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Kit for detecting D-dimer with spatial proximity chemiluminescence method and detection method of kit

A detection kit and dimer technology, which is applied in the direction of chemiluminescence/bioluminescence, analysis through chemical reaction of materials, and measurement devices, which can solve the problems of unstable detection results, many influencing factors, and poor repeatability, etc. problems, to achieve the effect of eliminating luminous interference, simple production process, and easy full automation

Inactive Publication Date: 2019-01-04
无锡壹闪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its operation is complicated and there are many influencing factors, resulting in unstable test results and poor repeatability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] 1. Preparation of calibrator

[0029] (1) Preparation of calibration product dilution: Weigh 14.1 g of potassium dihydrogen phosphate, sodium dihydrogen phosphate (2H 2 O) 3.0g, add ultrapure water to dissolve, Proclin-300 0.5 ~ 1mL, after mixing, add ultrapure water to make up to 1000mL, obtain the calibration product dilution, store at 2 ~ 8 ℃ for later use.

[0030] (2) Preparation of calibrator: the concentration of the calibrator is 0, 10ng / mL, 50ng / mL, 500ng / mL, 1000ng / mL, 10000ng / mL, and the purified D-dimer is diluted to the corresponding concentration with the calibrator diluent , Store at 2-8°C for later use.

[0031] 2. Preparation of enzyme markers

[0032] (1) Weigh 5mg HRP and dissolve it in 1mL distilled water, add 0.2mL freshly prepared 0.1M sodium periodate solution to the supernatant, stir for 20min in the dark at room temperature, put the above solution into a dialysis bag, and test the 1mM pH 4.4 The sodium acetate buffer was dialyzed overnight at...

Embodiment 1

[0050] This embodiment provides a D-dimer detection kit, including: enzyme markers, luminescent markers, auxiliary agents, triggers and calibrator; wherein, the calibrator includes no less than 6 different concentrations of D-Dimer Antigen calibrator and 0.1M phosphate buffer; enzyme markers include peroxidase-labeled D-Dimer detection antibody and 0.05M phosphate buffer; luminescent markers include 9,10-dihydroacridine-labeled D - Dimer capture antibody and 0.05M Tris buffer; the components of the auxiliary agent include luminescent auxiliary agent and pH 6.0 citrate buffer; the trigger agent is 0.05M pH 8.0 Tris-HCl buffer.

[0051] 1. Preparation of calibrator

[0052] (1) Preparation of calibration product dilution: Weigh 14.1 g of potassium dihydrogen phosphate, sodium dihydrogen phosphate (2H 2 O) 3.0g, add ultrapure water to dissolve, Proclin-300 0.8mL, after mixing, add ultrapure water to make up to 1000mL to obtain the calibration product dilution, store at 4°C for l...

Embodiment 2

[0067] This embodiment provides a method for detecting D-dimer using a D-dimer detection kit in spatially adjacent chemiluminescence, including steps:

[0068] S1: Add 25 μL of standard, 25 μL of peroxidase-labeled D-Dimer detection antibody and 25 μL of luminescent marker to the chemiluminescence reaction tube.

[0069] S2: React in a 37°C incubator for 15 minutes.

[0070] S3: Add 5 μL of auxiliary agent to the chemiluminescent reaction tube, shake and mix, and let it stand for 1-2 minutes; then add 75 μL of trigger agent, shake and mix, detect immediately, and read the signal value.

[0071] S4: Carry out logistic four-parameter fitting on the concentration and luminescence value of the calibrator, and calculate the sample concentration through the luminescence value of the sample.

[0072] The present invention adopts spatial proximity chemiluminescence method to detect D-dimer, and the sensitivity can reach 10 ng / mL; D-dimer has a diagnostic specificity of 86.3% for acut...

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Abstract

The invention relates to a kit for detecting D-dimer with a spatial proximity chemiluminescence method and a detection method of the kit. The kit comprises an enzyme marker, a luminescent marker, an adjuvant, a trigger and a calibrator, wherein the calibrator contains calibration products of D-dimer antigens with different concentrations and a 0.1 M phosphate diluent; the enzyme marker contains components of a peroxidase labeled D-dimer detection antibody and a 0.05 M phosphate buffer; the luminescent marker contains components of a 9,10-dihydracridine labeled D-dimer capture antibody and a 0.05 M Tris buffer; the adjuvant contains components of a luminescent adjuvant and a citrate buffer solution; the trigger is selected from a 0.05 M Tris buffer. The detection method as a true homogeneous chemiluminescence technology does not need a carrier, coating and washing processes are omitted, and the kit comprises few components, has high sensitivity and good repeatability and is easy to operate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting D-dimers by space proximity chemiluminescence method and a detection method thereof. Background technique [0002] D-dimer (D-Dimer) is a degradation product of cross-linked fibrin, and its formation mechanism is: under the action of thrombin, fibrinogen removes polypeptide A and polypeptide B from the α chain and β chain in turn, forming Fibrin I and fibrin II, fibrin is cross-linked on the blood vessel wall under the action of factor XII, and is cleaved by activated plasmin to produce various FDP fragments. Due to the cross-linking of the γ-chain, two D fragments connected by the γ-chain are produced, that is, the D-dimer, which has a molecular weight of about 180,000 Daltons and an in vivo half-life of 8 hours. [0003] D-dimer is one of the markers that specifically reflect secondary hyperfibrinolysis in vivo, and various diseases can cause the increase of D-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/535G01N33/577
CPCG01N21/76G01N33/535G01N33/577
Inventor 奚伟红朱丹丹廖鸳鸯
Owner 无锡壹闪生物科技有限公司
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