Serum anti- DNP antibodies assessed by ELISA

ELISA plates (Nunc, Copenhagen, Denmark) were coated with DNP-albumin (Albumin Human Dinitrophenyl, Sigma) and incubated overnight in refrigerator at 4⁰C.  Plates were washed with PBS pH7.35- tween20 (0.1%) BSA (1%). Serum was diluted in PBS pH7.35- tween20 (0.1%) BSA (1%) at 1:100 dilutions. Serum sample was loaded along with the strong positive and negative which was obtained from pooled sera of NMRI mice. The plate was incubated for 1.30 hours and washed as above. After was, goat anti-mouse-IgG-ALP(polyvalent IgG,IgA,IgM) (Sigma Aldrich chemie, Steinheim, Germany) was added. Again the plates were washed after incubation and the substrate was added to each well and after incubation for 20 mins, plates were read at OD 405nm. Then the plates were stopped when the positive value reached 1.5 with 3M NaOH.

Serum anti- ssDNA antibodies assessed by ELISA

ELISA plates (Nunc, Copenhagen, Denmark) were coated with single stranded DNA (ssDNA) and incubated overnight in refrigerator at 4⁰C. Plates were washed with PBSpH 7.35 and then they were blocked for 1 hour with PBS pH7.35- tween20 (0.2%) BSA (1%). Serum was diluted in PBS pH7.35- tween20 (0.2%) BSA (1%) at 1:150  dilutions. Serum sample was loaded along with the strong positive and negative which was obtained from pooled sera of NMRI mice. The plate was incubated for 1.30 hours and washed with PBS pH7.35- tween20 (0.2%) BSA (1%. After wash, goat anti-mouse-IgG-ALP (polyvalent IgG,IgA,IgM) (Sigma Aldrich chemie, Steinheim, Germany) was added and incubated for 2 hours. Again the plates were washed after incubation and the substrate was added to each well and after incubation for 20 mins, plates were read at OD 405nm. Then the plates were stopped when the positive value reached 1.5 with 3M NaOH.

Serum IgG1 concentration assessed by ELISA

ELISA plates (Nunc, Copenhagen, Denmark) were coated with Rat anti-mouse IgG1 Purified 1mg/ml (BD 559749Parmingen) and incubated overnight at 4⁰C. Plates were washed 3 times with PBS pH7.35- tween20 (0.1%). These plates after washing were blocked with fat free milk solution 5%. It was then incubated for 2 hours at 37⁰C. Meanwhile the sera were diluted in PBS at 1:200 dilutions. Plates were washed as above and the serum samples were loaded in double wells. It was again incubated for 2 hours at37⁰C and washed as above. IgG1 was detected with Peroxidase conjugate (LO-MG1-2 HR, Belgium).  Plates were again incubated at 37⁰C and washed as above. Substrate were added in each well and the plates were incubated for 3 minutes and read using ELISA plate reader at 450nm OD and stopped using 2M sulphuric acid. The concentrations of IgG1 in the serum were obtained by the standard curve using IGM standard (MADNP-1, Belgium).