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Method for the serological diagnosis of rheumatoid arthritis

A specific, red blood cell technology, applied in chemical instruments and methods, disease diagnosis, immunoglobulin against animals/humans, etc., can solve problems such as limited sensitivity

Active Publication Date: 2017-02-22
NOVIO TH BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In patients with rheumatoid arthritis, anti-Sa antibodies provided high specificity >98%, but limited sensitivity, 22% to 40% [17]

Method used

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  • Method for the serological diagnosis of rheumatoid arthritis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1: Preparation of citrullinated IgG

[0075] Rabbit IgG (25ug), citrullinated in vitro by rabbit skeletal muscle PAD (75mU; Sigma-Aldrich; EC 3.5.3.15) in deimination buffer (40mM Tris-HCl, pH 7.5, 5mM CaCl2 and 1mM DTT) Incubate at 37°C for 3h. The reaction was stopped by adding EGTA at pH 8.0 to a concentration of 50 mM. Citrullinated rabbit IgG was obtained after overnight dialysis against 10 mM Tris pH 7.6 with 2 mM EDTA.

[0076] Goat glycophorin A antibody (25ug), obtained from Abcam, UK, was prepared from rabbit skeletal muscle PAD (75mU; Sigma-Aldrich; EC 3.5.3.15) in deimination buffer (40mM Tris-HCl, pH 7.5, 5mM CaCl2 and 1 mM DTT) and incubated at 37°C for 3 h. The reaction was stopped by adding EGTA at pH 8.0 to a concentration of 50 mM. Citrullinated goat IgG was obtained after overnight dialysis against 10 mM Tris pH 7.6 with 2 mM EDTA.

[0077] Another glycophorin A antibody was obtained from Santa Cruz Biotechnology. R10sc-53905 is a mon...

Embodiment 2

[0079] Embodiment 2: Preparation of carbamylated IgG

[0080] Carbamylated IgG was obtained by incubating 1.18 mg of rabbit IgG dissolved in 1 mL of PBS with 1 mL of 0.2 M KCNO in 0.1 M Na2HPO4 buffer for three hours at 37 degrees Celsius. Carbamylated IgG was then dialyzed overnight at 4°C against 0.9% NaCl and stored at 4°C until use. This IgG was used as the antigen in an ELISA assay for the detection of anti-citrullinated antibodies in RA patient sera. Non-carbamylated IgG was used as a control therein.

Embodiment 3

[0081] Example 3: ELISA with citrullinated antibodies

[0082] An ELISA assay to detect antibodies against citrullinated antibodies was set up as follows. Citrullinated IgG, prepared as described above, was immobilized on NUNCELISA plates by incubating 50 μl / well of 100 μg / ml citrullinated IgG overnight at 37°C. Remaining binding sites were performed by adding 50 microliters of 100 micrograms / ml bovine serum albumin (BSA) in phosphate-buffered saline (PBS) to each well of the microtiter plate and incubating for 1 hour at 37 degrees Celsius. block. The plates were then washed 3 times with PBS+0.05% Tween 20 (PBS / Tween) and stored dry at 4°C if not used immediately.

[0083] Control plates were prepared in exactly the same manner, except that they contained non-citrullinated antibodies from species-matched controls immobilized on the solid phase.

[0084] Serial dilutions (1 / 10 to 1 / 100.000) of human sera from RA patients known to be negative for rheumatoid factor were used t...

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Abstract

The present invention is in the field of diagnostic methods. It provides a new marker antibody for rheumatoid arthritis as well as a new method for detecting rheumatoid arthritis. It also provides means and methods for predicting whether a subject will develop rheumatoid arthritis. The invention is based on the discovery that patients with rheumatoid arthritis have antibodies in their circulation that react specifically with citrullinated antibodies. In one aspect, the invention therefore relates to an antibody comprising a citrulline residue. In another aspect, the invention provides a method for the detection of antibodies specific for rheumatoid arthritis in a sample from a subject, wherein the sample is contacted with a citrullinated antibody and wherein it is determined whether the sample comprises antibodies specifically reactive with the citrullinated antibody.

Description

technical field [0001] The present invention is in the field of diagnostic methods. It provides new labeled antibodies for rheumatoid arthritis and new methods for detecting rheumatoid arthritis. It also provides means and methods for predicting whether a subject will develop rheumatoid arthritis. The present invention is based on the discovery that rheumatoid arthritis patients have in their circulation antibodies that specifically react with citrullinated antibodies. Background technique [0002] Serological diagnostic tests are of increasing importance in the early detection and differentiation of rheumatoid arthritis (RA). In addition to traditional rheumatoid factor testing, new specific autoantibodies to citrullinated antigens have made a significant contribution to the diagnosis of RA [26]. [0003] Rheumatoid factors are autoantibodies that can be IgM, IgG, or IgA and were first mentioned in 1922 [1]. It recognizes the CH2 and CH3 domains of the Fc fragment of hu...

Claims

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Application Information

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IPC IPC(8): C07K16/28G01N33/564
CPCC07K16/2896G01N33/564G01N2800/102C07K2317/40G01N33/80G01N2333/70596
Inventor 马丁努斯·胡贝图斯·莱昂纳德斯·萨尔登
Owner NOVIO TH BV
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