Preparation for protein suspending chip of bacillus anthracis spore and its quantitative determination method

A technology of bacillus anthracis and suspension chips, which can be applied to measuring devices, instruments, scientific instruments, etc., and can solve the problems of lack of models and evaluations

Inactive Publication Date: 2009-08-12
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether the suspension chip can detect anthrax and its spores, whether it is suitable for rapid and direct detection

Method used

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  • Preparation for protein suspending chip of bacillus anthracis spore and its quantitative determination method
  • Preparation for protein suspending chip of bacillus anthracis spore and its quantitative determination method
  • Preparation for protein suspending chip of bacillus anthracis spore and its quantitative determination method

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Experimental program
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Effect test

preparation example Construction

[0048] 2. Preparation of samples to be tested

[0049] 1. Preparation of target analyte samples

[0050] The target analyte is anthrax spores, but it involves the biological safety of severe infectious diseases. The anthracis vaccine strain Sterne spores is used as the target analyte sample. Bacillus anthracis vaccine strain (Sterne) was inoculated in spore medium DSM, cultured at 37°C for 2 to 3 weeks, stained with malachite green spore yellow spores, observed under a microscope, harvested spores when the spores accounted for more than 95% of the number of bacteria, and sterilized PB Buffer (0.01M, Ph7.2) was washed twice, resuspended in PB, diluted 10 times, and the number of spore copies was determined by plate viable count method, and stored at 4°C for future inspection. The concentration range of the diluted bacterial suspension is 10 7 cfu / mL.

[0051] Interfering samples or samples tested as method specificity are bacteria other than the target detection object, in...

Embodiment 1

[0055] Embodiment 1, the preparation of the protein suspension chip that detects anthrax spores

[0056] 1. Capture antibody-coated encoded microspheres

[0057] The No. 025 coded microspheres used in the present invention were purchased from BIO-RAD Company of the United States. The coded microspheres were used to label antibodies capable of capturing anthrax spores, that is, the microspheres were coated with goat anti-Sterne antibodies.

[0058] A. Activation of encoded microspheres

[0059] Take 100μL (1.25×10 6 pcs) encoded microspheres into a 1.5mL centrifuge tube, centrifuge at 14000g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere washing buffer to suspend, shake and sonicate, centrifuge at 14000g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere activation buffer, then add 10 μL of freshly prepared EDC (50 mg / mL), then add 10 μL of freshly prepared 50 mg / mL amino-active biotin (biotin-LC-hydrazide) or carboxyl-acti...

Embodiment 2

[0084] Embodiment 2, optimization of suspension chip preparation method conditions

[0085] 1. Selection of microsphere-coated antibody and antibody coating amount

[0086] 100 μL of microspheres coded as No. 025 were coated with 4 μg, 8 μg, 10 μg, 16 μg, 24 μg, 40 μg, and 48 μg, respectively. After testing the effect comparison, with 10μg / 1.25×10 6 A microsphere, that is, 20-40ng / 2500-5000 microspheres / test coating, has the best coating effect. After counting under a microscope, store it in a dark place and refrigerate it for later use. Such as figure 1As shown, the No. 025 microspheres coated with goat anti-Sterne antibody all fell in the correct detection area, and obtained high signal-to-noise ratio results (MFI value was much greater than 2000), indicating that the optimized suspension chip detection system can be successfully used for anthrax Spore detection.

[0087] 2. Optimization of biotinylated antibodies

[0088] In the present invention, amino active biotin (...

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Abstract

The invention relates to a preparation method, a detection method and a quantitative method for a suspension chip of a bacillus anthracis spore protein. The methods have the advantages of good detection capability, high sensitivity, strong specificity and wide dynamic range, and establish an open detection modeling platform for bacterial spores represented by bacillus anthracis.

Description

technical field [0001] The invention relates to a preparation method of a Bacillus anthracis (Bacillus anthracis) spore (endospore) protein suspension chip and a quantitative detection method thereof. Background technique [0002] Bacillus anthracis (Bacillus anthracis) is a pathogenic bacterium that causes zoonosis-anthrax. It is a Gram-positive aerobic bacterium that can form spores. The spores can infect humans through respiratory tract, digestive tract, and skin contact. Days of infection symptoms, the most common cutaneous anthrax. Inhalation of a large number of anthrax spores (more than 8000) can cause inhalational anthrax, also known as pulmonary anthrax. Because anthrax spores have strong resistance to the external environment and cause pollution to continue to exist, they have always been listed as one of the number one biological warfare agents in the military. It has been produced and stored as weapons in some countries, and now it has become the agent of choic...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/546
Inventor 王静周蕾孙肖红杨宇胡孔新杨瑞馥
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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