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
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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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