Figure 7

Monoclonal anti-ORF2p antibody inhibits L1 endonuclease activity in an in vitro endonuclease cleavage assay. (A) Schematic of in vitro endonuclease cleavage assay. Double-stranded DNA containing L1 ORF2 endonuclease consensus target sequence with 5′ tagged with fluorophore. L1 ORF2 endonuclease is added, DNA is cleaved releasing the fluorphore, which can be quantitated. (B) (Top) SDS-PAGE analysis of the products resulting from the in vitro endonuclease assay with or without the addition of the monoclonal anti-ORF2 antibody (0, 100, 150, 200 nM). Antibody (ORF2) denotes the addition of the monoclonal anti-ORF2 antibody, control indicates the addition of the same volume of the buffer used for the reactions containing monoclonal anti-ORF2p antibody, and L1 EN denotes bacterially purified human ORF2 endonuclease. (Bottom) Quantitation of the results of the in vitro endonuclease cleavage assay in A (see Methods). Results were normalized to 0 nM control (n = 3). (C) Same experimental approach as in B, but anti-hORF1p antibody was added to the in vitro endonuclease cleavage assay.