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TRFIA (time-resolved fluorescence immunoassay) method based on magnetic particles

An immunoassay method and time-resolved fluorescence technology, applied in the field of detection, can solve the problems of the influence of fluorescence synergist purity and low sensitivity, and achieve the effects of overcoming poor repeatability and instability, high reaction efficiency and sufficient reaction

Inactive Publication Date: 2019-01-08
GUANGZHOU BIOKEY HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of this patent is low, and the lower limit of detection is 10 -10 ~10 -11 At the mol / L level, a fluorescent synergist is still required, and the purity of the fluorescent synergist has a serious impact on the experimental results

Method used

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  • TRFIA (time-resolved fluorescence immunoassay) method based on magnetic particles
  • TRFIA (time-resolved fluorescence immunoassay) method based on magnetic particles
  • TRFIA (time-resolved fluorescence immunoassay) method based on magnetic particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Using the time-resolved fluorescent immunoassay method based on magnetic particles of the present invention to prepare a cTnI kit;

[0036] Wherein, the shape of the combined reagent strip is fan-shaped, and 1 to 13 holes are arranged sequentially from left to right. Wells 1 and 2 are test wells, wells 3 and 4 are fluorescent marker wells, wells 6 and 7 are diluent wells, holes 8 and 9 are sample diluent wells, 12th are enhancement solution wells, and 5th and 10th wells are diluent wells. , 11,13 are preparatory holes. A magnet can be stored between the 1st and 2nd wells for magnetic separation experiments; the 3rd and 4th wells can be disassembled from the entire reagent strip to form independent components, which is convenient for the storage of fluorescent markers. The structure of the combination reagent strip is shown in the appendix figure 1 .

[0037] The specific process is as follows:

[0038] (1) Preparation method of cTnI immunomagnetic beads:

[0039] ①...

Embodiment 2

[0076] In this embodiment, the components of the cTnI kit are packed into different reagent bottles, and the reaction place is a reaction cup, and the structure of the reaction cup is shown in the attached figure 2 .

[0077] (1) Preparation method of cTnI immunomagnetic beads:

[0078] Dispense the biotinylated cTnI monoclonal antibody with a concentration of 0.0050-0.0200 g / L into a reagent bottle, and dispense SA-magnetic beads into another reagent bottle. The two are linked together by streptavidin-biotin in the cuvette.

[0079] (2) Preparation method of europium-labeled cTnI antibody:

[0080] ① Preparation of europium-labeled cTnI monoclonal antibody

[0081] a. Take out 1 mg of cTnI monoclonal antibody and place it in a 30KD ultrafiltration centrifuge tube, centrifuge at 10,000 rpm for 10 min, and discard the filtrate.

[0082] b. Add 200 μL of labeling buffer (0.05M pH9.0 carbonate buffer), centrifuge at 10,000 rpm for 10 min, and discard the filtrate. Repeat th...

Embodiment 3

[0089] Embodiment 3 Application of the present invention to prepare cTnI kit and comparison of commercially available kits

[0090] In embodiment 1, the cTnI kit prepared by the present invention is used to measure calibrator cTnI, and its detection range is 0.03~50ng / ml. The concentration of the calibrator and the fluorescence value are fitted to the double logarithmic dose-response curve, and the standard curve is Y=1.011X+ 4.9268R=0.999, the performance of the kit is good, see image 3 .

[0091] Table 1 The standard curve of cTnI determined by the automatic magnetic particle time-resolved fluorescence immunoassay system

[0092] Concentrationng / ml

Fluorescence value

Fluorescence value

Fluorescence value

average

coefficient of variation

0

801

812

831

815

2％

0.03

2236

2501

2304

2347

6％

0.1

8439

8521

8301

8420

1％

0.5

40088

40056

41023

40389

1％

1

87569

...

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Abstract

The invention discloses a TRFIA (time-resolved fluorescence immunoassay) method based on magnetic particles. The method comprises the following steps: coating a magnetic bead with a specific antigen or antibody to form an immune magnetic bead; labelling a chelate formed by polyamino polycarboxy and rare earth ions with the specific antigen or antibody to form a fluorescent label; adding a sample for immune reaction to enable the immune magnetic bead, the tested sample and the fluorescent label to form a complex, adding a fluorescent enhancer after cleaning, enabling fluorescence value to be enhanced a million times with a dissociation-enhancement technology, and finally performing detection to obtain information such as concentration of the tested sample. One or more samples can be simultaneously detected, and multiple indexes can be detected in one experiment, so that operation is simple and quick. Sensitivity, repeatability and flexibility of detection are far better than those of traditional microporous plate TRFIA and chromatography methods.

Description

technical field [0001] The invention relates to the detection technical fields of biomedicine, animal epidemic disease, food safety, drug screening, etc., in particular, the invention relates to a time-resolved fluorescence detection method based on magnetic particles. Background technique [0002] Time-resolved Fluorescence immunoassay (TRFIA) technology is a new milestone in the development of markers after radioimmunoassay, and has become one of the most promising analysis methods in biomedical research and clinical ultra-micro biochemical testing. . TRFIA uses rare earth ions with long fluorescence lifetime and large Stokes shift as markers. It has the advantages of simple preparation, long storage time, no radioactive pollution, wide range of standard curve, no interference from natural fluorescence of samples, and multiple labels. It overcomes that radioimmunoassay is easy to pollute the environment; colloidal gold method and fluorescence immunochromatography have low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543G01N33/533
CPCG01N33/533G01N33/54326G01N33/577
Inventor 李根平
Owner GUANGZHOU BIOKEY HEALTH TECH CO LTD
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