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Monoclonal antibody of candida mannan and preparation method of monoclonal antibody

A monoclonal antibody, mannan technology, applied in the direction of antifungal/algae/lichen immunoglobulin, etc., can solve the problem of poor imaging detection specificity, and achieve the effect of high specificity, strong application prospect and high sensitivity

Inactive Publication Date: 2016-09-28
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity of imaging detection is poor. Vascular invasive infections such as Zygomycetes, Fusarium, Actinomycetes, Nocardia, and Staphylococcus aureus may be similar to invasive fungi on imaging, so imaging examinations are usually only can be used as an auxiliary diagnostic tool

Method used

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  • Monoclonal antibody of candida mannan and preparation method of monoclonal antibody
  • Monoclonal antibody of candida mannan and preparation method of monoclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0038] Example 1, the preparation and titer determination of mouse IgG type monoclonal antibody DNK-C6 against Candida mannan antigen

[0039] 1. The inactivated Candida was broken up by repeated freezing and thawing and ultrasonic disruption.

[0040] The specific method is:

[0041] 1) Add formaldehyde solution to the liquid medium to make the final concentration of formaldehyde 3.7%, and place it at 4° C. for 24 hours to inactivate the spores.

[0042] 2) The inactivated Aspergillus fumigatus cells were centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was removed.

[0043] 3) Resuspend the spores with pre-cooled physiological saline and wash repeatedly 6-10 times to fully remove formalin.

[0044] 4) Add liquid nitrogen and grind for 3-5 times with a sterilized mortar, add water, and use probe-type ultrasonic crushing for 30 minutes under ice bath.

[0045] 5) Carry out BCA protein quantification and phenol sulfate method sugar quantification.

[0046] 2. ...

Embodiment 2

[0062] Embodiment 2, the detection of the monoclonal antibody of anti-Candida mannan antigen

[0063] 1. SDS-PAGE electrophoresis detection

[0064] SDS-PAGE electrophoresis was performed on the antibody prepared in step 3, and the obtained gel was stained with Coomassie brilliant blue. See the experimental results figure 1 (GXM lane is anti-mannan monoclonal antibody, M lane is protein marker). It can be seen from the figure that there are clear and obvious bands in the 25KD and 50KD molecular weight regions, indicating that the purity of the antibody is very high.

[0065] 2. Potency determination

[0066] Antibody titers were determined by indirect ELISA. The enzyme-labeled secondary antibody used was goat anti-mouse IgG labeled with horseradish peroxidase, and the negative control was PBS solution. See the test results figure 2 . It can be seen from the results that the antibody titer is very high, not less than 1:1×10 6 .

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Abstract

The invention relates to an antibody, and a preparation method and application thereof, in particular to a mouse IgG type monoclonal antibody for candida mannan (Mn) antigen, a preparation method of the monoclonal antibody for resisting candida mannan (Mn) antigen, and application of the monoclonal antibody in detection of candida antigen. The invention provides a method which comprises the following steps: breaking candida, then immunizing animals by using the broken candida to prepare hybridoma cells; inoculating the animals by using the hybridoma cells screened by mannan; and separating and purifying to obtain the antibody. The monoclonal antibody prepared by the method has the features of good specificity, high valence and high purity. The monoclonal antibody is used for detecting candida in the field of medical health and scientific research, has the advantages of high sensitivity and good specificity, and is wide in application prospect.

Description

technical field [0001] The present invention relates to an antibody and a preparation method thereof, in particular to a monoclonal antibody for targeting Candida mannan (Mannan, MN) antigen and a preparation method thereof. Background technique [0002] Candida albicans is a kind of opportunistic pathogenic Candida. The human body will not be infected by Candida albicans under normal circumstances. Only when the body’s immune function or general defense is weakened or the mutual restriction of normal flora is out of balance, Candida albicans may be infected. , causing deep infection, and even systemic disseminated infection. [0003] In recent years, due to the aging population, the extensive application of immunosuppressants and broad-spectrum antibiotics, the increase in the incidence of AIDS and malignant tumors, the extensive development of organ and bone marrow transplantation and interventional therapy, the incidence of clinical invasive candida infection has been inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14
CPCC07K16/14
Inventor 彭洁李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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