Sera collected on days 10, 20, and 30 were used to measure Ab to the lung SAg, Col-V and Kα1T, by ELISA developed in our laboratory and detailed in earlier publications (8 (link)). Briefly, 1μg/ml Col-V (Sigma C3657) and Kα1T (prepared in the laboratory) were dissolved in 1X PBS and were coated on 96-well ELISA plates (Corning #3369) and stored at 4°C overnight. Sera were diluted (1:100) and were added to the wells and incubated at room temperature for 2 hours. Plates were then washed with PBS-tween (PBST 20), and 100 μl of goat-anti-mouse conjugated HRP (1:10000) was added to the wells. Thereafter, plates were developed with substrate tetra methyl benzidinec (Millipore ES022), and enzymatic reaction was stopped with 1N HCL. The colorimetric reading was obtained with ELISA reader at a 450nm wavelength. Finally, the Ab concentration of the sera was calculated with known concentrations of Ab to Col-V (Abcam ab7046) and Ka1T (Santa Cruz Biotechnology sc-12462-R) performed as standards in the same plates. Sera collected from naive C57BL/6 mice were used as negative control.