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. 2014 Oct 9;9(10):e109875. doi: 10.1371/journal.pone.0109875

Figure 2. In vitro characterization of scFv hB7A to AHP antigens.

Figure 2

(A) Sequence variability of AHP proteins amino acid sequence. ClustalW tree of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHP1-6). Amino acid sequence identity of AHP proteins (compared to AHP3 in brackets). (B) Far-western blot of recombinant AHP proteins. Recombinant protein expression with incorporated 6x-His-tag detected was confirmed by anti-6x-His-tag antibody immunodetection (top); AHP proteins were detected by recombinant scFv hB7A from the periplasmic extract of the bacterial expression, incorporated c-myc-tag detected by anti-c-myc-tag antibody (bottom). (C) The specificity of scFv hB7A against AHP proteins tested in indirect ELISA. Absorbance values of triplicates (±SD represented with error bars) at 450 nm are displayed for each AHP protein (500 ng/well). (D) The affinity of scFv hB7A to AHP3 tested in competitive ELISA. Absorbance values at 405 nm normalized to 490 nm of quadruplicates from two independent measurements were pooled (±SD represented with error bars). The AHP3 was coated at 20 nM and 2-fold dilutions of 1950 nM AHP3 was equilibrated with 45 nM scFv hB7A prior loading to wells. The data was fitted with DYNAFIT software.