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Anti-protease 3 antibody IgG chemiluminiscence immunodetection kit and preparation method thereof

A chemiluminescence immunoassay and detection kit technology, applied in measurement devices, scientific instruments, instruments, etc., can solve the problems of narrow detection linear range, low detection sensitivity, and high detection cost, and achieve wide linear range, high detection sensitivity, and easy operation. easy effect

Inactive Publication Date: 2017-03-15
SHENZHEN YHLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The current anti-proteinase 3 antibody IgG detection technology has the following disadvantages: high detection cost, low detection sensitivity, narrow detection linear range, low reproducibility, inability to quantify, complicated operation, etc.

Method used

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  • Anti-protease 3 antibody IgG chemiluminiscence immunodetection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation method of anti-proteinase 3 antibody IgG chemiluminescence immunoassay kit

[0022] (1) Preparation of nano-magnetic beads coated with protease 3 antigen:

[0023] Take 50mg of carboxylated magnetic particles (particle size: 0.05-1um) suspension, magnetically separate to remove the supernatant, resuspend with 0.02 M, pH 5.5MES buffer, add 0.5-2mL of newly prepared 10 mg / mL EDC Aqueous solution, activate the carboxyl group on the surface of magnetic beads, add 3-5mg protease 3 antigen, suspend at room temperature for 2-10 h, magnetically separate, remove the supernatant, and resuspend with 0.1 M Tris buffer at pH 8.0 containing 2% BSA to 1mg / mL, to obtain protease 3 antigen-coated magnetic particles, and store in 5mL per bottle at 4°C for later use.

[0024] (2) Preparation of acridinium ester labeled with mouse anti-human IgG monoclonal antibody:

[0025] Take 50 uL 25mg / mL mouse anti-human IgG monoclonal antibody, add 150 uL 0.1-0.2 M carbonate...

Embodiment 2

[0032] Example 2: Anti-Protease 3 antibody IgG chemiluminescent immunoassay method:

[0033] The present invention uses a fully automatic chemiluminescence immunoassay analyzer as a detection tool, and the methodology mode of the present invention is an indirect method, that is, the instrument sequentially adds 5uL of sample, 95uL of sample diluent, and 50uL of protease 3 antigen-coated magnetic particles to react for 10 After min, magnetic separation was performed. Add 100uL of acridinium ester coated with mouse anti-human IgG monoclonal antibody, react for 10 minutes, and perform magnetic separation. The instrument sends the reaction mixture into the darkroom, and sequentially adds 100uL of chemiluminescence pre-excitation solution and 200uL of chemiluminescence excitation solution for luminescence. React, and finally record the luminous intensity, and calculate the IgG content of the anti-protease 3 antibody of the tested sample from the standard curve.

Embodiment 3

[0034] Example 3: Performance evaluation of anti-proteinase 3 antibody IgG chemiluminescent immunoassay kit

[0035] The detection curve is attached figure 1 .

[0036] Sensitivity detection:

[0037] Refer to the recommended experimental protocol in the CLSI EP17-A document, calculate the sensitivity of the anti-protease 3 antibody IgG chemiluminescent immunochromatography kit, and the obtained sensitivity is 1AU / mL.

[0038] Linear detection:

[0039] Perform linear analysis on standard products with concentrations of 5 AU / mL, 20 AU / mL, 50 AU / mL, 90 AU / mL, and 165 AU / mL, and calculate the linear correlation coefficient, r=0.9996. In addition, the kit is resistant to protease 3 The linear range of antibody IgG sample detection is 3-300AU / mL.

[0040] Precision determination:

[0041] Take two anti-protease 3 antibody IgG samples with a concentration of 2 AU / mL and 100 AU mL, make 3 parallels for each concentration of each sample, and use three batches of kits for detection...

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Abstract

The invention discloses an anti-protease 3 antibody IgG chemiluminiscence immunodetection kit. The kit comprises magnetic particles coated with a protease 3 antibody, acridinium ester coated with a mouse anti-human IgG monoclonal antibody, an anti-protease 3 antibody IgG calibration product, a pre-exciting solution and an exciting solution. The invention further discloses a preparation method of the anti-protease 3 antibody IgG chemiluminiscence immunodetection kit. Compared with an existing kit, the kit has the advantages of being easy and convenient to operate, high in sensitivity, wide in detection range and the like.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic immunoassay, and specifically, the invention provides a chemiluminescence immunoassay kit for anti-protease 3 antibody IgG and a preparation method thereof. Background technique [0002] Protease 3 (Proteinase 3, PR3) is a serine protease in neutrophil cytoplasmic azurophilic granules, a glycoprotein with a molecular weight of about 29KDa. PR3 can degrade a variety of extracellular matrix such as elastin, hemoglobin, collagen type IV and other tissue components. Proteinase 3 promotes platelet activation through cathepsin G and inactivates C1 inhibitors. Antineutrophil cytoplasmic antibody (Antineut-rophiIcytoplasmic antibody, ANCA) is a serum marker in patients with Wegener's granulomatosis (WG), especially anti-proteinase 3 antibody. [0003] WG is a specific type of necrotizing granulomatous vasculitis. Antineutrophil cytoplasmic antibodies are the most diagnostic autoantibodies for WG. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/577
CPCG01N33/54326G01N33/577
Inventor 冯丽珠张昭李爽陈曼马晓雯
Owner SHENZHEN YHLO BIOTECH
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