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Lipoprotein (a) detection kit

A detection kit, lipoprotein technology

Active Publication Date: 2014-02-26
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of this, the object of the present invention is to propose a lipoprotein (a) detection kit applied to immunoturbidimetry, which solves the problem that the apo (a) molecules of different sizes are detected by the immunoturbidimetry. Enzyme cross-reactivity and susceptibility to rheumatoid factor interference

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of lipoprotein(a) detection kit

[0043] Reagent 1 is:

[0044]

[0045]

[0046] Reagent 2 is:

[0047]

[0048] Preparation of latex microspheres of monoclonal antibody against lipoprotein (a)

[0049] 1) Dilute the monoclonal antibody to 1mg / ml with 50mM HEPES buffer (pH7.5).

[0050] 2) Dilute 100nm latex microspheres with MES buffer (10mM pH5.0) to a mass concentration of 2%, 400μl in total.

[0051] 3) Prepare 400μl of EDC (2mg / ml) and 400μl of NHS (2mg / ml) with MES buffer (10mM pH5.0).

[0052] 4) Add 300 μl EDC solution dropwise to the latex microsphere solution, then add 300 μl NHS solution dropwise, and stir at room temperature for 15 minutes.

[0053] 5) Mix the activated microspheres and monoclonal antibody, and stir at room temperature for 2 hours.

[0054] 6) Centrifuge after the reaction, remove the supernatant, resuspend the pellet with 6ml of reagent 2 buffer, and disperse by ultrasonic.

[0055] The working calibrators are:

...

Embodiment 2

[0059] The assay method of lipoprotein (a) detection kit of the present invention and drawing standard curve

[0060] The measurement method used is the two-point endpoint method, the temperature is 37 ° C, the reaction direction is upward, the sample: reagent 1: reagent 2 is 2:180:60, the main / secondary wavelength is 600 / none, take 180 μl of reagent 1 and add 2 μl of sample Or after calibration, incubate stably at 37°C for 250 seconds, then add 60 μl reagent 2 and continue to incubate for 50 seconds, record the absorbance value A1, and record the absorbance value A2 after continuing the reaction for 300 seconds. ΔA=A2-A1

[0061] With the concentration of the standard as the abscissa and the corresponding ΔA as the ordinate, use Spline nonlinear fitting to draw the standard curve as figure 1 .

[0062] The experimental results show that: the standard curve is a straight line, indicating that there is no prozone and postzone phenomenon in the sample concentration range of 0-...

Embodiment 3

[0064] Since the antibodies used in the commonly used immunoturbidimetric method to detect lipoprotein (a) mainly act on K4, the detection deviation of apo(a) molecules of different sizes is relatively large. Lipoprotein(a)-cholesterol assay can overcome the problem of large bias caused by the polymorphism of apo(a) molecule. The present invention takes the lipoprotein (a)-cholesterol assay (ultracentrifugation-agarose gel electrophoresis) as a reference, respectively compares the lipoprotein (a) detection kit prepared in Example 1 of the present invention and similar products with lipoprotein ( a) - Correlation of cholesterol measurements. A similar product in the prior art is a lipoprotein (a) assay kit produced by DiaSys Diagnostic Systems GmbH, and the assay method is a particle-enhanced immunoturbidimetric method. Lipoprotein (a)-cholesterol detection kit is a lipoprotein cholesterol detection kit developed and produced by Beijing Leadman Biochemical Co., Ltd. The corre...

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Abstract

The invention discloses a lipoprotein (a) detection kit which is characterized by consisting of a reagent 1, a reagent 2 and a working calibration solution. The reagent 1 consists of a buffer solution, bull serum albumin (BSA), a surfactant, goat IgG and NaN3; the reagent 2 consists of a buffer solution, a lipoprotein (a) monoclonal antibody latex microsphere, BSA, the surfactant, the goat IgG, a stabilizer and NaN3. The lipoprotein (a) detection kit can be applied to a fully automatic biochemical analyzer, the detection result is high in accuracy, and the lipoprotein (a) has high comparability with absolute concentration of lipoprotein (a) of different crowds and different sizes. Moreover, the lipoprotein (a) detection kit does not have a cross reaction with plasminogen, is not interfered by rheumatoid factors and is high in reagent stability, convenient to use and convenient for large-scale popularization, only a monoclonal antibody of the lipoprotein (a) is needed, and the preparation cost and time of the kit are saved.

Description

technical field [0001] The invention relates to a lipoprotein (a) detection kit. Background technique [0002] Lipoprotein (a) is a protein in human serum with a structure similar to LDL (low-density lipoprotein), and its density is between HDL (high-density lipoprotein) and LDL. The core part of lipoprotein (a) is neutral lipid and apoB-100 molecules, surrounded by hydrophilic apo (a), the two are covalently linked by disulfide bonds; among them, apo (a) is highly glycosylated The hydrophilic protein, the length of the peptide chain is very inconsistent, and the molecular size is obviously polymorphic. The analysis of apo(a) cDNA found that apo(a) belongs to the plasminogen gene superfamily and has high homology with plasminogen. Similar to plasminogen, apo(a) contains a carboxy-terminal protease-like domain and a Kringle domain. Although the protease domain of apo(a) is 85% homologous to plasminogen, it has no catalytic activity, so Lipoprotein(a) can competitively inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N15/06
CPCG01N33/68G01N33/6893G01N2800/00
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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