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. 2017 Aug 11;7(14):3398–3414. doi: 10.7150/thno.20919

Figure 1.

Figure 1

Immuno-reactivities of the mJF5 and hJF5 antibodies and their DOTAGA and NODAGA chelator-labeled derivatives. A. Amino acid sequences of the heavy and light chains of the humanised antibody Ab633-HC3-LC2-T1-1B3-1G10 (hJF5); the signal peptides (cleaved off in the mature antibody) are highlighted in green, the variable domains are highlighted in blue, and the constant domains are highlighted in orange. B. Purity of the Protein A-purified antibody hJF5, with heavy and light chains of ~50kDa and ~31kDa respectively, determined by using SDS-PAGE under denaturing conditions followed by Coomassie staining. C. Western immunoblots using the purified hJF5 antibody (left) and mJF5 antibody (right) tested against purified Aspergillus mannoprotein antigen. Wells were loaded with 40 μl of a 0.5 mg/mL solution of antigen denatured in Laemmli buffer. Both antibodies show a characteristic smearing pattern, binding to glycoprotein antigen with molecular weights of between ~36 kDa and >210 kDa. D. ELISA of mJF5, hJF5, human IgG1 isotype control antibody ET901, and non-radiolabeled DOTAGA- or NODAGA-conjugated antibody derivatives, using microtiter plates coated with 1 mg/mL of purified Aspergillus mannoprotein antigen. No notable reductions in binding of the mJF5 and hJF5 antibodies are apparent as a result of conjugation to the chelators DOTAGA or NODAGA. Note, however, the increase in binding of the hJF5 and NODAGA-hJF5 antibodies compared to the mJF5 and NODAGA-mJF5 counterparts at equivalent antibody protein concentrations. The lack of binding of the NODAGA-conjugated human IgG1 isotype control ET901, demonstrates the specific binding of the mJF5 and hJF5 antibodies to the target antigen, and is confirmed by the molecular imaging studies in vivo. E. Immunofluorescence of germinating spores of Af293 using mJF5, hJF5, NODAGA-hJF5, and NODAGA-ET901. Brightfield images of cells are shown alongside their corresponding epifluorescence images following staining of the cells with the antibodies and FITC conjugates. Under epifluorescence, mJF5, hJF5 and NODAGA-hJF5 can be seen to bind to extracellular cell-wall bound antigen displayed on the surface of Af293 germ tubes, while the NODAGA conjugated human IgG1 isotype control antibody shows no fluorescence, consistent with hyphal-specific binding of the mJF5 and hJF5 antibodies. The scale bar that applies to all of the images = 6 μm.