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Homogeneous immunodetection kit for detecting target anti-Carp antibody and application thereof

A technology for immune detection and detection of targets, which is applied in the field of immune analysis, can solve the problems of selectivity, sensitivity, and response speed, cannot realize fully automatic high-throughput analysis, and the influence of reproducibility of experimental results. Small difference, mature and reliable extraction and purification process, and little interference

Pending Publication Date: 2022-03-22
CHEMCLIN DIAGNOSTICS CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] 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 reproducibility of the experimental results; 4. The subtypes of anti-Carp Ab detected are only IgA and IgG, and IgM and IgD antibodies are not detected; 5. Immunoblotting is used for the detection experiment The steps are relatively complicated, the experimental operation standards of different detection institutions are different, time-consuming, automatic high-throughput analysis cannot be realized, and the non-specific detection bands generated on the blot membrane when animal serum is used as the secondary antibody for detection have little effect on the experimental results. interference
[0007] In addition, the above two methods also have the common defect that both detection methods are heterogeneous reaction systems, compared with homogeneous reaction systems, their selectivity, sensitivity, and reaction speed are not as good as the latter

Method used

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  • Homogeneous immunodetection kit for detecting target anti-Carp antibody and application thereof
  • Homogeneous immunodetection kit for detecting target anti-Carp antibody and application thereof
  • Homogeneous immunodetection kit for detecting target anti-Carp antibody and application thereof

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preparation example Construction

[0113] 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.

[0114] 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

[0244] Reagents and experimental materials:

[0245] Reagent I is: carbamylated human serum albumin-coated receptors (luminescent particles) containing aldehyde active groups on the surface;

[0246] Reagent II: biotin-labeled rabbit anti-carbamylated protein antibody.

[0247] The preparation of various buffers is as follows:

[0248] 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.

[0249] 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.

[0250] Cross-linked dialysis buffer 1: Accurately weigh Na with a precision balance 2 CO 3 1.54 g, NaHCO ...

Embodiment 2

[0321] 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).

[0322] 1. Preparation of carbamylated synthetic peptide-BSA

[0323] 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.

[0324] 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.

[0325] 3. Accurately measure 40mg BSA and dissolve it in 800μL purified water to make the final concentration 50mg / ml.

[0326] 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.

[0327] 5. Add 100 ...

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Abstract

The invention relates to a homogeneous immunodetection kit for detecting a target anti-Carp antibody and application of the homogeneous immunodetection kit. The detection kit comprises a component a, a component b and a component c, wherein the component a comprises an antigen combined with a receptor; a component b, which comprises a second anti-Carp antibody; the second anti-Carp antibody is a biotinylated rabbit anti-carbamoylated protein antibody, and the second anti-Carp antibody is a rabbit anti-carbamoylated protein antibody; the second anti-Carp antibody is combined with biotin; the component c comprises a donor capable of generating singlet oxygen in an excited state; the donor is combined with streptavidin. The kit adopts a one-step competition method reaction mode, and the reaction mode can specifically detect anti-Carp Ab such as IgG, IgA, IgM and the like, so that a washing process in a traditional indirect method is omitted, and a one-step method is formed to complete the whole reaction. Compared with a traditional indirect method, the method is time-saving, free of cleaning and simple and convenient to operate.

Description

[0001] This application is a divisional application with the application number 201810142718.2, the application date is February 11, 2018, and the invention title is "Homogeneous immunoassay kit for detecting target anti-Carp antibody and its application". technical field [0002] 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 [0003] 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 monocyana...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/536G01N21/76
CPCG01N21/76G01N33/536G01N33/6854G01N2800/102
Inventor 饶星刘宇卉李临
Owner CHEMCLIN DIAGNOSTICS CO LTD
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