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Methods for preparing, quantifying and detecting protein suspension chip of ricin

A technology of ricin and suspension chips, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of lack of models and evaluations, and achieve good consistency results

Inactive Publication Date: 2009-09-30
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the suspension chip can detect plant toxins such as ricin, whether it is suitable for rapid and direct detection from powder, liquid and other samples, and its quantitative detection ability, there is still a lack of models and evaluations.

Method used

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  • Methods for preparing, quantifying and detecting protein suspension chip of ricin
  • Methods for preparing, quantifying and detecting protein suspension chip of ricin
  • Methods for preparing, quantifying and detecting protein suspension chip of ricin

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0045] 2. Preparation of samples to be tested

[0046] 1. Preparation of target analyte samples

[0047] The present invention uses ricin as the target analyte sample. Interfering samples or samples for method-specific testing are other toxins or antigenic proteins other than the target detection object, including BONT, recombinant HIV P24 antigen, BSA, casein, tryptone, avian influenza virus HA protein, avian influenza virus NH protein, etc. . All the above-mentioned samples to be analyzed were dissolved in the sample diluent and stored at 4°C. The stock solution concentration of ricin was 1 mg / mL. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0048] The ricin to be analyzed is serially diluted into samples of different concentrations by 4 times with the sample diluent, so as to draw a standard curve of sample detection dose-response, where several sample concentrations are lower than the sensitivity of detection,...

Embodiment 1

[0051] Embodiment 1, the preparation of the protein suspension chip that detects ricin virus

[0052] 1. Capture antibody-coated encoded microspheres

[0053] The No. 027 coded microsphere used in the present invention was purchased from BIO-RAD Company of the United States. The coded microsphere is used to label the antibody that can capture ricin, that is, the rabbit anti-ricin A antibody is used to coat the microsphere.

[0054] A. Activation of encoded microspheres

[0055] 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-active Biotin (ie S...

Embodiment 2

[0080] Embodiment 2, optimization of suspension chip preparation method conditions

[0081] 1. Selection of microsphere-coated antibody and antibody coating amount

[0082] 100 μL of microspheres coded as No. 027 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 1 As shown, the No. 027 microspheres coated with rabbit anti-ricin A antibody all fell in the correct detection area, and obtained high signal-to-noise ratio results (MFI value is much greater than 2000), indicating that the optimized suspension chip detection system can be successful For ricin detection.

[0083] 2. Optimization of biotinylated antibodies

[0084] In the present invention, amino active biotin (biotin-LC-hy...

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Abstract

The invention relates to a protein suspension chip of ricin and methods for preparing, quantifying and detecting the same. The detecting method comprises the following steps: adding a working solution and a sample to be tested into a hole; adding a detection antibody; adding SA-PE; adding a detection buffer solution and mixing the mixture evenly; and reading numerical values, analyzing data and judging the detection result. The quantifying method comprises the following steps: adding a positive test sample or a standard product for dilution through gradient multiple proportions, detecting a series of diluted samples in a suspension chip system, and reading a corresponding fluorescence value (MFI); making a dosage-reaction curve; and fitting the dosage-reaction curve and an equation. The suspension chip comprises a coded microsphere, a ricin capturing antibody coated on the microsphere, a biotin-marked ricin detection antibody, a streptavidin-phycoerythrin and the buffer solution. The methods have the advantages of good detectability, high sensitivity, strong specificity and wide dynamic range.

Description

technical field [0001] The invention relates to a protein suspension chip preparation and quantitative detection method for ricin. Background technique [0002] Ricin toxin (ricin toxin, hereinafter referred to as ricin) is a highly toxic glycoprotein contained in castor beans. It is a heterodimer composed of A and B polypeptide chains by disulfide bonds. RNA N-glycosidase activity can inactivate ribosomes; the B chain is the binding chain, which contains two galactose binding sites, and mediates the A chain into the cell to play a toxic role. Ricin can be poisoned by inhalation through the respiratory tract, ingestion through the digestive tract, and intramuscular injection. Oral lethal dose is 0.15-0.2g, intravenous lethal dose is 20mg, mouse abdominal cavity LD50 is about 3.0μg / kg. Ricin is highly toxic, stable in nature, wide in source and low in extraction cost. As the news about the development and use of ricin by many terrorist organizations and extremists has ente...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/546G01N21/76
Inventor 王静王景林杨宇孙肖红胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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