Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry.

Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 μm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1% Tween 20. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High (> 8000), medium (3000-8000) or low (1000-3000) antibody titre level for each of three infections could be quickly assessed in one probe visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.