Figure 2
From: Adenoviral targeting using genetically incorporated camelid single variable domains

Evaluation of anti-CEA VHH binding to hCEA protein. Each VHH was produced as soluble thioredoxin fusion protein in Escherichia coli cytosol. (a) Evaluation of VHH binding to hCEA protein by using dilution ELISA. The plates for ELISA were coated with purified hCEA protein and then purified VHH were added in wells at various concentrations. Bound VHHs were detected with HRP/anti-E-tag mAb. Each point represents a mean of six readings obtained in two separate experiments with the error bars showing standard deviations (s.d.). (b) The level of hCEA mRNA expression was determined by RT-PCR. Total RNA was extracted from human and mouse cancer cells, the first-strand cDNA was synthesized using random hexamer primers and used as the template for PCR. Products of PCR were analyzed by 1% agarose electrophoresis with ethidium bromide staining. One representative of three different experiments is shown. LLC, murine Lewis lung carcinoma cells; Ctr+, the hCEA gene-specific qPCR template standard (OriGene Technologies). (c) Evaluation of efficacy and specificity of the CEA-targeted VHHs binding to hCEA expressed on the cell surface. MC38CEA and MC38 cells were incubated with 100 ng/ml of JJB-A3, JJB-B2, JJB-B5, and JJB-D1 VHHs. Bound anti-CEA VHHs were detected by using anti-E-tag FITC-conjugated goat Ab using FASC analysis. One representative of two different experiments is shown.