Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Myeloperoxidase quantitative determination method and detection reagent

A technique of a marrow peroxidase and detection method, which is used in the field of medullaxyxoxidase and detection reagent field. It can solve problems such as affecting the test results and interference of spicy root peroxidase activity. The effect of repetitiveness, shortening testing time, and reducing funding expenditure

Active Publication Date: 2015-01-28
JIANGSU PROVINCE HOSPITAL
View PDF5 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing specific immunodetection ELISA method adopts two strains of anti-MPO antibodies, one as a capture antibody, and the other as an enzyme-labeled antibody (chromogenic antibody), the most commonly used is horseradish peroxidase (HRP). system, but the detected MPO has peroxidase activity, which can interfere with horseradish peroxidase activity and affect the detection results

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Myeloperoxidase quantitative determination method and detection reagent
  • Myeloperoxidase quantitative determination method and detection reagent
  • Myeloperoxidase quantitative determination method and detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation of Monoclonal Antibody

[0029] Step 1. Immunization of animals

[0030] On the first day, human MPO antigen (P257-5, purchased from SCIPAC Ltd., UK) was taken at 80 μg / mouse, mixed with an equal volume of Freund’s complete adjuvant and fully emulsified, and intraperitoneally injected into three 6-week-old mice with myeloma cells Female BALB / c mice of the same strain (Nanjing Medical University Experimental Animal Center). On the 21st day, mice were anesthetized intraperitoneally with 1% pentobarbital sodium 10 μL / g, the abdominal cavity was opened and the spleen was exposed, 40 μg MPO was dissolved in 100 μL sterile PBS, and the mesentery at the caudal end of the spleen was gently lifted with curved forceps. Insulin syringes were injected into the spleen from the tail of the spleen, stagnated for 30 seconds after each injection to prevent reflux, put back into the spleen and sutured in layers. On the 24th day, the tail-cut blood of the mice was...

Embodiment 2

[0040] Embodiment 2, establishment of MPO-ISA method and condition optimization

[0041] Step 1. Background of MPO-ISA method establishment

[0042] Active MPO also has peroxidase activity in vitro and can be directly used for enzyme activity detection (J Immunol Methods.1990; 126(1):125-133), so MPO in the ELISA detection method may interfere with the HRP system detection, affecting the detection results. We compared HRP (horseradish peroxidase) system and AP (alkaline phosphatase) system to detect MPO by indirect ELISA. Coat the plate with MPO antigen (P257-5, 1.4 μg / mL), use anti-human MPO monoclonal antibody 18B7 as the primary antibody, divide into four groups: 0ng / mL, 8.1ng / mL, 81ng / mL, 810ng / mL, 37℃ Incubate for 1 h, and then use HRP-labeled secondary antibody A0168 (1:2000) and AP-labeled secondary antibody A3562 (1:5000) to develop color. The results can be seen in the attached image 3 , the optical density value of the HRP system was significantly higher than th...

Embodiment 3

[0051] Embodiment 3, MPO-ISA method performance evaluation

[0052] Step 1. Standard curve

[0053] Dilute the MPO standard (8M80, derived from human leukocytes, purchased from Hytest, Ltd., Finland) with 0.5% bovine serum albumin in PBS, the concentration range is 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250ng / mL , detected with the MPO-ISA method established in Example 2. With concentration as the abscissa and A as the ordinate, draw a standard curve. The results show that the standard linear range of MPO is 0~250ng / mL, see attached Figure 7 , Y=0.0104X-0.0104, r 2 =0.9989, p<0.0001). The minimum detection limit concentration of MPO-ISA is 3.68ng / mL, which is calculated based on the MPO concentration corresponding to the average value of A value of 20 samples with zero value + 3SD.

[0054] Step 2. Method recovery

[0055] The samples with MPO concentrations of 31.25 and 125ng / mL were mixed with MPO standard substances (concentrations of 50, 200, and 500ng / mL) at a vol...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of applications of medical biotechnology, and relates to an enzyme-linked immunosorbent assay on the basis of immune response combining myeloperoxidase and specific monoclonal antibody as well as enzymatic activity thereof. According to the method disclosed by the invention, a screened myeloperoxidase monoclonal antibody is enveloped on an elisa plate, and the monoclonal antibody can realize specific combination with myeloperoxidase in a sample to be detected, but an enzymatic activity reaction of the myeloperoxidase is not affected; and through a catalytic action of the adsorbed myeloperoxidase directly to substrate tetramethylbenzidine (TMB), quantitative detection on the myeloperoxidase is realized. The detection method has good specificity as well as excellent stability and repeatability, and is simple and convenient to operate; and the method can avoid influence of a horseradish peroxidase detection system in a current ELISA detection method on enzyme detection of the myeloperoxidase, thus offering a new way for detection of the myeloperoxidase.

Description

technical field [0001] The invention belongs to the application field of medical biotechnology, and relates to a method for quantitatively detecting myeloperoxidase and a detection reagent. Background technique [0002] Myeloperoxidase (MPO) is a heme enzyme derived from white blood cells, which mainly exists in the azurophilic granules of myeloid cells (mainly neutrophils and monocytes). Stimulation can lead to the aggregation of neutrophils and the release of MPO. 95% of MPO in the blood is released into the blood by activated neutrophils degranulation. The relative molecular mass of MPO is about 150kDa, which is a dimer composed of two subunits, and each subunit consists of a heavy chain (α chain, with a relative molecular mass of about 59kDa) and a light chain (β chain) , with a relative molecular mass of about 15kDa), the two subunits are connected by a disulfide bond at the α chain. [0003] At present, the commonly used MPO assay methods are colorimetric activity de...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N33/577G01N33/573C12N5/20C07K16/40C12R1/91
CPCG01N33/577
Inventor 卞智萍陈相健顾春荣吴恒芳徐晋妉杨笛
Owner JIANGSU PROVINCE HOSPITAL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com