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Calprotectin chemiluminescence immunoassay kit and preparation method thereof

A technology of chemiluminescent immunity and detection kits, which is applied in biological testing, measuring devices, scientific instruments, etc., and can solve the problems of long operation time, only qualitative, low degree of automation, etc.

Inactive Publication Date: 2020-07-07
DIRUI MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of poor sensitivity and high cost of the existing latex-enhanced immunoturbidimetric method, poor quantitative accuracy of the enzyme-linked immunosorbent method, long operation time, low degree of automation, and low sensitivity of the colloidal gold method, which can only be qualitative but not quantitative, the present invention Provided is a calprotectin chemiluminescent immunoassay kit and a preparation method thereof

Method used

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  • Calprotectin chemiluminescence immunoassay kit and preparation method thereof
  • Calprotectin chemiluminescence immunoassay kit and preparation method thereof
  • Calprotectin chemiluminescence immunoassay kit and preparation method thereof

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preparation example Construction

[0045] The preparation method of the calprotectin chemiluminescent immunoassay kit of the present invention mainly comprises the following steps:

[0046] Step 1. Preparation of streptavidin magnetic particles

[0047] Take streptavidin magnetic particle solution, add TBST solution, mix thoroughly, place on a magnetic separator until the supernatant is free of turbidity, discard the supernatant, and keep the magnetic particles; repeat washing 3 times, wash with 50-100mM MES , 0.05-0.15% Tween-20, 0.05-0.1% Proclin300, resuspended in buffer solution with pH 6.5-7.5;

[0048] Step 2. Preparation of calprotectin antibody labeled with chemiluminescence marker

[0049] Replace the calprotectin antibody buffer with PBS, add a chemiluminescent marker, mix well, and purify the labeled calprotectin antibody after standing at room temperature for 2 to 4 hours; place the collected chemiluminescent marker-labeled calprotectin antibody in Store at 2-8°C; use 50-100mM MES or PBS, 0.05% Twee...

Embodiment 1

[0056] Preparation of embodiment 1 calprotectin chemiluminescent immunoassay kit

[0057] (1) Preparation process of streptavidin magnetic particles

[0058] Take 1 mL of streptavidin magnetic particle solution with Tosyl functional group at a concentration of 100 mg / mL (streptavidin magnetic beads were purchased from Agilent Technologies Co., Ltd.), add 5 mL of TBST solution and mix well for 10 min, then place in On a magnetic separator, until the supernatant is free of turbidity, discard the supernatant, and keep the magnetic particles; after repeated washing 3 times, resuspend with 50mM MES, 0.15% Tween-20, 0.10% Proclin300, pH6.5 buffer to prepare The solid-phase reagent with a final concentration of 0.1% into magnetic beads is the R1 reagent.

[0059] (2) Preparation process of calprotectin antibody labeled with chemiluminescence marker

[0060] Use an ultrafiltration centrifuge tube to replace the calprotectin antibody buffer with PBS (the mass of calprotectin antibody...

Embodiment 2

[0063] Example 2 Preparation of Calprotectin Chemiluminescent Immunoassay Kit

[0064] (1) Preparation process of streptavidin magnetic particles

[0065] Take 1 mL of streptavidin magnetic particle solution with Tosyl functional group at a concentration of 100 mg / mL (streptavidin magnetic beads were purchased from Agilent Technologies Co., Ltd.), add 8 mL of TBST solution and mix well for 15 min, then place in On a magnetic separator, until the supernatant is free of turbidity, discard the supernatant, and keep the magnetic particles; after repeated washing 3 times, resuspend with 75mM MES, 0.10% Tween-20, 0.05% Proclin300, pH7.0 buffer to prepare The solid-phase reagent with a final concentration of 0.05% into magnetic beads is the R1 reagent.

[0066] (2) Preparation process of calprotectin antibody labeled with chemiluminescence marker

[0067] Use an ultrafiltration centrifuge tube to replace the calprotectin antibody buffer with PBS (the mass of calprotectin antibody i...

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Abstract

The invention discloses a calprotectin chemiluminiscence immunoassay kit and a preparation method thereof, belongs to the field of in-vitro detection, and solves the problems of low sensitivity, highcost, poor accuracy, long operation time, low automation degree and incapability of quantification of an existing kit. The chemiluminescence immunoassay kit comprises streptavidin magnetic particles,a chemiluminescence marker labeled calprotectin antibody, a coupling marker labeled calprotectin antibody, a chemiluminescence substrate solution and a calprotectin calibrator, wherein the concentration of the magnetic beads is 0.02-1%, and the particle size is 1.0-3.0 microns, the molar ratio of the calprotectin antibody to the chemiluminiscence marker is 1: (3-20), the molar ratio of the calprotectin antibody to the coupling marker is 1: (5-20), the chemiluminescence marker is acridinium ester, luminol, isoluminol or terpyridyl ruthenium. And the coupling marker is biotin. The method is highin sensitivity, high in accuracy, strong in specificity, high in automatic measurement degree, low in cost, small in error, short in measurement time and wide in application range.

Description

technical field [0001] The invention belongs to the technical field of in vitro detection, and in particular relates to a calprotectin chemiluminescence immunoassay kit and a preparation method thereof. Background technique [0002] Calprotectin is a calcium and zinc binding protein with a molecular weight of 36kD, which contains two 14kDa (mrp14, s100 a9) and one 8kDa (mrp8, s100 a8) amino acid chains, each of which can bind two calcium ions , has heat resistance and anti-protease activity. [0003] Calprotectin is widely distributed in human tissues, body fluids and cells, and is the main protein of neutrophils, monocytes and keratinocytes. The content of calprotectin in different parts of the body is different. The content of plasma calprotectin in healthy adults is 0.1-0.6mg / L; the content of virus-infected patients is 0.1-11.4mg / L; the content of bacterial infection is 0.6-11.0mg / L. In healthy adults, plasma calprotectin levels are significantly higher in males than ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533
CPCG01N33/533G01N33/68G01N2333/4727
Inventor 高智玲翟涛李冬梅韩美玉张旭东高威孙成艳何浩会
Owner DIRUI MEDICAL TECH CO LTD
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