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

This ability of anti-bird IgY secondary antibody to react with the antiglobulins of many bird species makes it a useful secondary antibody for assays that utilize long incubation periods; the anti-bird IgY secondary antibody has been used in ABV testing using, Western blot analysis, microplate ELISA, and indirect immunofluorescence assays. 8, 9, [17] [18] [19] In our Western blots, strong signals were obtained using the anti-bird IgY secondary antibody with the positive samples from Blue and gold macaw, Cockatiel, and Mallard (Figure 1 ), however when used in our dot-blot ELISA, the use of anti-bird IgY secondary antibody produced weak signals with Blue and gold macaw and Cockatiel positive plasma (Figure 2 ). The polyclonal nature of the anti-bird IgY antibodies make it an ideal secondary antibody for immunodiagnostic assays due to its ability to recognize multiple epitopes on the target antiglobulin and on the different species antiglobulins. However, the varying affinities of the polyclonal IgY antibodies can cause differences in the signal strength obtained from assays with long or short incubation times, such as seen between the Western blot and dot-blot ELISA, respectively, in our study. In the dot-blot ELISA, mainly high affinity secondary antibodies, which bind quickly and with a greater stability than lower affinity secondary antibodies, produce the signal. Additionally, the polyclonal nature of the anti-bird IgY secondary antibody produced a higher avidity in the dot-blot ELISAs for Monk parakeet and Mallard antiglobulins, as observed by the stronger dot visibility and signal intensity, than the avidity for Blue and gold macaw and Cockatiel antiglobulins ( Figure 2 ). Exactly what factors were involved and how they differed was beyond the scope of this project, but the issue of reaction incubation time and its effect on avidity should be explored further if a dot-blot ELISA is to be optimized as a rapid, patient-side diagnostic assay. Background noise in all tests must also be considered. In this study, a faint signal was observed in the negative samples of Blue and gold macaw and Cockatiel samples when the anti-duck and anti-chicken IgY secondary antibodies were used in the Western blot and dot-blot ELISAs; the signals of the corresponding negative samples were visibly similar to those of the positive samples (Figures 1 and 2) . However, when analyzing the signal intensities of the dot-blot ELISA, there was no significant difference between the signal intensity of the negative and positive Blue and gold macaw or Cockatiel samples when anti-duck or anti-chicken IgY secondary antibody was used (Table 1 ). This background signal may have been due to cross-reactions with antibodies to E. coli antigens in the samples. Antibodies against E. coli are found in human serum and can cause background noise in serologic assays that use recombinant proteins. 31, 32 Similar studies in birds show non-specific signals due to E. coli antibodies in plasma samples and to recombinant E. coli antigens in serologic tests. 33, 34 Affinity purification of the His-tagged recombinant N-protein with the Ni-NTA Agarose column may not have removed all E. coli antigens from the recombinant E. coli/N-protein solution. E.coli proteins can have some histidine conformation or during the generation of the recombinant N-protein,some His-tagged E.coli proteins may have been generated with the insertion of the pET21a vector into the E. coli. Thus, some E. coli ant