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Glycosylated CD59 detection kit based on chemiluminescence method and application thereof

A chemiluminescent method and a detection 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, strong selectivity, and accurate results

Pending Publication Date: 2021-06-22
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

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 (products of G Biosciences, not registered in China) to 400 minutes (PamelaGhosh, A Specific And Sensitive Assay For Blood Levels Of Glycated Cd59 :ANovel Biomarker For Diabetes)
The detection method based on the chemiluminescence method using the anti-gCD59 monoclonal antibody as the capture antibody, the anti-CD59 monoclonal antibody as the labeled secondary antibody, and the detection kit based on the chemiluminescence immunosorbent method have not been reported yet.

Method used

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  • Glycosylated CD59 detection kit based on chemiluminescence method and application thereof
  • Glycosylated CD59 detection kit based on chemiluminescence method and application thereof
  • Glycosylated CD59 detection kit based on chemiluminescence method and application thereof

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 detection kit based on chemiluminescence method according to the present invention

[0067] 1. Preparation of luminescent plate:

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

[0069]

[0070] Sterilize by filtration and store at 4°C;

[0071] Prepare the washing liquid, the formula and the preparation method of the washing liquid are:

[0072]

[0073] Sterilize by filtration and store at 4°C;

[0074] Prepare the blocking solution, the formula and preparation method of the blocking solution are:

[0075]

[0076] Sterilize by filtration and store at 4°C;

[0077] Dilute the anti-gCD59 mouse monoclonal antibody Mab1 with the coating solution to a working concentration of 5 μg / mL, mix well, and let stand for 15 minutes;

[0078] Take the marked ELISA plate, spot the plate with an 8-well row gun, so that the coated antibody solution reaches 200 μL / w...

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Abstract

The invention discloses a glycosylated CD59 detection kit based on a chemiluminescence method. The glycosylated CD59 detection kit is composed of a luminescent plate coated with an anti-gCD59 antibody, a gCD59 standard substance gradient solution, a sample diluent, a peroxidase-labeled anti-CD59 monoclonal antibody enzyme conjugate, a 20 * concentrated washing solution, a luminescent solution, a plate sealing film, a sealing bag and a specification, wherein the luminescent plate, the gCD59 standard substance gradient solution, the sample diluent, the peroxidase-labeled anti-CD59 monoclonal antibody enzyme conjugate, the 20 * concentrated washing solution, the luminescent solution and the plate sealing film are arranged in the kit body. The invention also discloses an application of the kit in detection of a biological sample containing gCD59. Experiments prove that the kit is high in stability, high in selectivity, high in detection speed, low in cost and easy to operate, the defects that in the prior art, when gCD59 is detected, sensitivity is not high, operation time is long and the like are overcome, the operation time is shortened to 40 minutes from 400 minutes, the limit of quantitation is increased to 5 pg / mL from 18.75 pg / mL, and the kit has excellent clinical application prospects.

Description

technical field [0001] The invention relates to the detection of glycosylated CD59 (gCD59), in particular to a glycosylated CD59 detection kit based on a chemiluminescence method and an application thereof, 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 compl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/535G01N21/76
CPCG01N33/6893G01N33/577G01N33/535G01N21/76G01N2800/042
Inventor 马万山陈振欧兰香李文靖高丽鹤苏真真
Owner 山东省大健康精准医疗产业技术研究院