Candida mannan antigen immunoassay kit and preparation method and application thereof

A detection kit and mannan technology, applied in the biological field, can solve the problems of time-consuming, difficult to distinguish, and large limitations.

Inactive Publication Date: 2016-08-17
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) Histopathological examination, which is an invasive examination and difficult to obtain materials;
[0006] (2) Blood culture method, which has poor sensitivity, takes a long time, is easy to contaminate, some fungal samples are difficult to collect, and the positive detection rate is not high;
[0

Method used

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  • Candida mannan antigen immunoassay kit and preparation method and application thereof
  • Candida mannan antigen immunoassay kit and preparation method and application thereof
  • Candida mannan antigen immunoassay kit and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] The preparation of embodiment 1 anti-Mn polyclonal antibody

[0092] 1. Immunization of animals

[0093] Three adult female New Zealand white rabbits were immunized with the mannan antigen of Candida albicans by multi-point subcutaneous injection on the back. One week before the immunization, blood was removed from the rabbit's ear veins, and the serum was separated as a negative control. The immunization dose was 1.0mL / time 1.0 mL of Freund's adjuvant was added to each immunization as an immune adjuvant, and a booster immunization was performed on the 7th, 14th, 21st and 28th days after the initial immunization.

[0094] 2. Obtaining polyclonal antibodies

[0095] 1) Titer determination: After 3 times of immunization, blood was collected from the vein of the rabbit's ear, with a volume of 1 mL / rat, the serum was separated, and the antibody titer in the serum was detected by indirect ELISA. The polyclonal antibody titer is very high, greater than 1:1×10 5 .

[0096]...

Embodiment 2

[0109] The detection of embodiment 2 anti-Mn polyclonal antibodies

[0110] 1. SDS-PAGE electrophoresis detection

[0111] The antibody prepared in Example 1 was subjected to SDS-PAGE electrophoresis, and the resulting gel was stained with Coomassie brilliant blue. See the experimental results figure 1 . 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.

[0112] 2. Potency determination

[0113] Antibody titers were determined by indirect ELISA. The coating used was originally Candida albicans mannan antigen, and the enzyme-labeled secondary antibody used was horseradish peroxidase-labeled goat anti-rabbit IgG. See the test results figure 2 . It can be seen from the results that the titer of the polyclonal antibody is very high, greater than 1:1×10 5 .

Embodiment 3

[0114] Example 3 Preparation of Candida mannan antigen immunoassay kit

[0115] 1. Preparation of ELISA plate

[0116] 1. Preparation of coating solution:

[0117] For the Mn coating solution, use 0.01M PBS solution to dilute Mn to 25ng / 100μL pH7.0-pH7.4.

[0118] 2. Preparation of blocking solution:

[0119] 1) The blocking solution, using 0.01M PBS solution, its pH7.0-pH7.4;

[0120] 2) Add 3% skimmed milk powder to the above solution to prepare a blocking solution.

[0121] 3. Coating method of ELISA plate:

[0122] 1) Add the prepared coating solution to the wells of the microtiter plate, and add 60 μL of the coating solution to each well;

[0123] 2) The above-mentioned microtiter plate was coated at 2-8°C for 8 hours;

[0124] 3) Add the prepared blocking solution to the wells of the microplate, add 60 μL of blocking solution to each well, and place in a 37°C incubator for 30 minutes;

[0125] 4) After removing the microplate from the incubator, discard the blocki...

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Abstract

The invention discloses a candida mannan antigen immunoassay kit, comprising a Mn-coated ELISA plate and an anti-Mn polyclonal enzyme-labeled antibody. According to the kit of the invention, the ELISA plate is coated with Mn, a sample or standard antigen to be detected and a coating antigen are subjected to competitive binding with a limited antibody binding site and then to rendering reaction via horse radish peroxidase and a substrate, a standard curve is drawn according to an Mn standard article, and concentration value of the antigen to be detected is calculated according to the standard curve. The kit of the invention has good sensitivity and specificity, results show high accordance rate with a reference reagent, more accurate and more reliable detection results can be provided, the kit is simple and easy to operate, detecting is fast and sensitive, an ELISA apparatus used herein is simple and popular and is low in cost, and the kit of the invention is an effective tool for quantitative detection of Mn.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Candida mannan (Mn) antigen immunodetection kit and a preparation method and application thereof. Background technique [0002] Invasive fungal disease (IFD) or invasive fungal infection (IFI) refers to fungal infectious diseases caused by pathogenic fungi invading subcutaneous tissue, mucous membrane, muscle and internal organs. Candida is the most common fungus among opportunistic fungi or conditionally pathogenic fungi. It mainly invades the mucous membrane and can further spread to the blood, causing disseminated infection; sisease, IFD) occupies the first place. Candida can easily multiply in the respiratory system, causing fungal pneumonia. The clinical manifestations of fungal pneumonia are basically the same as those of bacterial pneumonia, and it is easy to be misdiagnosed as bacterial pneumonia, which will further aggravate the fungal infection and even spread to the wh...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 彭洁刘春龙李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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