Homogeneous immunoassay kit for detecting target anti-Carp antibodies and application thereof

A technology for immune detection and target detection, which is applied in the field of immune analysis, and can solve problems such as complex experimental steps, long time-consuming, and influence on the reproducibility of experimental results

Pending Publication Date: 2018-08-24
CHEMCLIN DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The defects of the detection method in the patent WO2012/105838A1 are: 1. The anti-Carp Ab in the human body is an antibody produced against carbamylated proteins in the body, and carbamylated fetal calf serum (Car-FCS) is used as Animal-derived antigens are not exactly the same as the epitopes of carbamylated proteins in the human body; 2. Carbamylated fetal bovine serum contains a large number of anti-Carp Ab antibodies and other antibodies that can be compared with human serum samples Proteins and other complex components of immunoglobulin non-specific interactions lead to high background values ​​in the experiment, which interferes with the analysis of results; 3. Carbamylated fetal bovine serum is difficult to repeatedly prepare, and there are batch-to-batch differences, which may cause It will affect the reproducibili

Method used

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  • Homogeneous immunoassay kit for detecting target anti-Carp antibodies and application thereof
  • Homogeneous immunoassay kit for detecting target anti-Carp antibodies and application thereof
  • Homogeneous immunoassay kit for detecting target anti-Carp antibodies and application thereof

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preparation example Construction

[0112] In the present invention, there is no particular limitation on the preparation method of the polyclonal antibody, which can be prepared by conventional preparation methods in the art. For example, the preparation method of the polyclonal antibody includes: immunizing animals with carbamylated antigens, and obtaining The animal serum of the polyclonal antibody; the animal serum is purified by affinity chromatography to obtain a polyclonal antibody that specifically recognizes the carbamylated antigen.

[0113] In the present invention, the preparation method of the monoclonal antibody is not particularly limited, and can be prepared by conventional preparation methods in the art. For example, the preparation method of the monoclonal antibody includes: the preparation method of the monoclonal antibody includes: adding ammonia The rabbit spleen cells immunized with the formylated antigen were fused with rabbit myeloma cells and then cultured, and the cell culture supernatan...

Embodiment 1

[0243] Reagents and experimental materials:

[0244] Reagent I is: carbamylated human serum albumin-coated receptors (luminescent particles) containing aldehyde active groups on the surface;

[0245] Reagent II: biotin-labeled rabbit anti-carbamylated protein antibody.

[0246] The preparation of various buffers is as follows:

[0247] Calibrator buffer: Accurately weigh 4.77g of HEPES and 1.7g of NaCl with a precision balance, add 160mL of purified water and mix for 30min, adjust the pH value to 7.4±0.2, continue to add Proclin300 0.1g, BSA 30g, 1M MgCl 2 0.5ml, 0.1M ZnCl 2 0.1ml, after stirring for 30min, add purified water to make the volume to 200g, retest the pH value, and set aside at 2-8C.

[0248] KOCN solution: Accurately weigh KOCN 8.112g, Na 2 HPO 4 12H 2 O 5.9g, KH 2 PO 40.488g, dilute purified water to 100mL, and adjust the pH value to 7.2±0.05.

[0249] Cross-linked dialysis buffer 1: Accurately weigh Na with a precision balance 2 CO 3 1.54g, NaHCO 3...

Embodiment 2

[0319] The difference from Example 1 is that the carbamylated antigen in reagent I is carbamylated synthetic polypeptide-BSA, and reagent I is: carbamylated synthetic polypeptide-BSA coated receptor (luminescent particles).

[0320] 1. Preparation of carbamylated synthetic peptide-BSA

[0321] 1. Dissolve 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide sodium salt (Sulfo-SMCC) in 800 μL dimethyl sulfoxide ( DMSO) to a final concentration of 20 mM.

[0322] 2. Accurately measure 3 mg of dry powdered synthetic peptide and dissolve it in 600 μL of purified water to make the final concentration 5 mg / ml.

[0323] 3. Accurately measure 40mg BSA and dissolve it in 800μL purified water to make the final concentration 50mg / ml.

[0324] 4. The synthetic peptide solution and BSA solution were thoroughly mixed and dissolved in 2.25ml of 0.01M PBS buffer at a mass ratio of 1:1 (2 mg each), and left to stand at room temperature for 1 hour.

[0325] 5. Add 100 ...

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Abstract

The invention relates to a homogeneous immunoassay kit for detecting target anti-Carp antibodies and application thereof, wherein the kit adopts a one-step competition reaction mode, and the reactionmode can specifically detect the anti-Carp Ab such as IgG, IgA and IgM, thereby omitting the process of washing in a conventional indirect method, forming a one-step process to complete the entire reaction. Compared with the traditional indirect method, the homogeneous immunoassay kit is time-saving, washing-free, and easy to operate.

Description

technical field [0001] The invention belongs to the technical field of immune analysis, and in particular relates to a homogeneous immunoassay kit for detecting a target anti-Carp antibody, a preparation method and a use method thereof. Background technique [0002] Anti-carbamylated protein antibody (anti-Carbamylated protein antibody, anti-Carp Ab) is a new biological agent that is closely related to the diagnosis and course monitoring of rheumatoid arthritis (RA) discovered in recent years. landmark. Carbamylation is a non-enzyme-mediated protein post-translational modification. The specific process is that the lysine residue of the protein is converted into homocitrulline under the action of urea derivative monocyanate or cyanate acid residues. This process can break human immune tolerance and induce the production of autoimmune antibody anti-Carp antibody (anti-Carp Ab). [0003] The detection methods of anti-Carp Ab disclosed in patents WO2012 / 105838A1 and WO2016 / 01...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/536G01N21/76
CPCG01N21/76G01N33/536G01N33/6854G01N2800/102
Inventor 廖智星饶星李会强刘宇卉李临
Owner CHEMCLIN DIAGNOSTICS CO LTD
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