Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species

Escandon, Paulina; Heatley, J Jill; Berghman, Luc R; Tizard, Ian; Musser, Jeffrey MB

Semi-Quantitative Signal Intensity Of Dot-Blot ELISA image analysis software program (ImageJ, National Institutes of Health; https://imagej.nih.gov/ij/) was used to obtain semi-quantitative intensity values of the dotblot ELISA signals. Digitally formatted images were converted into 2-bit grey-scale by selecting Image, Type, 32-bit. Image brightness and contrast was adjusted by selecting Image, Adjust, Brightness/Contrast, Auto, Apply. An area of interest was delineated over individual signal dots using the rectangular selection tool, then selecting Analyse, Gel, Select First Lane. This delineated rectangular area was dragged to each of the other signal dots and then selecting Analysis, Gel, Select Next Lane. Use of a consistent delineated area provided equally sized areas of interest for analysis. A graphical depiction of dot signal intensity for each dot selected was acquired by selecting Analysis, Gel, Plot Lanes. The area representing the dot signal intensity was delineated using the straight line tool from the bar menu. After all dot areas were delineated, the wand tool from the bar menu was used to highlight these areas, which generated a signal intensity value. For each dot, three dot signal intensity values were independently generated with Image J and a mean dot signal intensity value was reported.