Immulon 1b elisa plates

Recombinant human MMP-10 (R&D Systems) was diluted (0.1 μg/ml) in carbonate coating buffer, added to Immulon 1B ELISA plates (Thermo Scientific), and incubated overnight at 4 °C. All remaining incubations were conducted on a platform shaker set at 200 revolutions per minute at room temperature. The plates were incubated with a 3% bovine serum albumin (BSA; Equitech-Bio, Inc.) in PBS–0.05% Tween 20 (PBST) blocking buffer. Plates were incubated with patient serum or joint fluid samples (1:200) or the positive control anti-MMP-10 MAb910 (1:200; R&D Systems). Following washes with PBST, horseradish peroxidase–conjugated goat anti-human IgG (Santa Cruz Biotechnology) or horseradish peroxidase–conjugated donkey anti-mouse IgG (Santa Cruz Biotechnology) was added, followed by TMB substrate (BD Biosciences). For interplate standardization, the positive control MAb910 was included on each plate. A positive antibody response was defined as >3 SD above the mean in 58 healthy subjects; this mean + 3 SD value characteristically corresponded to >0.51 OD₄₅₀.
To examine specificity of the antibody response to MMP-10, serum samples were also tested with MMP-3 (R&D Systems), using the same methods detailed for MMP-10. Seropositivity responses to the B. burgdorferi were analyzed by ELISA and Western blot as previously described [49 (link)–51 (link)].

All antibody responses were determined by ELISA. B. burgdorferi sonicate (strain G39/40) (5 μg/ml), or recombinant human MMP-10 (R&D Systems), human apoB-100 (Millipore), or ECGF (R&D Systems) (in each instance, 2.5 μg/ml) diluted in carbonate coating buffer was added to Immulon 1B ELISA plates (Thermo Scientific), and incubated overnight at 4°C. After washing with PBS with 0.05% Tween 20 (PBST) between each step, the plates were incubated with a 3% bovine serum albumin (BSA; Sigma Aldrich) in PBS blocking buffer, followed by patient serum or synovial fluid samples (1:100), in each instance, for 1 hour on an orbital shaker (200 rpm). The secondary antibodies were horseradish peroxidase (HRP)-conjugated mouse anti-human IgG1, 2, 3, or 4 Fc antibodies (Life Sciences), which were incubated for 2 hours on a shaker, followed by TMB substrate (BD Biosciences). For interplate standardization, one positive patient control and two negative controls were included on each plate. A positive antibody response was defined as >3 SD above the mean value in healthy control subjects.