detection of cryptococcal infection. The antibody titer is up to more than one million, and the antibody has excellent affinity and good specific binding capability. The detection limit of a colloidal gold-labeled immunodiagnostic reagent developed with the antibody as the raw material is 0.5 ng / ml, and the antibody has excellent performance in sensitivity, specificity, stability and all other aspects.

Description

technical field [0001] The invention belongs to the field of monoclonal antibodies, and in particular relates to the preparation and application of an anti-cryptococcus capsular polysaccharide monoclonal antibody and a hybridoma cell line thereof. Background technique [0002] Immunodiagnostic reagents are the fastest-growing segment of in vitro diagnostic reagents, which use the specific combination between antigens and antibodies for qualitative or quantitative detection. [0003] In recent years, with the clinical application of broad-spectrum antibiotics, glucocorticoids, antineoplastic drugs, and immunosuppressants, the development of various invasive diagnosis and treatment techniques, and the increasing number of HIV-1 infected people, the incidence of deep fungal infections has also increased. increasing year by year. Cryptococcus is one of the common pathogens of clinical deep fungal infection. The cryptococcus that causes human infection is mainly Cryptococcus neo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N5/20C12N15/13G01N33/577
Inventor 苑庆华何永胜周跃辉王玉芳臧丹戎黄炎彬张鹏远
Owner GENOBIO PHARM CO LTD
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