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Kit for detecting tumor necrosis factors with spatial proximity chemiluminescence method and detection method of kit

A tumor necrosis factor, detection kit technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc., to achieve the effect of easy full automation, easy to scale up production, and 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

IL-6 is a Th2-type cytokine. In children with acute intussusception, the increase of IL-6 has immunomodulatory, anti-inflammatory, anti-viral effects, and at the same time resists the effect of TNF-α, but TNF-α increases Superior to IL-6, resulting in continuous contraction of intestinal smooth muscle leading to intussusception

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0026] 1. Preparation of calibrator

[0027] (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.

[0028] (2) Preparation of calibrator: the concentration of the calibrator is 0, 10pg / mL, 50pg / mL, 100pg / mL, 500pg / mL, 1000pg / mL, and the purified tumor necrosis factor is diluted to the corresponding concentration with the calibrator diluent, 2 Store at ~8°C for later use.

[0029] 2. Preparation of enzyme markers

[0030] (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 ov...

Embodiment 1

[0048] This embodiment provides a tumor necrosis factor-α detection kit, including: enzyme markers, luminescent markers, auxiliary agents, triggers and calibrator; wherein, the calibrator includes no less than 6 different concentrations of TNF-α Antigen calibrator and 0.1M phosphate buffer; enzyme markers include peroxidase-labeled TNF-α detection antibody and 0.05M phosphate buffer; luminescent markers include 9,10-dihydroacridine-labeled TNF - α 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.

[0049] 1. Preparation of calibrator

[0050] (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...

Embodiment 2

[0065] This embodiment provides a method for detecting tumor necrosis factor-alpha using a tumor necrosis factor-alpha detection kit in the spatial proximity chemiluminescence method, including steps:

[0066] S1: Add 25 μL of standard substance, 25 μL of peroxidase-labeled TNF-α detection antibody and 25 μL of luminescent marker to the chemiluminescence reaction tube respectively.

[0067] S2: React in a 37°C incubator for 15 minutes.

[0068] 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.

[0069] 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.

[0070] The invention adopts the spatial proximity chemiluminescence method to detect the tumor necrosis factor, and the s...

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Abstract

The invention relates to a kit for detecting tumor necrosis factors 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 TNF-alpha antigens with different concentrations and a 0.1 M phosphate diluent; the enzyme marker contains components of a peroxidase labeled TNF-alpha detection antibody and a 0.05 M phosphate buffer; the luminescent marker contains components of a 9,10-dihydracridine labeled TNF-alpha capture antibody and a 0.05 M Tris buffer; the adjuvant contains components of a luminescent adjuvant and a citrate buffer. A reagent spatial proximity luminescence analysis detection technology is adopted serves as a true homogeneous chemiluminescence technology, cleaning and a carrier are not needed, a coating process is omitted, and detection sensitivity and specificity are high, so that detectionresults are more real and reliable; meanwhile, reaction time and reaction steps are optimized, and operation is simpler.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for detecting tumor necrosis factor by space proximity chemiluminescence method and a detection method thereof. Background technique [0002] In 1975, E.A. Carswell et al. found that after the mice inoculated with BCG were injected with bacterial lipopolysaccharide, a substance that could cause hemorrhagic necrosis of various tumors appeared in the serum, and named it tumor necrosis factor (tumor necrosis factor, TNF) ). In the 1980s, it was discovered that it played an important role in wasting disease, also known as cachectic factor. TNF is mainly produced by activated macrophages, NK cells and T lymphocytes. In 1985, Shalaby named the TNF produced by macrophages as TNF-α, and the lymphotoxin (LT) produced by T lymphocytes as TNF-β. Although TNF-α and TNF-β have only about 30% homology, they have a common receptor. The biological activity of TNF-α accounts for 70% to 95% ...

Claims

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Application Information

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