Electrochemical luminescence kit for detecting anti-LRP4 antibody and manufacturing method thereof

An electrochemical and kit technology, applied in the field of immunoassay, can solve the problems such as no method or kit for quantitatively detecting anti-LRP4 antibodies with high sensitivity and high specificity, and achieves fast detection speed, improved work efficiency, and linear range. wide effect

Pending Publication Date: 2020-04-24
江苏三联生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved in the present invention is: in order to solve the technical problem that there is no method or kit for quantitatively detecting anti-

Method used

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  • Electrochemical luminescence kit for detecting anti-LRP4 antibody and manufacturing method thereof
  • Electrochemical luminescence kit for detecting anti-LRP4 antibody and manufacturing method thereof
  • Electrochemical luminescence kit for detecting anti-LRP4 antibody and manufacturing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation method of electrochemiluminescence kit for detecting anti-LRP4 antibody

[0023] 1) Preparation of working solution of magnetic beads coated with streptavidin

[0024] The suspension of streptavidin-coated magnetic bead particles was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.75mg in PBS buffer (0.1M, pH 7.4) with pH=7.4 and a concentration of 0.1mol / L / mL, the buffer contains 0.5 wt% BSA, 0.05 wt% Triton X-100, 0.05 wt% proclin 300, and 0.05 wt% sodium azide.

[0025] 2) Preparation of biotin-labeled LRP4 antigen

[0026] Accurately weigh 1 mg of LRP4 antigen, add an appropriate amount of PBS buffer (0.1M, pH 7.4) to adjust the total antibody concentration to 1 mg / mL, and add it to a dialysis bag for dialysis. Change the dialysate every 3 to 4 hours. ~4 times, transfer the antibody to the centrifuge tube or cryopreservation tube after dialysis;

[0027] Accurately weigh 1 mg of N-hydroxysuccinimide-activated ...

Embodiment 2

[0043] Example 2 The method for detecting anti-LRP4 antibodies using the kit of the present invention uses an automatic electrochemiluminescence immunoassay analyzer as the detection instrument, and the kit is loaded on the instrument for detection. The steps are as follows:

[0044] Add the sample (or calibrator or quality control), biotin-labeled LRP4 antigen working solution and terpyridine ruthenium-labeled anti-human IgG antibody working solution to the reaction cup, and incubate at 37°C for 10 minutes to form antigen-antibody-antibody Antibody complex solution;

[0045] Add streptavidin-coated magnetic beads working solution to the antigen-antibody-anti-antibody complex solution, and incubate at 37°C for 10 minutes to form a magnetic complex suspension;

[0046] Placing the magnetic composite suspension in a magnetic field, and the cleaning fluid flows through and washing the magnetic composite;

[0047] Inject the electrochemiluminescence substrate solution into the washed magn...

Embodiment 3

[0048] Example 3 Performance evaluation of the kit of the present invention

[0049] 1) Research on reagent sensitivity

[0050] The reagent sensitivity is determined according to the minimum detection limit (Limit of Blank, LOB), and the minimum detection limit is performed according to the following experimental method. Check the zero-concentration calibrator 20 times, get the signal value (RLU) of the 20-time measurement results, calculate the average value M and standard deviation SD, and get the RLU value corresponding to M+2SD, according to the zero-concentration calibrator and the adjacent concentration The concentration between the calibrator (1ng / mL) and the average value of RLU are obtained by two-point regression fitting to obtain a linear equation. The RLU value corresponding to M+2SD is brought into the above equation to calculate the corresponding concentration, namely LOB .

[0051] The measurement results in Table 1 below show that the LOB of the reagent detected by...

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Abstract

The invention relates to a novel electrochemical luminescence kit for detecting a low-density lipoprotein receptor associated protein 4 (LRP4) antibody from a detected sample and a manufacturing method thereof. The manufactured kit comprises a streptavidin-coated magnetic bead particle working solution, a biotin-labeled LRP4 working solution, a terpyridyl ruthenium-labeled anti-human IgG antibodyworking solution, an anti-LRP4 antibody calibration product and/or quality control product working solution, a tripropylamine-containing electrochemiluminescence substrate solution and a cleaning solution. A luminescence system of the manufactured kit is electrochemiluminescence, a streptavidin-biotin biological reaction signal amplification system is used, a detection speed is high, sensitivity is high, a linear range is wide, detection result repeatability is good, and accurate quantitative detection of the anti-LRP4 antibody in a sample can be achieved.

Description

Technical field [0001] The invention relates to an electrochemiluminescence kit for detecting anti-LRP4 antibodies and its usage, belonging to the technical field of immunoassay. Background technique [0002] Myasthenia gravis (MG) is an autoimmune disease, which is a signal transmission disorder of neurotransmitter at the neuromuscular junction (NMJ) mediated by autoantibody, complement participation, and cellular immunity dependent. The incidence of MG population is (32~64) / 100,000. There are currently about 600,000 MG patients in my country [1] . The main clinical manifestations are skeletal muscle weakness and fatigue, which aggravate after exercise. The symptoms are significantly relieved and relieved after rest and application of cholinesterase inhibitors. It is characterized by high morbidity, high disability, poor prognosis, and easy relapse. It has seriously threatened human life and health, and has received extensive attention from medical and scientific research worke...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531G01N21/76G01N27/26
CPCG01N21/76G01N27/26G01N33/531G01N33/68G01N2800/24
Inventor 卜梅杰泮锋纲丁俊杰施启尧
Owner 江苏三联生物工程股份有限公司
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