Chemiluminescence detection kit for N-MID osteocalcin and preparation method of chemiluminescence detection kit

A chemiluminescence detection, osteocalcin terminal technology, used in chemiluminescence/bioluminescence, biological testing, analysis by chemical reaction of materials, etc., can solve the problem of easy inactivation of enzyme markers, complicated operation, low sensitivity, etc. problem, to achieve the effect of stability, good specificity and high sensitivity of the kit

Inactive Publication Date: 2018-03-23
太原瑞盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, although radioimmunoassay and radioimmunoassay have the advantages of high sensitivity and high specificity, there are many operating steps, special testing equipment is required, reagents are expensive, matching instruments are required and there is radioactive contamination, which makes the operation difficult. People are exposed to radiation hazards; in addition, radioactive isotopes are easy to decay, have a short validity period, and are not easy to store
The ELISA method uses horseradish peroxidase or alkaline phosphatase markers. The enzyme markers are easily inactivated, and the chromogenic substrate is easily decomposed when exposed to light. The sensitivity is low, the operation is complicated, and the repeatability is poor. It is not suitable for emergency and clinical use. Patients ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0025] Embodiment: the formation of kit and the preparation of specific components thereof

[0026] 1. Assembly of the kit

[0027] A chemiluminescent detection kit for N-terminal osteocalcin, which contains the following components:

[0028] Carboxyl magnetic beads coupled N-terminal osteocalcin monoclonal antibody;

[0029] Acridine ester-labeled N-end osteocalcin monoclonal antibody;

[0030] N-Middle Osteocalcin Series Standard Solution

[0031] Chemiluminescence pre-excitation solution A and chemiluminescence excitation solution B;

[0032] Cleaning fluid.

Embodiment 2

[0033] Embodiment 2: the preparation of concrete component

[0034] 1. Preparation of magnetic bead-conjugated antibodies

[0035] (1) Take 1 mg of carboxyl magnetic particles in a 0.5 mL centrifuge tube, add 0.1 mol / L MES buffer, vortex and mix well, place on a magnetic stand, let stand for 5 min to separate the magnetic particles from the liquid, discard Wash the supernatant 3 times, then add 200 μL of MES (pH 6.0) buffer, and vortex.

[0036] (2) Add 15 μg N-middle osteocalcin monoclonal antibody, so that the molar ratio of carboxyl group to antibody is 150:1: Vortex, rotate the reaction tube, and incubate at room temperature for 30 min.

[0037] (3) Add 10 μL of coupling reagent EDC with a concentration of 10 mg / mL, vortex on the rotary reactor, and incubate at room temperature for 2 h.

[0038] (4) Take 200 μL of glycine buffer (concentration: 50 mmol / L) containing 1% BSA for blocking for 1 h.

[0039] (5) Remove the supernatant, add 200 μL of washing buffer (TBS+0.05%...

Embodiment 3

[0059] Embodiment 3: the implementation of specific kit

[0060] (1) Add 50 μL of the sample to be tested into the cuvette, then add 150 μL of magnetic particle coupling suspension, shake and mix, and incubate at 37°C for 8 min.

[0061] (2) Separation and washing 3 times, fully shake the washed reaction vessel to disperse the magnetic particles.

[0062] (3) Add 150 μL of acridinium ester marker into the cuvette, shake to mix, and incubate at 37°C for 7 min.

[0063] (4) Separation, washing 3 times, fully shaking the washed reaction vessel to disperse the magnetic particles.

[0064] (5) Add 100 μL of chemiluminescence pre-excitation solution A, add 100 μL of chemiluminescence excitation solution B after 1 s, and measure its relative luminous intensity. The content of N-terminal osteocalcin in the sample is proportional to its relative luminous intensity .

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Abstract

The invention discloses a chemiluminescent detection kit for N-middle osteocalcin and a preparation method thereof. The kit includes: magnetic particles coupled with N-terminal osteocalcin capture antibody, acridinium ester-labeled N-terminal osteocalcin detection antibody, N-terminal osteocalcin series standards, chemiluminescent pre- Excitation solution A, chemiluminescence excitation solution B, cleaning solution. The kit of the invention uses the magnetic separation chemiluminescence technology as the detection means, and simultaneously combines the acridinium ester labeling technology. Compared with the existing technology for detecting N-terminal osteocalcin, this kit has the advantages of high sensitivity, high signal-to-noise ratio and rapid detection.

Description

technical field [0001] The invention belongs to the technical field of immunoassay and analysis, in particular to an N-middle end osteocalcin chemiluminescent immunoassay kit and a preparation method thereof. Background technique [0002] Osteocalcin is the most important specific non-collagen protein in the bone matrix, synthesized and secreted by osteoblasts in bones and teeth, and its binding to bone calcium depends on vitamin K. Osteocalcin molecule contains 49 amino acids with a molecular weight of about 5800D. In bone synthesis, osteoblasts produce osteocalcin, which depends on vitamin K, and vitamin D3 can promote the production of osteocalcin. The use of vitamin K antagonists can reduce the content of this protein in bone, but it does not affect its proline content, nor does it affect the mechanical strength of bone. After the production of osteocalcin, part of it enters the bone matrix, and the other part is released into the peripheral blood. Therefore, the cont...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N21/76G01N33/577
CPCG01N33/68G01N21/76G01N33/54326G01N33/577
Inventor 胡雪婷常燕刘丽青曹晶杜爱铭徐兵
Owner 太原瑞盛生物科技有限公司
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