Detection kit and detection method for anti-mitochondrial antibody IgG (Immunoglobulin G)

A technology of anti-mitochondrial antibody and detection kit, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of not being suitable for laboratories, high fluorescence measurement background, and insufficient objectivity of analysis results, so as to improve clinical sensitivity As well as the effect of linear range and shortened response time

Inactive Publication Date: 2018-03-30
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has the following disadvantages: (1) it is impossible to distinguish non-specific recognition according to the size of the molecular weight when analyzing the results; (2) the operation is relatively complicated and requires a more expensive fluorescence microscope, which is difficult to promote in many primary hospitals and is not suitable for specimens (3) The background in the fluorescence measurement is high, and it is difficult to apply the fluorescence immunoassay technique to the quantitative measurement; (4) The judgment of the results requires experienced professionals, and the objectivity of the analysis results is insufficient
[0008] For example, China Patent Application No. 201510783072.2, the invention patent of the patent name "a kit for quantitative detection of anti-M2-3E antibody IgG using magnetic particle chemiluminescence and its preparation method and detection method", which applies the magnetic particle chemiluminescence quantitative detection technology In the detection of anti-M2-3E antibody, the sensitivity and linear range were improved, but the source of the antigen was not disclosed

Method used

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  • Detection kit and detection method for anti-mitochondrial antibody IgG (Immunoglobulin G)
  • Detection kit and detection method for anti-mitochondrial antibody IgG (Immunoglobulin G)
  • Detection kit and detection method for anti-mitochondrial antibody IgG (Immunoglobulin G)

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Embodiment 1

[0037] The preparation of the first reagent of embodiment one

[0038] 1. Materials and instruments:

[0039] Materials: Naturally extracted M2 antigen, N-hydroxysuccinimide activated biotin, tris buffer, glycerol, recombinant M2 antigen, phosphate buffer

[0040] Instrument: Reagent cryogenic storage box

[0041] 2. Preparation steps:

[0042]Step 1: Mix 0.75mg of naturally extracted M2 antigen with 0.04mg of biotin activated by N-hydroxysuccinimide, and let stand at 30°C for 25min;

[0043] Step 2: Add 12uL of tris buffer solution with a substance concentration of 0.05mol / L, let stand at 30°C for 15min, then add 350uL of glycerol to obtain biotinylated naturally extracted M2 antigen, Store at -20°C for later use;

[0044] Step 3: Mix 0.75 mg of biotin activated by recombinant M2 antigen and 0.04 mg of N-hydroxysuccinimide, and let stand at 30°C for 25 minutes;

[0045] Step 4: Add 12uL of tris buffer solution with a substance concentration of 0.05mol / L, let it stand at ...

Embodiment 2

[0047] The preparation of the second reagent of embodiment two

[0048] 1. Materials and instruments:

[0049] Materials: anti-human IgG antibody, 2-iminosulfane hydrochloride coupling agent, glycine, alkaline phosphatase, tris buffer

[0050] Instruments: Reagent cryogenic storage box, G-25 gel column, Supperdex200 gel purification column

[0051] 2. Preparation steps:

[0052] Step 1: Add 3mg of anti-human IgG antibody to 40mL of 2-iminosulfane hydrochloride coupling agent with a concentration of 10mg / mL, and let stand at 20°C for 20min;

[0053] Step 2: Add 2mL of 0.08mol / L glycine solution, let it stand at 20°C for 4min, use G-25 gel column to desalt, collect the activated anti-human IgG antibody, and store it at 5°C for later use;

[0054] Step 3: Add 3 mg alkaline phosphatase solution to 4 mg / mL 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester solution at 25°C Let it stand for 30 minutes, use G-25 gel column to desalt, collect the activated alkali...

Embodiment 3

[0057] The preparation of embodiment three calibrator

[0058] 1. Materials and instruments:

[0059] Materials: anti-mitochondrial antibody, phosphate buffer saline, standard;

[0060] 2. Preparation steps:

[0061] Select the anti-mitochondrial antibody, dilute it with a phosphate buffer solution with a pH of 7-7.5 and a substance concentration of 0.01mol / L according to a certain ratio, and prepare a calibration solution with a concentration of 20RU / mL and 200RU / mL with reference to the standard. Taste.

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Abstract

The invention discloses a detection kit for an anti-mitochondrial antibody IgG (Immunoglobulin G). The detection kit is characterized by comprising a first reagent, a second reagent, a magnetic particle separation reagent, a chemiluminescent substrate, a calibration material, a quality control material and a cleaning solution, wherein the first reagent is a solution containing an M2 antigen coupled with biotin; part of the M2 antigen is a naturally-extracted M2 antigen, and the other part of the M2 antigen is a recombinant M2 antigen; the second reagent is a solution containing an anti-human IgG antibody coupled with alkaline phosphatase. According to the detection kit disclosed by the invention, by using the complementarity of antigens from multiple sources in the first reagent, clinicalsensitivity is greatly improved, and linear range is greatly widened; standard time of coming out the result after all the flows are finished is 50 minutes; compared with an enzyme linked immunosorbent assay, the detection kit has the advantage that reaction time is greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis, and in particular relates to a detection kit for anti-mitochondrial antibody IgG and a detection method using the detection kit. Background technique [0002] The main target antigen of anti-mitochondrial antibody (Anti-Mitochondrial Antibody; AMA) is the pyruvate dehydrogenase complex on the mitochondrial respiratory chain. Nine molecules are considered to be the target antigens of AMA, namely M1-M9, among which M2 is the main target antigen. [0003] The AMA-M2 antibody is highly specific for primary biliary cirrhosis (PBC), and can be detected in about 90% of PBC patients. Highly specific antibody for liver cirrhosis (PBC). [0004] At present, the detection methods for AMA-M2 antibody are mainly indirect immunofluorescence method and enzyme-linked immunosorbent assay. [0005] The basic principle of indirect immunofluorescence (Indirect Immunofluorescence; IIF) is to use a speci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/531G01N33/535G01N35/00G01N21/76
CPCG01N33/54326G01N21/76G01N33/531G01N33/535G01N35/00
Inventor 李庆春崔利歌柳乐赵婷黎静雯杨苏清徐乐
Owner SUZHOU HAOOUBO BIOPHARML
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