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A magnetic microsphere detection kit for detecting glycosylated CD59 and its application

A detection kit and magnetic microsphere technology, applied in the field of clinical testing, can solve the problem of low sensitivity of the kit, and achieve the effects of improving antibody coupling efficiency, direct detection method, and overcoming cumbersome operation steps.

Active Publication Date: 2022-03-18
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 magnetic particle chemiluminescence method using anti-gCD59 monoclonal antibody as capture antibody, anti-CD59 monoclonal antibody as labeled secondary antibody, and related magnetic microsphere detection kits for detecting glycosylated CD59 have not been reported yet.

Method used

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  • A magnetic microsphere detection kit for detecting glycosylated CD59 and its application
  • A magnetic microsphere detection kit for detecting glycosylated CD59 and its application
  • A magnetic microsphere detection kit for detecting glycosylated CD59 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Preparation of gCD59 protein

[0071] 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.

[0072] 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.

[0073] CD59 protein can also be prepared by a similar method.

Embodiment 2

[0074] Example 2: Preparation of anti-gCD59 mouse monoclonal antibody Mab1 and anti-CD59 mouse monoclonal antibody Mab2

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Fu...

Embodiment 3

[0079] Example 3: Preparation of the magnetic microsphere detection kit for detecting glycosylated CD59 according to the present invention

[0080] 1. Preparation of magnetic microspheres:

[0081] 1.1) Prepare coupling buffer: prepare 0.01mol / L MES buffer solution and adjust the pH value to 6.0;

[0082] MES 1.95g

[0083] Purified water to 1000mL

[0084] Sterilize by filtration and store at 4°C;

[0085] 1.2) prepare washing liquid, the formula of washing liquid and the preparation method are:

[0086]

[0087] Sterilize by filtration and store at 4°C;

[0088] 1.3) Preservation solution is prepared, the formula and preparation method of the preservation solution are:

[0089]

[0090]

[0091] Sterilize by filtration and store at 4°C;

[0092] 1.4) Coupling operation

[0093] (1) Antibody acidification treatment

[0094] Acid treatment: Take 1mL of antibody Mab1 solution with a concentration of 5mg / mL to a 2.0mL low-adsorption EP tube, add 0.2mL acidificat...

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Abstract

The invention discloses a magnetic microsphere detection kit for detecting glycosylated CD59, which consists of magnetic microspheres coupled with anti-gCD59 monoclonal antibody, gradient solution of gCD59 standard substance, sample diluent, peroxide Consists of enzyme-labeled anti-CD59 monoclonal antibody enzyme conjugate, 20× concentrated washing solution, and luminescence solution. The invention also discloses the application of the kit in detecting biological samples containing gCD59. Experiments have proved that the kit of the present invention has high stability, strong selectivity, fast detection speed, low cost, and easy operation, and overcomes the shortcomings of low sensitivity and long operation time in the detection of gCD59 in the prior art, and the operation time is shortened from 400 minutes Within 30 minutes, the limit of quantification was increased from 18.75pg / mL to 5pg / mL, which has excellent clinical application prospects.

Description

technical field [0001] The invention relates to the detection of glycosylated CD59 (gCD59), in particular to a magnetic microsphere detection kit for detecting glycosylated CD59 and its 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 complement re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/577G01N33/543G01N33/531
CPCG01N33/581G01N33/577G01N33/54326G01N33/531G01N2333/70596G01N2800/042
Inventor 陈振欧兰香武建伟张绍明王岩韩淑毅
Owner 山东莱博生物科技有限公司