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Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof

A technology for promoting luteinization and chemiluminescence immunity, which is applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc. problems, to achieve cost advantages, improve detection results, and high sensitivity

Active Publication Date: 2015-02-04
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the kit can achieve rapid and accurate detection, the preparation process of horseradish peroxidase-labeled luteinizing hormone monoclonal antibody is very cumbersome, the process stability is not good, and the labeling rate is low, which limits its detection. Effects especially on inter-analytical precision and lead to increased cost

Method used

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  • Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof
  • Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof
  • Luteinizing hormone nano-magnetic particle chemiluminescence assay kit and preparation method thereof and assay method thereof

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Embodiment 1

[0037] Embodiment 1 The preparation of the first reagent

[0038] (1) Materials and instruments: anti-LH monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL, hereinafter referred to as LH antibody); fluorescein isothiocyanate (FITC), sodium bicarbonate Reagents should be chemically pure; G-25 gel purification column was purchased from GE.

[0039] (2) Preparation steps:

[0040] ① Use 0.1~0.2mol / L carbonate buffer solution with pH9.0~10.0 to prepare 0.5mg / mL FITC solution;

[0041] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of LH antibody to FITC molecular ratio of 1:20, mix well, let stand at room temperature for 12 hours, and react to generate LH antibody-FITC conjugate;

[0042] ③Separate the reaction liquid after step ② through a G-25 gel column to remove unreacted FITC to obtain a solution containing the LH antibody-FITC conjugate (i.e. FITC-labeled LH antibody);

[0043] ④ Dilute th...

Embodiment 2

[0044] Embodiment 2 Preparation of the second reagent

[0045] (1) Materials and instruments: anti-LH monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL, hereinafter referred to as LH antibody solution); alkaline phosphatase (ALP) preserved in phosphate buffer solution, the ALP purity is about 99%, the specific activity is about 1500U / mg, and the concentration is 10mg / mL); 5mg / mL SMCC solution, 10mg / mL2IT solution (SMCC and 2IT solid powder, use DMF to make the above solution) ( purchased from THERMO company); TRIS and other chemical reagents should be chemically pure; G-25 gel purification column, AKTA-purifier100 protein purification instrument and Supperdex200 gel purification column are all products of GE Company.

[0046] (2) Preparation steps:

[0047] ① Take 1mg LH antibody solution, add 2~4μl of 10mg / mL 2IT solution, let stand at room temperature for 20min, add 10μl of 0.1mol / L glycine solution, let stand at room temperature for...

Embodiment 3

[0051] The preparation of embodiment 3 magnetic separation reagents

[0052] (1) Materials and instruments:

[0053] Suspension of magnetic particles: the content of magnetic particles is 5wt%, and the magnetic particles contain carboxyl (COOH) active groups. The carboxyl group content per gram (g) of magnetic particles (dry weight) is not less than 0.4 millimoles (mmol), with superparamagnetism, The diameter is between 0.5-2μm;

[0054] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90% by weight and a dilution titer of more than 1:1 million;

[0055] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0056] (2) Preparation steps:

[0057] ①Take 100mg of the suspension of magnetic particles, magnetically separate to remove the supernatant, and resuspend in 10mL of 0.05mol / L, pH4.5~5MES buffer;

[0058] ②Add 2~4mg of anti-FITC antibody and suspend at r...

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Abstract

The invention relates to a luteinizing hormone nano-magnetic particle chemiluminescence assay kit and a preparation method thereof and an assay method thereof. The luteinizing hormone nano-magnetic particle chemiluminescence assay kit comprises a solution containing a fluorescein-labeled luteinizing hormone antibody, a suspension coated with magnetic particles of an anti-fluorescein antibody, and a solution containing an alkaline phosphatase-labeled luteinizing hormone antibody, wherein the alkaline phosphatase-labeled luteinizing hormone antibody is formed by connecting an alkaline phosphatase with a luteinizing hormone antibody through SMCC (4-(N-maleimidomethyl) cyclohexyl-1-succinimide carboxylate) and 2IT (2-imido sulfane hydrochloride). The luteinizing hormone nano-magnetic particle chemiluminescence assay kit can be used for quantitative determination of the luteinizing hormone at lower cost and higher accuracy and precision.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chemiluminescence immunoassay kit for luteinizing hormone combined with immunomagnetic particle separation technology and chemiluminescence immunoassay technology and a preparation method thereof. The present invention also particularly relates to the preparation method of the kit. Background technique [0002] Luteinizing Hormone (LH) is a glycoprotein hormone secreted by basophils in the anterior pituitary gland, with a molecular weight of about 30,000 Daltons and composed of two non-covalently bound different subunits. Its α subunit has 92 amino acid residues, which are the same as those of LH, TSH, and hCG. The amino acid sequence of the β subunit is very different from that of LH and TSH, but very similar to that of hCG. [0003] LH is regulated by gonadotropin-releasing hormone (Gn-RH) produced by the hypothalamus. Studies on normal women have shown that pituit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N21/76
Inventor 于大为程晓蕾李冬冬
Owner SUZHOU HAOOUBO BIOPHARML
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