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A kind of glycosylated CD59 ELISA kit and its preparation method and application

An enzyme-linked immunoassay detection and kit technology, which is applied in the field of clinical testing, can solve the problem of low sensitivity of the kit, and achieve the effects of reducing non-specific adsorption, increasing sensitivity and improving coating effect.

Active Publication Date: 2022-05-10
山东省大健康精准医疗产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The relevant enzyme-linked immunoassay methods reported in the literature are: the anti-CD59 monoclonal antibody is the capture antibody, the anti-gCD59 monoclonal antibody is the detection antibody, and the enzyme-labeled secondary antibody is goat anti-rabbit IgG-horseradish peroxidase (HRP) marker or SA-HRP, the sensitivity of the kit is not high, and the operation time is as long as 200 minutes (product of G Biosciences, not registered in China) to 400 minutes (Pamela Ghosh, A Specific And Sensitive Assay For Blood Levels Of Glycated Cd59 :A Novel Biomarker For Diabetes)
There is no report about the detection method using the anti-gCD59 monoclonal antibody as the capture antibody and the anti-CD59 monoclonal antibody as the labeled secondary antibody

Method used

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  • A kind of glycosylated CD59 ELISA kit and its preparation method and application
  • A kind of glycosylated CD59 ELISA kit and its preparation method and application
  • A kind of glycosylated CD59 ELISA kit and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Preparation of gCD59 protein

[0058] Using the published amino acid sequence of gCD59 (DACLITKAGLQVYNKCWKFEHC) and CD59 amino acid sequence (LQCYNCPNPTADCKTAVNCSS), the corresponding nucleotide sequences of gCD59 and CD59 were respectively obtained by using protein expression techniques well known to those skilled in the biological field.

[0059] A plasmid expressing gCD59, ie pcDNA3.1-gCD59 expression vector, was constructed, and after transfection into CHO cells, positive clones were screened, cultured and purified to obtain gCD59 protein.

[0060] CD59 protein can also be prepared by a similar method.

Embodiment 2

[0061] Example 2: Preparation of anti-gCD59 mouse monoclonal antibody Mab1 and anti-CD59 mouse monoclonal antibody Mab2

[0062] Healthy BALB / c mice (8 weeks old) were immunized with the obtained gCD59 and CD59 proteins respectively. After the mice produced antibodies, anti-gCD59 mouse monoclonal antibody Mab1 and anti-CD59 mouse monoclonal antibody Mab1 were prepared by conventional myeloma fusion cell technology. Monoclonal antibody Mab2.

[0063] Experimental verification shows that the amino acid sequence of the prepared antibody Mab1 against the epitope is: DACLITKAGLQVYNKCWKFEHC, and the amino acid sequence of the antibody Mab2 against the epitope is: LQCYNCPNPTADCKTAVNCSS.

[0064] Among them, the obtained Mab1 has been verified and confirmed not to cross-react with CD59, and Mab2 has been verified to have no cross-reaction with gCD59. The specific process adopts the hybridoma antibody preparation technology commonly known to those in the biological field.

[0065] Fu...

Embodiment 3

[0066] Example 3: Preparation of glycosylated CD59 ELISA kit of the present invention

[0067] 1. Preparation of ELISA plate:

[0068] Prepare coating solution: prepare 0.01mol / L phosphate buffer solution, and adjust the pH value to 7.4;

[0069]

[0070] Prepare the washing liquid, the formula and the preparation method of the washing liquid are:

[0071]

[0072] Prepare the blocking solution, the formula and preparation method of the blocking solution are:

[0073]

[0074] Dilute the anti-gCD59 mouse monoclonal antibody Mab1 to a working concentration of 10 μg / mL with the coating solution, mix well, and let stand for 15 minutes;

[0075] Take the marked ELISA plate, spot the plate with an 8-well row gun, so that the coated antibody solution reaches 200 μL / well, and cover with a cover film;

[0076] Place in a refrigerator at 2°C-8°C, coat for 4-6 hours, then add 5 μL of 0.1% SDS aqueous solution to each well, and coat overnight for 12-16 hours;

[0077] Take o...

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Abstract

The present invention disclosed a kind of sugar -based CD59 enzyme -linked immune testing kit. The bags located in the box are enzyme plates with antibody GCD59 antibodies, GCD59 standard gradient solution, sample diluted solution, peroxidase standardAnti -CD59 monoclonal antibody enzyme binding, 20 × concentrated washing fluid, color rendering agent, termination liquid and sealing film, sealing bag and instructions.The invention also disclosed the application of the kit in the GCD59 biological sample.The experiment confirmed that the kit of the present invention has high stability, strong selectivity, fast detection speed, low cost, and easy operation. When the existing technology detects GCD59, the sensitivity is not high, the operation length is short, and the operation time is shortened by 400 minutes.By 45 minutes, the quantitative limit was increased from 18.75pg / ml to 12.5pg / ml, which has great clinical application prospects.

Description

technical field [0001] The invention relates to the detection of glycosylated CD59 (gCD59), in particular to a glycosylated CD59 enzyme-linked immunoassay kit and its preparation method and application, belonging to the technical field of clinical testing. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Long-term high blood sugar leads to chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. In the population of diabetic patients, 70% of the patients have proliferative lesions in the small blood vessels and microvessels of the whole body. Substance deposition is associated with thickening of the vascular basement membrane caused by subendothelium. [0003] Glycosylated CD59 (gCD59) is a novel biomarker. CD59 is a complement regulatory protein that protects 'self' cells from complement-mediated damage (Davies CS et al. Glycation of CD59 impairments complemen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/58G01N33/545G01N33/543
CPCG01N33/6893G01N33/577G01N33/581G01N33/545G01N33/54306G01N2333/70596G01N2800/042
Inventor 韩淑毅陈振汪运山欧兰香李文靖苏真真高丽鹤
Owner 山东省大健康精准医疗产业技术研究院