RA patients and healthy controls (HC)
In this cross-sectional and case-control study, serum samples were collected from 111 patients with RA who met the ACR classification criteria [14] and from 81 HC. All of the patient sera were leftovers and 5 sera were shortage for testing IgA class of anti-hinge antibodies. All the samples were stored at -80°C until use.
Characterizations of the RA patients and the HC were shown in Table 1. The RA was significantly older than the HC group, although no gender difference between the two groups. Many RA patients had long disease duration and treated with biologics. Positive for rheumatoid factor and anti-CCP2 antibodies due to routine laboratory examination were 67/111 (60.4%) and 77/111 (69.4%), respectively.
Proteolytic cleavage for generating F(ab’)2 fragments
The following biologics were used: tocilizumab (TCZ;IgG1), infliximab (IFX;IgG1), panitumumab (PAN;IgG2) and natalizumab (NTZ;IgG4). All the biologics except IFX were humanized monoclonal IgG. Although IFX is chimeric, the hinge region and CH2 domain were derived from human IgG1 according to the database from the national center for biotechnology information (NCBI, USA). We used pepsin (Sigma-Aldrich, USA) and human MMP-3 as proteases to cleave the biologics. The pro-MMP-3 was a kind gift from Daiichi-Fine Chemical, Toyama, Japan.
The biologics were dialyzed in the 0.1 M sodium citrate buffer (SCB, pH3.5), and then 100 μg of pepsin (Sigma-Aldrich, USA) in SCB was incubated with 10 mg of TCZ or NTZ overnight and of PAN for 2 hours. For stopping the digestion, 1M Tris was added to the IgG solution until the pH increased to 7.4. Proteolysis by MMP-3 was performed after activation of pro-MMP-3 by incubation at 55°C for 25 min [15]. The activated MMP-3, 50μg, in 50mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 10 mM CaCl2 was mixed with 5mg of each biologic at 37°C. After 2 hours incubation, each small amount of the reaction mixture (20 μL) was removed and the reaction was stopped by rapid freezing. The remaining reaction mixture was continued for 24 hours and stopped by adjustment to 20 mM ethylenediaminetetraacetic acid (EDTA).
Detection of human IgG fragments
Cleaved human IgG fragments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine buffer using 10% gels under non-reducing conditions. Samples were heated at 100°C for 2 min in 50 mM Tris-HCl buffer (pH 6.8) with final concentration of 20% glycerin and 1% (w/v) SDS. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 (0.25% (w/v); Nacalai Tesque, Kyoto, Japan) in 50% (v/v) methanol and 10% (v/v) acetic acid.
Purification of IgG F(ab’)2
At the outset, human IgG digested by pepsin or MMP-3 was separated by gel filtration on Sephadex G-150 (Pharmacia Fine Chemicals AB, Uppsala, Sweden). Estimated IgG F(ab’)2 fractions were concentrated by Vivaspin 20 (Sartorius Stedium, Goettingen, Germany). Finally to remove IgG possessing Fc, the concentrated crude IgG F(ab’)2 were applied to a Protein G Mag Sepharose (GE Healthcare, Uppsala, Sweden) in 50 mM Tris, 150 mM NaCl pH 7.5. Purification of the IgG F(ab’)2 was confirmed by the SDS-PAGE described above. Each purified F(ab’)2 fragment was denoted by addition of an italic subscript meaning the protease responsible for the cleavage (e.g. IgG1 F(ab’)2MMP-3).
Measurement of AHA by enzyme linked immunosorbent assay (ELISA)
ELISA plates (Sumilon, Sumitomo-Bakelite, Tokyo, Japan) were coated overnight at 4°C with 100 μL/well of IgG F(ab’)2 of 0.5 μg/ml in 0.1 M carbonate/bicarbonate buffer, pH 9.6. After washing with 10mM Tris buffer containing 0.9% NaCl, pH 7.4,with 0.05% Tween-20 (TBST), serum samples diluted at 1:200 in the TBST were added to the plate (100 μL/well). Plates were incubated for 2 hours at room temperature (RT). After washing, 100 μL/well of alkaline phosphatase (ALP)-conjugated anti-human IgG Fc (Sigma-Aldrich) diluted 1:10,000 with the TBST, or ALP-conjugated anti-human IgA (Sigma-Aldrich) diluted 1:10,000 with the TBST was added and incubated for 1 hour at RT. After washing, the AHA was visualized with 100 μL/well at 1mg/mL of nitrophenyl phosphate tablets (Sigma-Aldrich) in diethanolamine buffer, pH 9.8, for 30 minutes except 2 hours for IgA AHA measurement. Absorbance was measured at 405 nm using a microplate reader.
Levels of IgG AHA were calculated by a calibration curve using pooled human IgG purified by 40% ammonium sulfate and DEAE sephadex. We arbitrarily defined 1 mg/mL of the pooled IgG as containing 800 arbitrary units (AU)/mL of IgG AHA to IgG F(ab’)2pepsin. We also used this calibration curve for measuring IgG or IgA AHA to other IgG F(ab’)2 fragments.
Inhibition study for specificities of IgG AHA against IgG1- or IgG4 F(ab’)2pepsin
Same volume of various concentrations of inhibitors (5,000, 1,000, 200, 40, 8, 1.6, 0.32, 0 μg/mL) and 1:200 diluted IgG AHA positive serum from RA patient was mixed well in the tube, followed by incubation for 2h at RT. The mixtures were added to ELISA plate (100 μL/well) coated with IgG1- or IgG4 F(ab’)2pepsin, and then allowed to react for 2h at RT. The subsequent procedure was the same as the measurement of AHA described above. The extents of inhibition by inhibitors were expressed as percent inhibition of the AHA responses, calculated as follows:
Percent inhibition
=1-(absorbance in the presence of inhibitors/absorbance in the absence of inhibitors) x 100
Statistical analysis
We used the Mann-Whitney U test and the Kruskal-Wallis test to compare the difference between 2 groups and among multiple groups, respectively. We also used Fisher’s exact χ2 test for nominal characteristic. To elucidate the independent variables associated with RA diagnosis, univariate logistic regression followed by multivariate logistic regression analysis was performed. Age and gender were forcedly entered into the model. Two-tailed P value <0.05 was considered significant. All of the data were analyzed on a personal computer using SPSS version 19 (IBM Japan, Tokyo, Japan) and StatFlex version 6 (Osaka, Japan).