Chemiluminescence immunoassay kit and preparation method thereof

A chemiluminescence immunoassay and proinsulin technology, applied in the field of immunoassay medicine, can solve the problems of inability to achieve biotin avidin system-level amplification, difficult to ensure the repeatability of results, and no beneficial effect on sensitivity, so as to achieve easy capture. Optical signal, improving detection sensitivity and accuracy, the effect of improving sensitivity

Inactive Publication Date: 2014-01-15
北京惠中医疗器械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this proinsulin chemiluminescence immunoassay kit uses chemiluminescence immunoassay, its published sensitivity and other indicators have no obvious advantages compared with ELISA. At the same time, the detection fluctuation is large and the repeatability is poor. The main disadvantages of its design are as follows : 1. The luminescence efficiency of direct labeling antibodies with isoluminol is low, and the markers are unstable. This direct labeling method belongs to the instantaneous luminescence type (that is, the entire luminescence process is completed in less than 1 second), and it is not easy to guarantee the repetition of results. 2. Using FITC and goat anti-FITC system, goat anti-FITC as polyclonal antibody, taken from goat serum, complex components

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  • Chemiluminescence immunoassay kit and preparation method thereof

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Effect test

Embodiment 1

[0042] Example 1 Preparation of Human Proinsulin Chemiluminescent Immunoassay Kit

[0043] 1) Preparation of proinsulin anti-reagent 1

[0044] (1) Coupling of proinsulin monoclonal antibody and biotin: Dissolve N-hydroxysuccinimide biotin in dimethyl sulfoxide to a final concentration of 5.677 mg / ml, and prepare N-hydroxysuccinimide The amine biotin solution and the proinsulin monoclonal antibody with a concentration of 2 mg / ml were mixed in a ratio of 1:10 (mg: mg), reacted at room temperature for 4 hours, and used the molecular sieve chromatography method to utilize the large molecular weight of the conjugate, from Purification from the reaction mixture yielded the final conjugate with high purity.

[0045] (2) Preparation of proinsulin anti-reagent 1: Add about 400 g of purified water, 6.05 g of Tris, 1.85 g of bovine gamma globulin, and 1M MgCl into the container 2 0.369ml, 0.1M ZnCl 2 0.369ml, BSA11.55g, Proclin3000.185ml, after mixing, adjust the pH value to 8.0 with...

Embodiment 2

[0058] The using method of embodiment 2 kit of the present invention:

[0059] 1) Pipette 40 μl of serum to be tested or proinsulin calibrator, 60 μl of proinsulin anti-reagent 1 and 60 μl of proinsulin anti-reagent 2, and add to the bottom of the corresponding marked test tube.

[0060] 2) Gently reciprocate the test tube rack with a multifunctional vortex machine.

[0061] 3) The test tubes were placed in a 37°C water bath for 16 minutes.

[0062] 4) Add 30 μl of separation reagent to each test tube.

[0063] 5) Gently reciprocate the test tube rack with a multifunctional vortex machine.

[0064] 6) The test tubes were placed in a 37°C water bath for 10.5 minutes.

[0065] 7) Put the test tube rack into the magnetic separator, and settle in the magnetic field for 2 minutes.

[0066] 8) Invert the magnetic separator with a large, slow circular motion and decant the supernatant, place the inverted tube on the filter paper, and tap the bottom of the magnetic separator in a ...

Embodiment 3

[0074] The analytical performance of embodiment 3 kits of the present invention

[0075] 1) Sensitivity: ≤5pg / ml.

[0076] 2) Accuracy: the average recovery test of low, middle and high three concentrations of serum (50pg / mL, 200pg / mL and 1000pg / mL respectively) is 98.6%, 99.1% and 102.3% (three averages) respectively.

[0077] 3) Range of standard curve: 0~5000pg / ml

[0078] 4) Precision: both intra-assay and inter-assay variation are less than 10%.

[0079] 5) Specificity: the cross-reactivity rate with analogues (insulin, C-peptide) is less than 0.01%.

[0080] 6) Stability: each component was placed at 37°C, and after 8 days of investigation, each component was still stable.

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Abstract

The invention provides a human proinsulin chemiluminescence immunoassay kit and a preparation method thereof. The kit provided by the invention comprises an anti-proinsulin reagent 1, an anti-proinsulin reagent 2, a magnetic separation reagent, a proinsulin calibrator, a chemiluminescence substrate, and a washing liquid. The invention also provides a preparation method of the kit. The preparation method comprises the following steps: coupling antibody and biotin, preparing the anti-proinsulin reagent 1, coupling enzyme and antibody, preparing the anti-proinsulin reagent 2, preparing the magnetic separation reagent, preparing the proinsulin correction sample, and preparing the chemiluminescence substrate. Compared to the proinsulin kit existing in the present market, the kit provided by the invention has the characteristics of high detection sensitivity, good repeatability, and low cost.

Description

technical field [0001] The invention relates to the field of immunoanalysis medicine, in particular, the invention provides a human proinsulin chemiluminescence immunoassay assay kit and a preparation method thereof. Background technique [0002] Proinsulin (PI) is the immature insulin initially secreted by β cells, which is a long peptide chain composed of 86 amino acids, referred to as proinsulin. It is the precursor substance of insulin, which is composed of insulin and C-peptide. It has dual immune activity, which can be combined with insulin antibody and C-peptide antibody. Proinsulin is synthesized and secreted by pancreatic β cells and mainly catabolized by the kidneys. Under physiological conditions, only a very small amount of proinsulin is released into the blood. Under pathological conditions, the release of proinsulin from pancreatic β cells increases, and the level of proinsulin in the blood increases. The biological effect of proinsulin is basically the same ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/76
CPCG01N33/74G01N2333/62
Inventor 韩子华刘向祎路晟
Owner 北京惠中医疗器械有限公司
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