Antibody for detecting aspergillus

A technology of Aspergillus and antibody capture, applied in the field of biochemistry, can solve the problems of popularization and application limitations, and achieve the effect of simple operation, low cost and high specificity

Active Publication Date: 2012-05-23
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the popularization and application of this kit is limited

Method used

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  • Antibody for detecting aspergillus
  • Antibody for detecting aspergillus

Examples

Experimental program
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Effect test

example 1

[0025] Example 1 Preparation and identification of monoclonal antibody of the present invention

[0026] 1. Preparation of monoclonal antibody 9D47A1 and monoclonal antibody 7E11A1

[0027] 1) Preparation of Aspergillus fumigatus mycelium antigen:

[0028] Aspergillus fumigatus strains were grown on Sabouraud plates at 37°C for several days to form single colonies. Harvest a typical single colony, add it to a 500ml Erlenmeyer flask containing 50ml RPMI-1640, shake the bacteria at 37°C for 3-7 days, and filter the mycelia and filtrate. The collected mycelium was resuspended in PBS, ultrasonically lysed, and the supernatant was collected after centrifugation, and stored at -80°C for future use.

[0029] 2) Immune mice

[0030] Take female BALB / c mice aged 4-6 weeks, mix and emulsify with Freund's complete adjuvant and equal volume of Aspergillus fumigatus mycelia antigen for the first time, inject 100 μg / mouse subcutaneously, and then inject Freund's complete adjuvant every 1...

example 2

[0063] Example 2 The present invention detects the double-antibody sandwich ELISA kit of Aspergillus

[0064] 1. The double antibody sandwich ELISA kit for detecting Aspergillus consists of the following reagents:

[0065] Microwell reaction plate coated with monoclonal antibody 9D47A1

[0066] Sample treatment solution: 4% ethylenediaminetetraacetic acid (PH 7.0), that is, weigh 40g of ethylenediaminetetraacetic acid, dissolve it in 1L of distilled water, adjust the pH value to 7.0 with NaOH, and store at 4°C;

[0067] Enzyme Conjugate: Mab 7E11A1 labeled with horseradish peroxidase;

[0068] Concentrated lotion: 20×PBS containing 2% Tween-20, i.e. 4.56g NaH in 1L solution 2 PO 4 , 58.02gNa 2 HPO 4 .12H 2 O, 175.3g NaCl, 15 pounds of 20min after autoclaving, add 20ml Tween-20 and stir well, dilute 20 times when used;

[0069] Positive control: Aspergillus fumigatus culture filtrate diluted 1:1000,

[0070] Negative control: 10mM PH7.4 PBS containing 0.1% Tween-20, i.e...

example 3

[0094] Example 3 Colloidal gold rapid immunochromatographic test paper for detection of Aspergillus

[0095] 1, the colloidal gold rapid immunochromatography test paper that detects aspergillus of the present invention constitutes as figure 2 As shown, the test paper is composed of sample pad 1, polyester cellulose pad 2, nitrocellulose membrane 3 and water-absorbing pad 4, which are partially overlapped in sequence, wherein the polyester cellulose membrane 2 is sprayed with a layer of colloidal gold-mAb 9D47A1 complex , the particle size is 18-20nm, and it is dark red; the nitrocellulose membrane 3 is distributed with a detection zone 5 and a quality control zone 6, wherein the detection zone 5 is formed by spraying monoclonal antibody 7E11A1 on the nitrocellulose membrane 3, quality control Band 6 was formed by spraying goat anti-mouse IgG1 antibody on nitrocellulose membrane 3 .

[0096] The preparation method of the test paper of the present invention is: the Aspergillus...

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Abstract

This invention provides the capture antibody and the detection antibody to detect the aspergillus, the two antibody is the single clone antibody of the cross oncocyte CCTCC NO.C200731 and CCTCC NO.C200730, they can unites the glucoprotein whose molecular weight is 25-100kDa in the cell wall of the aspergillus, the reaction with the Penicillium marneffei, white oidiomycosis, smoothness oidiomycosis, tropic oidiomycosis, close smoothness oidiomycosis, Candida krusei are all negative. The antibody in this invention can make into the immunity reagent to detect the aspergillus in common use.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a novel antibody for detecting aspergillus. Background technique [0002] Aspergillus is the most common saprophyte in nature, there are more than 100 species, of which at least 10 species of Aspergillus are opportunistic pathogens, often pollute food, the environment and can infect immunosuppressed people, seriously threatening human health. Invasive Aspergillosis (IA) caused by Aspergillus has a high mortality rate of 95% due to the lack of early effective diagnosis and treatment methods. IA is mainly caused by Aspergillus fumigatus and Aspergillus flavus, and a small part is caused by Aspergillus terreus, Aspergillus niger and Aspergillus nidulans. Among them, Aspergillus fumigatus is the main pathogen, accounting for about 80% to 90% of human infections caused by Aspergillus fungi . [0003] In terms of food testing, the current method for detecting Aspergillus contamination in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N33/577G01N33/543C12N5/12
Inventor 车小燕郝卫潘玉先王艳芳
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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