Elx405 automatic plate washer

ELISA evaluation was performed with the use of commercial IBV ELISA kit (IDEXX, USA) according to manufacturer recommendation with minor modifications. Serum samples collected from birds 14 and 21 DPV were diluted 1:100 (instead of 1:500) in order to increase low antibody level detection. TW were tested undiluted and the level of specific IgA in TW was detected as described previously [15 (link)] with minor modifications. Anti-IBV IgA antibodies were detected by replacing the conjugated antibodies of ELISA kit with goat anti-chicken IgA HRP antibodies (AbD Serotec, UK) dilluted 1:10 000. The following steps of ELISA procedure were performed with Eppendorf epMotion 5075 LH automated pipetting station (Eppendorf, Germany), BioTek ELx405 automatic plate washer (BioTek, USA) and BioTek ELx800 plate reader. Based on sample ODs the sample to positive (S/P)-ratios were calculated and used to express the mean serum and TW anti-IBV antibody (S/P)-ratio +/− SD per group and day post vaccination. Mean OD values +/− SD per group and day post vaccination were used to express the level of anti-IBV IgA antibodies in TW.

Sera and tracheal washings were tested for specific IgY anti-aMPV antibodies with the APV Ab ELISA kit (IDEXX Laboratories, USA). Sera of MDA- birds were diluted 100-fold rather than 500-fold (manufacturer’s recommendations) to enhance detection of low antibody levels. 100-fold dilution, as a comparative to 500-fold, was also used in analyses of MDA+14/V and NV serum. Tracheal washings were incubated undiluted. Successive steps of the procedure were performed according to the manufacturer’s recommendations. ELISA was carried out with the use of the Eppendorf epMotion 5075 LH automated pipetting station (Eppendorf, Germany), BioTek ELx405 automatic plate washer (BioTek, USA) and BioTek ELx800 plate reader. The sample to positive (S/P)-ratio was calculated based on the ODs and used to express the mean (S/P)-ratio +/− SD per group and per DPV.