Pfuse migg2a fc vector

The pFUSE-mIgG2A-Fc vector, expressing the CH2 and CH3 domains of the murine IgG2a heavy chain, was purchased from InvivoGen (San Diego, CA). The cDNA for peptide p5 with a five amino acid spacer added to the N-terminal of the peptide was synthesized and purchased from Integrated DNA Technologies (Coralville, IA). The p5 cDNA was cloned into the vector using In-Fusion cloning techniques. The vector was transiently transfected into HEK293T/17 and CHOK1 cell lines that were cultured in serum free medium. Secreted Fcp5 was purified from the culture medium by affinity chromatography using a protein A-conjugated matrix. Binding of the purified Fcp5 with human AL and ATTR amyloid deposits in formalin-fixed paraffin embedded tissues was demonstrated immunohistochemically. Additionally, reactivity with synthetic fibrils and AL and ATTR amyloid extracts was assessed by using a pulldown assay [see e.g. 4 (link)]. Reactivity with systemic inflammation-associated (AA) amyloid deposits in a murine model of the disease [4 (link)] was assessed microautoradiographically at 4 h post IV injection of ¹²⁵I-labeled Fcp5.