Protein suspension chip method capable of quantitatively determining yersinia pestis and method for making same

A technology of Yersinia pestis and suspension chips, which can be used in measuring devices, biological testing, material inspection products, etc., can solve problems such as cumbersome methods, low sensitivity, and inconvenient nucleic acid detection methods

Inactive Publication Date: 2009-07-29
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The indirect hemagglutination method (IHA) for detecting Yersinia pestis specific antibody (F1 antibody) has low sensitivity and poor specificity (Williams, J.E., Arntzen, L., Robinson, D.M.&et al.Comparison of passive haemagglutination and enzyme-linkedimmunosorbent assay for serodiagnosis of plague. Bull WHO 1982, 60, 777-781.)
For the nucleic acid detection method, although PCR technology is used to detect and analyze Yersinia pestis (Hinnebusch, J.&Schwan, T.G. New method for plaguesurveillance using polymerase chain reaction to detect Yersiniapestis in fleas. J Clin Microbiol 1993, 31 (6), 1511 -1514.; Higgins, J.A., Ezzell, J., Hinnebusch, B.J., Shipley, M., Henchal, E.A. & Ibrahim, M.S. 5'nuclease PCR assay to detect Yersinia pestis. JClin Microbiol 1998, 36(8), 2284-2288 .; Neubauer, H., Meyer, H., Prior,...

Method used

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  • Protein suspension chip method capable of quantitatively determining yersinia pestis and method for making same
  • Protein suspension chip method capable of quantitatively determining yersinia pestis and method for making same
  • Protein suspension chip method capable of quantitatively determining yersinia pestis and method for making same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the preparation of the protein suspension chip that detects Yersinia pestis

[0054] 1. Capture antibody-coated encoded microspheres

[0055] First, the coded microspheres (Bead, microsphere) used in the present invention can be purchased from the products of LUMINEX company, each coded microsphere can be labeled with an antibody that can capture the corresponding target molecule, and the microspheres (100 μL Contains 1.25×10 6 encoded microspheres).

[0056] A. Activation of encoded microspheres

[0057]Take 100 μL of encoded microspheres into a 1.5 mL centrifuge tube, centrifuge at 14,000 g, 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 sulfo-NHS (50 mg / mL), and shake at room tem...

Embodiment 2

[0071] Embodiment 2, the improvement of suspension chip preparation conditions

[0072] 1. Selection of microspheres coated with different antibodies and coating amount

[0073] Three kinds of monoclonal antibodies (2F6, 5G12, 2H12) and three kinds of polyclonal antibodies (rabbit anti-EV76, goat anti-EV76, rabbit anti-F1) against Yersinia pestis F1 were selected respectively, with 4 μg, 8 μg, 10 μg, 16 μg, 24 μg, 40 μg , The amount of 48 μg is coated with 100 μL of microspheres coded as No. 28.

[0074] After testing, 4 μg, 8 μg, 16 μg of monoclonal antibody 2F6, 48 μg of rabbit anti-EV76 and 40 μg of goat anti-EV76 coated microspheres were not effective, and could not meet the needs of detection after coating; 16 μg of monoclonal antibody 5G12, 24 μg of monoclonal antibody 2H12 and 16μg rabbit anti-EV76, 10μg goat anti-EV76, 10μg, 40μg rabbit anti-F1 coating effect is very good, the MFI value is far greater than 2000, after counting under the microscope, store in dark and r...

Embodiment 3

[0087] Embodiment 3, preparation and detection of sample

[0088] 1. Preparation of samples to be tested

[0089] Prepare serial concentrations of F1 antigen standards to draw a standard curve for sample detection dose-response: dilute the F1 antigen with a 4-fold gradient with the sample diluent to form a serial concentration of the same sample for ELISA and suspension chip detection.

[0090] 2. Artificial contamination of "white powder" samples

[0091] Mix a certain amount of F1 antigen into milk powder, starch, flour, instant fruit and other powder samples according to different concentrations, add 0.5g powder into 5mL sample dilution buffer, mix well, let stand for 2 hours, let the test The sample is fully adsorbed with the white powder.

[0092] For the above-mentioned contaminated "white powder" samples, choose cotton, thin filter paper, thick filter paper, 0.45μm filter membrane, 0.22μm filter membrane to filter, and 2000rpm, 5 minutes and 1000rpm, 4 minutes low-spe...

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Abstract

The invention aims at providing a protein suspension chip which can quantitatively detect the bacteria and a preparation method thereof, particulary relates to a protein suspension chip which is suitable for quantitative detection and analysis of Yersinia pestis (Y.pestis). The method has high sensitivity, strong specificity, good detection capacity and wide dynamic range, and establishes a new detection mode platform.

Description

technical field [0001] The invention relates to a protein suspension chip capable of quantitatively detecting bacteria and a preparation method thereof, in particular to a protein suspension chip suitable for quantitatively detecting and analyzing Yersinia pestis. Background technique [0002] Yersinia pestis (Yersinia pestis) is a Gram-negative, non-motile, non-spore-forming coccus bacillus, with Gimusa, Wright's or Wesson's staining showing bipolar dense staining, and can be infected at 4-40°C. For internal growth, the optimum growth temperature is 28-30°C, the optimum growth pH value is 7.2-7.6, and the upper and lower limits of pH tolerance are pH5.0 and pH9.6 respectively. Yersinia pestis evolved from pseudotuberculosis bacteria and is in an active period of evolution. Its main host is rodents, and it is mainly transmitted among animals through the bite of fleas. Occasionally, human diseases can be caused by flea bites or contact with animals and animal fur with Yersin...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/546G01N33/533
Inventor 王静孙肖红周蕾杨宇胡孔新张晓龙
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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