Elx405 microplate washer

From the phage display selections, 20 clones were chosen to be examined for ERBB2 affinity and specificity in binding assays. Individual phages were amplified in 2 mL of NZY amine overnight and the supernatant containing the amplified phage was used for binding analysis. To Immulon^™ 2HB 96-well microtiter EIA plates, 100 ng purified ERBB2 diluted in sodium bicarbonate buffer (pH 9.6) was added and incubated overnight at 4° C. The ERBB2-immobilized plates were aspirated and subsequently blocked for 2 h at room temperature with BioFx synthetic bock. Blocking buffer was removed, and phage supernatant was added to wells for incubation at room temperature for 1 h. Plates were washed 10x with 0.1% TBST using an ELx405 Microplate Washer (BioTek Instruments, Inc., Winooski, VT) and were then incubated 1 h at room temperature with polyclonal rabbit anti-phage antibody diluted 1:1000 in 0.1% TBST. The plates were again washed with 0.1% TBST, and polyclonal goat anti-rabbit horse radish peroxidase conjugated antibody (Santa Cruz Biotechnology, Dallas, TX) diluted 1:1000 in 0.1 % TBST was added and incubated for 1 h at room temperature. After this final incubation, plates were washed with 0.1% TBST before the addition of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic-acid) to visualize captured phage. Following a 15 min incubation, absorbance at 405 nm was analyzed by plate reader.

M2e-specific antibodies in serum of the immunized mice were measured by ELISA as described before [18 (link)]. To determine M2e-specific serum antibody responses in a ferret, ninety-six well plates (Maxisorp, Nunc) were coated with 50 μl of 5 μg/ml M2e peptide in phosphate buffered saline (PBS) and stored overnight at 4 °C. Plates were blocked with 200 μl of 2x ELISA plate blocking buffer concentrate in distilled water for 2 h at room temperature. Serum from individual ferrets was diluted to 1:8, added to wells, and incubated for 1.5 h at room temperature. Next, plates were incubated with 50 μl of 1:500 dilution of HRP-labeled anti-IgG antibody for 1.5 h at room temperature. Plates were washed three times with PBST (0.05 % tween 20 in PBS) between each step using ELx405 microplate washer (BioTek, VT). Color was developed with OPD dissolved in phosphate-citrate buffer (pH 5.0) containing 0.03 % H₂O₂, as substrate. After 10 minutes, 50 μl of 3 M phosphoric acid was added to terminate the reaction. Absorbance at 492 nm was recorded using SpectraMax Plus384 microplate reader (Molecular Devices LLC., CA)

M2e-specific immunoglobulin G (IgG) in mouse sera was determined using enzyme-linked immunosorbent assay (ELISA) as described earlier.^{35 (link)} A 96-well plate (MaxiSorpff -Nunc, Sigma Aldrich) was coated with 50 µL of 5 µg/mL M2e peptide in PBS and incubated at 4°C overnight. 100 µL of 3% bovine serum albumin in PBS was used to block the plate and the plate was incubated for 2 h at room temperature. Plate was washed thrice with PBST using ELx405 microplate washer (BioTek, VT, USA). Serum samples were then analyzed by adding 50 µL of serum (individual or pooled) to the plate and incubating the plate at room temperature for 1 h. Plate was then washed thrice with PBST and 50 µL of goat anti-mouse IgG or anti-mouse IgG1 or anti-mouse IgG2a labeled with HRP (1:4000 dilution in PBST) was added per well. Following incubation for 1 h at room temperature, the plate was washed thrice with PBST and color was developed with OPD as substrate. After 5– 10min, 50 µL of 3% phosphoric acid was added to terminate the reaction. Absorbance at 492 nm was recorded by SpectraMax Plus384 microplate reader (Molecular Devices, LLC, CA).