The design of the gradient immunochip (the 6*6 spots array on a well bottom) was based on three rows of viral antigens, namely NDV (upper row), IBV (middle row), IBDV (bottom row), and the concentration of each viral antigen used was decreased gradually from spot to spot left to right in a row (Fig. 1). The number of coloured spots after completion of analyses of chicken sera should indicate the level of post-vaccinal antibodies to the particular infection (low, medium or high). First, all components of the multiplex test system were characterized by ELISA, including the optimization of reagents concentration and assay conditions, pH value, and composition of absorption and reaction buffers. The NDV, IBV and IBDV antigens as major components for immunochip development and fabrication were purified by dialysis against distilled water, which was the most effective among other approaches used (Online Resource Fig. S1). The resulting NDV, IBV and IBDV antigen preparations were sufficiently pure to be utilized as coating antigens, however, an unspecific reaction towards anti-IgY Ab-HRP was observed. To optimize antigen absorption conditions, a range of buffers was investigated across pH value from 4.0 to 9.6. The best result in terms of low unspecific binding and signal development was obtained for slightly acidic buffer (pH 6.0) (Online Resource Fig. S2). As a result, PBS buffer with pH 6.0 was chosen as an optimal for all three viral antigens absorption taking into account the difference of resulting optical density between hyperimmune serum, negative control serum (SPF-chicken serum) and conjugate control (Online Resource Fig. S3).
Chicken IgY has a strong ability to absorb on polystyrene wells due to its hydrophobicity, so, particular attention should be paid to the choice of reaction buffer (Miers et al. 1983). The presence of detergent (Tween 20) was necessary to reduce IgY unspecific binding during the first 30-min incubation of absorbed antigens with chicken sera (Online Resource Fig. S4). In terms of lower unspecific binding the most favorable results were obtained for PBS pH 6.0 with optimal concentration of Tween 20 to be 0.1%. We found that the combination of particular buffer composition for viral antigens absorption and assay steps, together with the necessary level of detergent in chicken serum dilution buffer provided low unspecific binding of IgY and HRP conjugate and improve the differentiation of post-vaccinal chicken sera by different titre groups.
The chosen optimal conditions were used to create a gradient immunochip for multiplex test system (Fig. 1). High binding polystyrene ELISA plate was used as a standardized solid support for a well-based immunochip. To ensure adequate colour intensity of microspots, the NDV, IBV, and IBDV antigen concentration was increased ten times and HRP conjugate concentration was increased two times against ELISA conditions. An array of 6*6 spots was placed on flat bottom of a well, where NDV, IBV and IBVD antigens were absorbed in gradually decreased concentrations (in duplicates) from left to right (Fig. 1). The gradient assay required the choice of NDV, IBV, and IBVD antigen concentrations that would meet certain threshold intensities of the immunochip microspots during a subsequent immunochemical reaction with virus-specific antibodies. Therefore, each viral antigen concentrations were chosen to provide a different number of coloured circular zones depending on the titre of specific antibodies present in the test sample (post-vaccinal chicken serum). An illustration of this selection for the NDV antigen is presented in Fig. 2. As a result of NDV, IBV, and IBVD antigen dilution optimisation, it was possible to correlate the number of developed coloured spots with the titre value of the anti-NDV, IBV or IBDV antibodies in the analysed chicken serum, i.e. evaluate the result semi-quantitatively. To create positive control, a purified IgY of chicken SPF-serum was used. An assessment of the intensity of coloured microspots when varying the concentration of absorbed IgY demonstrated the possibility of quantitative determination at least 1–2 µg/ml chicken antibodies on the surface (Online Resource Fig. S5).
A variety of chicken sera was selected to cover the full spectrum of anti-NDV, IBV and IBVD post-vaccinal antibody titres. Comparable data were obtained using commercial ELISA kit and the developed gradient multiplex assay in immunochip-in-a well format for 63 post-vaccinal chicken sera simultaneously for three infections in one probe (Fig. 3). Quick assessment of immunochip results can be done with the help of smartphone to enlarge or make photo of a coloured array after analysis (Fig. 3A and B). It should be noted that SPF-sera showed no visible unspecific reaction and no coloured microspots were observed (n = 20). The design of the multiplex assay makes it possible to assess the immune status of chickens semi-quantitatively by the number of observed coloured microspots (Fig. 1). As a result, the post-vaccinal antibody titres (ELISA results) and corresponding number of coloured microspots (gradient immunochip results) can be subdivided into a few ranges, namely zero, low, medium and high: no specific anti-pathogen antibodies (NDV, IBV and IBDV) – no coloured microspots; 0–1 coloured microspot correspond to antibody titre up to 1000, 2–3 coloured microspots – antibody titres from 1000 to 3000; 3–4 coloured microspots – antibody titres 3000–8000, 5 coloured microspots - high and very high level of immune response, antibody titres higher than 8000 (Fig. 3, Online Resource Table S1). The developed gradient multiplex analysis can be utilized to quickly semi-qiantitatively distinguish tested sera based on the level of post-vaccinal antibodies (titre group) and evaluate the immunity status of flocks on poultry farms following preventive measures against Newcastle disease, infectious bronchitis and bursal disease. The method that has been developed possesses advantages in comparison to arrays that provide a yes/no answer (Wang et al. 2010; Yan et al. 2018; Li et al. 2021) and analytical systems that necessitate costly equipment, such as the Luminex method (Pinette et al. 2014; Wang et al. 2018, 2019).