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

A technology of hybridoma cell line and monoclonal antibody, which is applied in the direction of anti-fungal/algae/lichen immunoglobulin, biochemical equipment and methods, instruments, etc., which can solve the problems of high price and achieve good affinity and specificity The effect of binding ability

Active Publication Date: 2019-06-14
天津喜诺生物医药有限公司
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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-cryptococcus capsular polysaccharide monoclonal antibody, preparation of hybridoma cell strain thereof and application
  • Anti-cryptococcus capsular polysaccharide monoclonal antibody, preparation of hybridoma cell strain thereof and application
  • Anti-cryptococcus capsular polysaccharide monoclonal antibody, preparation of hybridoma cell strain thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Preparation method of monoclonal antibody against cryptococcal capsular polysaccharide

[0086] 1. Polysaccharide extraction

[0087] Cultivate cryptococcus (the strain number is ATCC 208821) with liquid Sabouraud medium, cultivate at 20-25°C for 3-4 days, centrifuge the supernatant after the bacterial 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, weigh (or calculate the mass by concentration) ) precipitate was dissolved by 0.2M NaCl, dialyzed in 50KD dialysis bag for 24h, 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 still, the obtained precipitate was wash...

Embodiment 2

[0104] Embodiment 2 colloidal gold immune test strip preparation

[0105] 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:

[0106] Step 1 Gold-labeled antibody preparation

[0107] 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 precipitat...

Embodiment 3

[0114] Embodiment 3 colloidal gold immune test strip clinical sample comparison

[0115] 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, 296 cases had the same positive test results of the assessment reagent and the comparison reagent, 18 cases had positive test results of only the test reagent, 4 cases had positive test results of only the comparison reagent, and 4 cases had negative results. The number of cases with consistent results ...

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Abstract

The invention relates to an anti-cryptococcus capsular polysaccharide hybridoma cell strain and an anti-cryptococcus capsular polysaccharide monoclonal antibody secreted thereby. 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 has good specific binding capability. The detection limit of a colloidal gold-labeled immunodiagnostic reagent developed by using the antibody is 0.5 ng / ml, and the colloidal gold-labeled immunodiagnostic reagent has better performance in aspects of sensitivity, specificity, stability and antibody.

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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IPC IPC(8): C12N5/20C07K16/14G01N33/577G01N33/569
Inventor 苑庆华何永胜周跃辉王玉芳臧丹戎黄炎彬王兴
Owner 天津喜诺生物医药有限公司
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