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Kit for detecting cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody

A technology of anti-cyclic citrullinated peptide and bispecific antibody, applied in the field of clinical laboratory science

Inactive Publication Date: 2013-08-07
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports of anti-CCP and anti-IgG bispecific antibodies in the serum of RA patients and no kits for this detection

Method used

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  • Kit for detecting cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody
  • Kit for detecting cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody
  • Kit for detecting cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1: the preparation of rabbit IgG-HRP

[0081] 1. Materials

[0082] 1. Rabbit immunoglobulin G (IgG): SIGMA, I5006-10mg, batch number: 017K7430, purity ≥ 95%.

[0083] 2. Horseradish peroxidase: SIGMA, P8375-5KU, RZ=3.1, batch number: 078K7675.

[0084] 3. SephacrylTM S-200 molecular sieve filler: Amersham Biosciences, batch number: 298473.

[0085]4. ELISA plate: 96 wells, Costar.

[0086] 5. Rheumatoid factor positive serum.

[0087] 6. Coating buffer: 50mmol / l carbonate buffer solution (pH9.5), 0.32g sodium carbonate, 0.58g sodium bicarbonate, diluted with water to 200ml.

[0088] 2. Method

[0089] 1. Labeling reaction

[0090] (1) 5.5mg horseradish peroxidase is fully dissolved in 1.32ml distilled water;

[0091] (2) Add 0.1M NaIO dropwise to the dissolved HRP solution 4 , and keep stirring to fully oxidize the HRP, and react at room temperature for 20 minutes;

[0092] (3) Dialysis bag processing

[0093] The dialysis bag was cut into small pi...

Embodiment 2

[0125] Example 2: A kit for detecting anti-cyclic citrullinated peptide (CCP) and anti-rabbit IgG bispecific antibody

[0126] 1. Kit composition

[0127] 1. Cyclic citrullinated peptide-coated microplates: source IgG anti-CCP ELISA kit, EURO-DIAGNOSTICAImmunoscan Switzerland;

[0128] 2. Enzyme-labeled antigen: horseradish peroxidase-labeled rabbit immunoglobulin G, derived from the rabbit IgG-HRP prepared in Example 1;

[0129] 3. Sample diluent: PBS solution containing 1% gelatin; (use PBS to prepare gelatin solution with 1% mass concentration, gelatin needs to be heated to dissolve, and must be cooled before use)

[0130] 4. Washing solution (PBST): PBS solution containing 0.05% Tween-20; (add 500 μl Tween-20 to 1L PBS, mix well)

[0131] 5. Substrate solution: 3', 3', 5', 5', -tetramethylbenzidine (TMB);

[0132] 6. Positive control solution: the serum of rheumatoid arthritis patients whose bispecific antibody has been confirmed to be positive;

[0133] 7. Negative ...

Embodiment 3

[0135] Example 3: Detection kit for anti-cyclic citrullinated peptide (CCP) and anti-rabbit IgG bispecific antibody

[0136] 1. Kit composition

[0137] 1. Cyclic citrullinated peptide-coated microwell plate: synthesized cyclic citrullinated peptide-coated microwell plate;

[0138] According to the literature Schellekens GA, Visser H, de Jong BA, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide (diagnostic properties of cyclic citrullinated peptide antibodies in rheumatoid arthritis). Arthritis Rheum 2000; 43: The CCP sequence disclosed in 155-63. should be synthesized with a purity of more than 95% after purification by reverse-phase high-performance liquid chromatography, and the correctness of the synthesized polypeptide sequence should be confirmed by amino acid sequencing and mass spectrometry analysis.

[0139] The sequence contains a total of 21 amino acids: cfc1-cyc:

[0140] Wherein, X represents citrul...

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Abstract

The invention discloses a kit for detecting a cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody, which belongs to the field of clinical laboratory science. The kit of the invention is developed based on an enzyme-linked immunosorbent assay technology, utilizes a double-antigen sandwich method, comprises two antigens of CCP and IgG and can detect a naturalbispecific antibody existing in the serum of a rheumatoid arthritis patient, is convenient for use and can simply and easily detect the bispecific antibody.

Description

technical field [0001] The invention relates to a kit for detecting anti-cyclic citrullinated peptide (CCP) and anti-immunoglobulin G (IgG) bispecific antibody, which belongs to the field of clinical laboratory science. Background technique [0002] Rheumatoid arthritis (RA) is a common systemic autoimmune disease with synovial inflammation and symmetrical and destructive joint disease as the main clinical features. The incidence of RA is about 0.4% in my country and 0.5%-1.0% in the world. In the early stage of RA, patients mainly present with swelling and pain of proximal interphalangeal joints and metacarpophalangeal joints. As the disease progresses, large joints such as knees, elbows, wrists, and ankles are successively affected. At this time, a large amount of activated leukocyte infiltration can be seen in the synovial tissue, which leads to tissue hyperplasia and inflammation, and finally causes progressive destruction of the joint. Since RA is a systemic disease,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N21/31
Inventor 李金明汪维王露楠张括
Owner BEIJING HOSPITAL
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