Editing Alpaca Antibody (section)

== Competitive ELISA ==

<br/>Nunc Maxisorb plates were coated overnight at 4° C with 5 μg/ml soluble ovTNFα.in PBS, and then blocked with 4% MPBS. After washing three times with PBS, serially diluted soluble VHH antibodies were added and incubated at room temperature for 90 min to determine saturation curves. To detect bound VHH, an anti-E-Tag/HRP antibody (Amersham) was added at 1:8000 dilution in 4% MPBS for 90 min at room temperature. The wells were washed and developed with 3,3’,5,5’-tetramethyl benzidine (Applichem). Competitive ELISA was performed by preparing plates as described above and then a saturating amount of soluble expressed VHH was added and incubated for 60 min at room temperature. PBS was added to control wells containing no expressed soluble protein. Serial dilutions of bacterial supernatants containing VHH-displaying phage were added for 90 min at room temperature. Plates were then washed three times with PBST and three times with PBS. To detect bound phage, an anti-M13/HRP antibody (Amersham) was added at 1:8000 dilution in 4% MPBS for 90 min at room temperature. The wells were washed and developed as described above. Percent binding was calculated as the ODtest/ODcontrol x 100.