Tnt system kit

Full-length cDNAs for mouse IL-17A, IL-17F, and IL-22 were used as templates for in vitro transcription and translation and labeled with [³⁵S]methionine using the TNT system kit (Promega). Radio-labeled proteins were immunoprecipitated in duplicate or triplicate with serum or control antibodies in 96-well PVDF filtration plates (Millipore). In each well, 20,000 counts per min (cpm) of ³⁵S-labeled proteins and 2.5 μl of WT and Aire^−/− serum were used for immunoprecipitation. The radioactivity of the immunoprecipitated material was quantified with the use of a liquid scintillation counter. For each assay, a polyclonal rabbit anti-mouse IL-17A antibody (Sigma-Aldrich, prs4887), a polyclonal rabbit anti-mouse IL-17F antibody (Abnova, pab16929), or a polyclonal goat anti-mouse IL-22 antibody (Santa Cruz Biotechnology, sc-14436) were used as positive controls. Serum from NOD WT mice served as negative standards. The autoantibody index was calculated as follows: [(cpm in the unknown sample) – (cpm in the negative standard)] / [(cpm in the positive standard) – (cpm in the negative standard)] × 100. RLBAs were performed in two independent laboratories using the same antigen construct. The upper limit of the normal range for each assay was defined as the mean value obtained for the WT mice tested plus three standard deviations.

The autoantibody assay was described previously [7 ]. Briefly, full-length cDNA for mouse IRBP (Thermo Scientific, #MMM1013) was used for in vitro transcription and translation and labeling with ³⁵S-methionine using the TNT system kit (Promega). The ³⁵S-IRBP was immunoprecipitated with serum samples in 96-well PVDF filtration plates (Millipore). Serum samples were analyzed in triplicate with 20 000 cpm of ³⁵S-IRBP per well. Radioactivity of immunoprecipitated material was evaluated with a liquid scintillation counter (1450 MicroBeta TriLux, Perkin Elmer). Serum samples from Aire^+/+ and Aire^−/− mice were used as negative and positive standards, respectively (data not shown). The IRBP autoantibody index for each serum sample was found by the following calculation: (cpm in unknown sample–cpm in negative standard) ÷ (cpm in positive standard–cpm in the negative standard) × 100).