Antigens were spotted in triplicates of three dilutions (1x, 5x, 125x) onto home made nitrocellulose chip. Dilutions were carry out in 0.05% Tween 20 containing PBS. BioOdyssey Calligrapher (Bio-Rad) was used for the printing with the following program: sourceselect Run = No substrate Type = Slides Number of Slides in Y= 4 Number of Slides in X= 4 Slide Size = standard Substrate Size in Millimeters in X= 75.500 Substrate Size in Millimeters in Y= 25.350 [Grid Layout] Source plate Type= 384 Number of Source plate Rows= 8 Number of Source plate columns= 12 Number of Dips per Source plate= 96 Pin Type= solid Pin Distance = 4.5mm Number of pins in Y= 2 Number of pins in X= 2 Number of samples = 256 Number of Spots to be printed = 1536 Replicate Spots per Grid in X= 1 Replicate Spots per Grid in Y= 3 Use Advanced parameters = Yes [Grid Advanced] Top Left Grid Margin in x= 5.000 (mm) Top Left Grid Margin in Y= 5.000 (mm) Bottom Right Grid Margin in x= 8.150 (mm) Bottom Right Grid Margin in Y= 5.000 (mm) Distance between Spots in x= 550.000 (micron) Distance between Spots in Y= 550.000 (micron) No. of Spots per Grid in X= 8 No. of Spots per Grid in Y= 6 No. of Total Spots per Grid= 48 No. of Reprints of the Same Spot= 1 Grid printing Direction= Left-right, Top-down No. of Different supergrids in x= 2 No. of Different supergrids in Y= 2 Total Number of supergrids= 4 Distance between supergrids in x= 4.500 (mm) Distance between supergrids in Y= 4,500 (mm) Supergrid printing Direcrion= Left-right, Top-down No. of Duplicate supergrid clusters ln x= 2 No. of Duplicate supergrid clusters in Y= 1 Distance between Duplicate supergrid clusters in x= 17.675 (mm) - Distance between Duplicate supergrid clusters in Y= 4.500 (mm) No. of Full plates= 0 No. of Dips in the Last plate= 64 Total No. of Spots in a Grid= 48 Sample picking Direction= Top-down, Left-right SubDips Exist= 0 SubDip Rows= 1 SubDip Columns= 1 Total SUbDips= 1 [Blotting] Use blots= No Blot slides Blot Distance= 450 (micron) All Blotting slides are sufficient for= 11.666667 (plates) [washing] wash selection= Different samples Pre-wash Step1= 2 Pre-wash Step1 Time= 2.500 (s) Pre-wash step2= 1 Pre-wash Step2 Time= 2.000 (s) Pre-wash Step3= 0 Pre-wash Step3 Time= 0.000 (s) Main-wash cycles= 5 Main-wash Step1= 3 Main-wash Step1 Time= 3.000 (s) Main-wash Step2= 1 Main-wash Step2 Time= 0.500 (s) Main-wash step3= 0 Main-wash Step3 Time= 0.000 (s) Post-wash Step1= 3 Post-wash Step1 Time= 3.000 (s) Post-wash Step2= 2 post-wash Step2 Time= 5.000 (s) Post-wash step3= 1 post-wash step3 Time= 0.500 (s) [options] Z-Down offset in sourceplate = 0.000 (mm) Source plate Approach speed= 20.000 (mm/s) Source plate Withdraw speed= 15.000 (mm/s) Source plate Dwell Time = 1000.000 (ms) oscillation in water-bath1 = Yes oscillation in Water-bath2 = Yes Water-bath1 Oscillation Distance in x= 4.000 (mm) water-bath2 Oscillation Distance in x= 1.500 (mm) water-bath1 Number of oscillations in Bath= 3 water-bath2 Number of oscillations in Bath= 3 Z-Down offset on slides = 0.000 (mm) Slide/Membrane/Blot Approach speed= 10.000 (mm/s) Dwell Time for printing= 25.000 (ms) Number of substrates between Redips = 0 Number of Redips per sample = 1
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