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. 2013 Mar 22;3:1525. doi: 10.1038/srep01525

Figure 3. Functionality of the CEA-specific sscFv analogues.

Figure 3

(a) SDS-PAGE of variously NHS-based PEGylated sscFv. The reaction was quenched after indicated times with an excess of glycine. (b) SDS-PAGE of maleimide-based PEGylated sscFv. Processed antibody was treated with PEGylation reagent but no reducing agent. (c) ELISA of purified maleimide- and NHS-based PEGylated sscFv against full length CEA. (d) Fluorescence read-out of a dilution series of CEA expressing (CAPAN-1) and non-expressing (A375) cancer cell lines after treatment with fluorescein-sscFv (ex. 488 nm; em. 518 nm). (e) Comparison of the ELISA-activity of commonly formed reporter complexes (secondary antibody conjugated to HRP) with on-plate formation by addition of HRP-Strep conjugate to antigen-bound (full length CEA) biotin-sscFv. In the 1:4600 dilution of the HRP-Strep conjugate no non-specific background was observed. (f) One-step ELISA of preformed and purified HRP-sscFv conjugate against full length CEA. Experiments (c–f) were performed in triplicates; error bars show s.d.