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Preparation and application of an anti-cryptococcal capsular polysaccharide monoclonal antibody and its hybridoma cell line

A hybridoma cell line, monoclonal antibody technology, applied in applications, anti-fungal/algae/lichen immunoglobulin, botanical equipment and methods, etc., can solve problems such as high price and achieve good specific binding ability. Effect

Active Publication Date: 2022-04-26
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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  • Preparation and application of an anti-cryptococcal capsular polysaccharide monoclonal antibody and its hybridoma cell line

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 (strain number: ATCC 208821) with liquid Sabouraud medium, culture at 20-25°C for 3-4 days, centrifuge the supernatant after inactivation of the bacterial liquid, and add sodium acetate to the supernatant to a final concentration of 5%, adjust pH=5.0; add 3 times the volume of absolute ethanol, overnight at 4°C, creamy precipitation can be seen at the bottom; after discarding the supernatant, the precipitation becomes Cryptococcus capsular polysaccharide, and the precipitation is air-dried and weighed (or calculated by concentration). ) The precipitate was dissolved in 0.2M NaCl, dialyzed in a 50KD dialysis bag for 24h, and suspended at 4°C for concentration; slowly add 0.3% CTAB three times the mass of the polysaccharide precipitate, and centrifuge after standing, the obtained precipitate was washed twice...

Embodiment 2

[0103] Example 2 Preparation of colloidal gold immunoassay strips

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

[0105] Step 1 Gold-labeled antibody preparation

[0106] Measure 50mL of colloidal gold solution, add it to a beaker that has been sterilized and dried, place it on an electrothermal magnetic stirrer for stirring, at 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; blocking: add 5 mL of 10% bovine serum albumin (BSA) solution, 2 %PEG20000 2.5ml, the speed is the same as above, stirring for 1h; low-speed centrifugation: 1500 rpm, 4°C for...

Embodiment 3

[0113] Example 3 Comparison of clinical samples of colloidal gold immune test strips

[0114] The Cryptococcus capsular polysaccharide detection card (colloidal gold method) prepared by Example 2 was used as the assessment reagent to carry out the clinical test, and the Cryptococcus antigen detection kit (colloidal gold immunochromatography) of the American Immuno-Mycologics company was selected as the contrast reagent. , Blind detection of serum and cerebrospinal fluid samples into the group, and statistical analysis of the detection results to examine the consistency of the detection results of the above test strips and comparative reagents. Among the cerebrospinal fluid samples, 321 cases had the same positive results of the test reagents and comparison reagents, 22 cases had positive test results of only the assessment reagents, 10 cases had positive test results of only the comparison reagents, and 10 cases were negative. The number of cases with consistent results was 50...

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Abstract

The invention relates to an anti-cryptococcus capsular polysaccharide hybridoma cell line and the anti-cryptococcus capsular polysaccharide monoclonal antibody secreted therein, the antibody can specifically bind to the cryptococcal capsular polysaccharide, and can be used for in vitro detection of cryptococcal infection , its antibody titer is as high as more than one million, has excellent affinity, and has good specific binding ability; the detection limit of the colloidal gold-labeled immunodiagnostic reagent developed with this antibody as a raw material is 0.5ng / ml, in terms of sensitivity, Specificity, stability, all aspects of the antibody have better performance.

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 Patents(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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