Alkaline phosphatase conjugated goat anti mouse igg

Total serum IgG levels were measured by ELISA as previously described [19 ]. In brief, 96-well PVDF plates were coated with anti-mouse IgG at 10μg/ml in 1x PBS. Serum samples were added to plates blocked with 1% BSA in 1x PBS. Plates were then washed and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The plates were read with an automated ELISA reader (Molecular Devices, Sunnyvale, CA). Total IgE levels were determined with the BD OptEIA mouse IgE ELISA Set (BD Biosciences, San Jose, CA). For isotype-specific anti-goat Ab levels, plates were coated with goat IgG (10μg/ml) and binding detected with AP-conjugated anti-mouse immunoglobulin isotype Abs.

Five full thickness OA and five control cartilage samples were powdered under liquid N₂, then extracted in 4 M guanidine HCl, 0.05MTris/HCl, pH 7.5, at 4°C for 24 h to remove proteoglycans and other matrix proteins. The residues were stored at −20°C for pepsin digestion. Collagen was solubilized by pepsin in 3% (v/v) acetic acid for 24 h at 4°C and then run on SDS-6%PAGE to resolve collagen types II and III for quantitation by densitometry.
SDS-polyacrylamide electrophoresis was run according to Laemmli^{17 (link)} with 6% and 12.5% gels. Proteins bands were stained with Coomassie Brilliant Blue or transblotted to PVDF membrane for Western blot analysis, using the anti-type II and anti-type III collagen-specific mouse monoclonal antibodies. mAb 1C10 binds to a sequence-specific epitope in the denatured α1(II) triple-helical domain (res 934–945)^{18 (link)} and mAb 4G9 binds to a conformational epitope in the collagen type III N-propeptide globular domain^{8 (link)}. Westerns were developed after primary antibody incubation using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Avondale, PA) and 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium as substrate for alkaline phosphatase.

Multiscreen cellulose filter plates (Millipore) were coated overnight with 10 μg of histone from calf thymus (Sigma-Aldrich). Cells from the mLN were collected and plated starting at 3 × 10⁶/well and in 2-fold serial dilutions in complete RPMI 1640 containing 10% FBS. After 5 h, the wells were washed with PBS containing 0.5% BSA and 0.05% Tween 20, and IgG was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson immunoresearch). Spots were counted using a dissecting microscope. Images were taken using a CTL-immunoSpot S5 analyzer.