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Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof

A galactomannan and detection kit technology, applied in the biological field, can solve the problems of difficulty in collecting fungal samples, unfavorable promotion and popularization of kits, and low positive detection rate.

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. This method has poor sensitivity and takes a long time. It is difficult to collect some fungal samples, and the positive detection rate is not high. The most common clinical sputum culture positive for Aspergillus cannot diagnose Aspergillus infection;
[0007] (3) Imaging examination, the sensitivity of this method is poor, and it is difficult to see the characteristic manifestations in the early stage of the disease;
However, this method uses a sandwich immunoassay for detection, the detection steps are cumbersome, and the cost of monoclonal antibody preparation is high, which is not conducive to the promotion and popularization of the kit in clinical detection
At present, there is no immunological detection product for the clinical detection of Aspergillus in China. Therefore, in the field of clinical detection of Aspergillus, there is an urgent need for a quantitative detection of GM antigen independently developed in China, which is simple to use, low in cost and high in specificity. diagnostic kit products

Method used

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  • Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof
  • Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof
  • Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Example 1 Preparation of Aspergillus galactomannan (GM) antigen

[0145] Aspergillus was used to prepare GM antigen, and the Aspergillus strain used in the present invention was purchased from China Medical Microbiology Culture Collection Center (CMCC).

[0146] 1. Extract GM, the steps are as follows:

[0147] Prepare PDA solid medium 2.0L, its composition is the supernatant after boiling 600g potatoes, D-glucose 80.0g, agar powder 30.0g, put the Aspergillus strain in the medium, cultivate at 25°C for 3 days, until the medium grows Full of green spores. Wash the spores with sterile physiological saline, filter the spore suspension through 8 layers of sterile gauze three times to remove mycelium, add formaldehyde with a final concentration of 3.7% to the spore suspension and let it stand at 4°C for 24 hours to inactivate the bacteria and spores. The spores were collected by centrifugation at 12,000 g for 30 min at 4°C, and washed 6 times with sterile saline to remove ...

Embodiment 2

[0168] Example 2 Preparation of Anti-GM Antigen Polyclonal Antibody

[0169] 1. Preparation of polyclonal antibody against GM antigen

[0170] 1. Immunization of animals

[0171] Mix equal volumes of GM antigen and Freund's complete adjuvant to an appropriate volume, fully emulsify and inject New Zealand big-eared rabbits subcutaneously at multiple points, and the immunization dose of each rabbit is controlled at 0.01-0.1mg. Ear blood was collected 3 days before immunization, and serum was separated as a negative control. After the initial immunization, immunize once every 2 weeks, and the method is the same as the first time.

[0172] 2. Obtaining polyclonal antibodies

[0173] 1) Titer determination: During the immunization process, blood was collected every 7 days to measure the titer once after immunization, and the number of immunizations was 6 times.

[0174] 2) Separation of antiserum: When the serum titer reaches a high level, a large amount of blood is collected b...

Embodiment 3

[0190] Example 3 Preparation of Aspergillus galactomannan (GM) antigen immunoassay kit

[0191] 1. Preparation of GM antigen-coated microtiter plates

[0192] 1. Prepare GM antigen coating solution:

[0193] GM antigen was diluted to 100ng / mL with 0.1mol / L Tris-HCl buffer solution, pH6.0-pH9.0.

[0194] 2. Prepare blocking solution:

[0195] Add 2% newborn bovine serum to normal saline to prepare blocking solution.

[0196] 3. Coating microtiter plate:

[0197] 1) Add the prepared GM antigen coating solution to the wells of the microtiter plate, and add 100 μL of the coating solution to each well;

[0198] 2) The above-mentioned microtiter plate was coated at 12-18°C for 6 hours;

[0199] 3) Add the prepared blocking solution into the wells of the microplate, add 100 μL of blocking solution to each well, and place in an incubator at 12-18°C for 2 hours;

[0200] 4) Take out the enzyme label plate from the incubator, discard the blocking solution, and keep the temperature...

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Abstract

The invention provides an Aspergillus galactomannan (GM) antigen immunoassay kit, comprising a solid phase carrier coated with GM antigen, an anti-GM antigen polyclonal antibody and a GM antigen standard product, the kit first GM antigen is coated on a solid phase carrier. During detection, the sample to be tested or GM antigen standard competes with the coated antigen for limited antibody binding sites, and then a color reaction occurs between the enzyme and the substrate, and the color depth of the measurement result It is negatively correlated with the concentration of the antigen to be tested. A standard curve can be drawn according to the GM antigen standard, and the concentration of the antigen to be tested can be calculated according to the standard curve equation. The kit has good sensitivity and specificity, the result coincides with the reference reagent in a high rate, and can provide more accurate and reliable test results. Its operation is simple and easy, and the detection is fast and sensitive. Inexpensive, the detection kit provides an effective tool for the quantitative detection of GM antigen.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an Aspergillus galactomannan (GM) antigen immunoassay kit and a preparation method and application thereof. Background technique [0002] Aspergillus (Aspergillus) is a saprophytic bacterium widely present in nature and a resident fungus of normal human skin and mucous membranes. Aspergillus spores are small, 2-3μm in diameter, can float in the air, and enter the human body through the respiratory tract. Since Aspergillus enters the human body mainly through the respiratory tract, Aspergillus infection mainly occurs in the lungs. [0003] The incidence of invasive aspergillosis (IA) in immunosuppressed patients has been increasing year by year due to the abuse of antibiotics, and it has become the main cause of death. Aspergillus fumigatus is the most common cause of severe deep Aspergillus infection in immunosuppressed patients Pathogens, followed by Aspergillus flavus, Aspergillu...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/56961G01N33/543G01N2400/38
Inventor 刘春龙彭洁张舟李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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