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A kind of myeloperoxidase quantitative detection method and detection reagent

A kind of myeloperoxidase, myeloperoxidase technology

Active Publication Date: 2016-06-22
JIANGSU PROVINCE HOSPITAL
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  • 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

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  • A kind of myeloperoxidase quantitative detection method and detection reagent
  • A kind of myeloperoxidase quantitative detection method and detection reagent
  • A kind of myeloperoxidase quantitative detection method and detection reagent

Examples

Experimental program
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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 SCIPA Ltd., UK) was mixed with an equal volume of Freund's complete adjuvant at 80 μg per mouse and fully emulsified, and three 6-week-old mice at the same time as myeloma cells were injected intraperitoneally. Germ-line female BALB / c mice (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, the mesentery at the caudal end of the spleen was gently lifted with curved forceps, and insulin The syringe was injected into the spleen from the end of the spleen, stagnant for 30s after each injection to prevent reflux, and the spleen was put back 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 (JImmunolMethods.1990; 126(1):125-133), so MPO in the ELISA detection method may interfere with the detection of the HRP system , affecting the test 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 t...

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...

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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

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Application Information

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Patent Type & Authority Patents(China)
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
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