In this study, 108 patients with (thus far) seropositive RA fulfilling the revised ACR criteria, 122 patients with seronegative RA and other rheumatic disorders, and 200 healthy blood donors were analyzed for anti-MCV and RF-IgG seropositivity using the POCT as well as commercially available ELISAs (See Table for patients' characteristics).
Main diagnoses in the control group were ankylosing spondylitis (n = 21), psoriatic arthritis (n = 21), seronegative course of rheumatoid arthritis (n = 20) and Sjögrens' syndrome (n = 9), polymyalgia rheumatica (n = 8), systemic vasculitis (n = 7), systemic lupus erythematosus (n = 7), Lyme borreliosis (n = 6) and osteoarthritis (n = 6) (all patient diagnoses are listed in Additional file 1
All patients were recruited from the in- and outpatient clinics of the Department of Rheumatology at the Charité-Universitätsmedizin Berlin and at the Rheumaklinik Berlin-Buch. The study was approved by the local Ethics Committee, and blood samples were obtained after written informed consent.
Lateral-flow immunochromatographic device
Lateral-flow immunochromatographic assay (LFIA) was manufactured as double antigen direct sandwich assay. Devices (DCN, Carlsbad, CA, USA) for testing of up to 10 μl of biological samples were produced by mounting a nitrocellulose membrane (Thickness, 205 ± 1 μm) (Millipore, Billerica, MA, USA) to a plastic support. Purified recombinant MCV and purified Fc-part of human immunoglobulin (approximately 1 mg/ml each) were striped in two test line (MCV and RF) positions, while protein L (0.5 mg/ml) (Sigma, St. Louis, MO, USA) was striped in the control line position C. Gold particles (40 nm, British BioCell International), were individually conjugated to goat anti-human IgG and IgM (Dianova, Hamburg, Germany) and mixed. Anti-human immunoglobulin colloidal gold conjugate was dispensed onto a conjugate pad (Arista Biologicals, Allentown, PA, USA). The conjugate pad was then affixed to the test strip by overlapping the nitrocellulose membrane at its proximal end. The assembly was completed by addition of a sample pad onto the conjugate pad. Assay buffer consists of 20 mM Tris, 0.01% sodium azide, 250 mM NaCl, 0.05% Tween 20. Test performance was stable for at least 24 months after manufacture by storage at room temperature.
Direct antibody sandwich format
A blood drop (approximately 20 μl) was placed in the sample port at A on the device. After adding six drops of assay buffer into the buffer port B, patients' antibodies migrated down to the nitrocellulose membrane by capillary action. At the test line T anti-MCV or RF bound to their respective immobilized antigens. By adding an assay buffer, the anti-human IgG gold conjugate was resuspended, and after migration on the nitrocellulose membrane indicated the autoantibody-antigen complexes formed as a red line. Non-MCV and RF specific antibodies migrated to the control line C and were visualized by gold-conjugated anti-human IgG.
During development of the assay, the amount of gold-conjugated anti-human IgG, the number of conjugated colloidal gold particles, and the amount of anti-human IgG were empirically titrated to yield a distinct line at the test positions using a serum sample with a reactivity of approximately 100 U/ml in both standardized anti-MCV and RF-ELISA (Orgentec, Mainz, Germany).
The ratio of applied antigens and serum anti-MCV antibodies and/or RF was such that monodentate binding of autoantibodies to the epitopes was favoured on the basis of steric and other conditions. Subsequently, bidentate antibody binding was favoured at the two test lines, an anti-MCV and a RF-IgG binding sites, due to the extremely high concentration of antigens (approximately 2 mg/ml). Once optimized, this process became independent of the concentration of serum anti-MCV and RF. The colour formation for all reactions was completed after 10 to 15 minutes. The device provides an integrated control system indicating correct test performance or invalid test results. See Figure for possible result constellation.
Serum samples and whole blood samples were run in the LFIA device, and the values were compared to ELISA-derived anti-MCV and RF concentrations, respectively. Results were dichotomized on the basis of being above or below the limit of quantification of the ELISA (cut-off anti-MCV 40 U/ml, cut-off RF-IgG 30 U/ml).
Anti-MCV antibodies (cut-off 20 U/ml) and RF-IgG (cut-off 20 U/ml) were determined by an ELISA (Orgentec).
Sensitivity, specificity and predictive values were calculated according to the appropriate formula. Sensitivity was exclusively calculated within the RA group. Specificity was calculated against rheumatic diseases and a healthy control group. In this study, prevalence was considered as the ratio of seropositive RA patients against all patients with rheumatic diseases (n = 230).