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. 2009 Jan 21;4(1):e4241. doi: 10.1371/journal.pone.0004241

Figure 5. Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies.

Figure 5

Biotherapeutic agents, human IgG (Jackson Immunoresearch), human serum and bovine serum samples were run on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with 0.5% gelatin from cold water fish skin (Sigma) in TBST. The membranes were incubated at room temperature for 2 hr with the affinity-purified chicken anti-Neu5Gc diluted 1∶100,000 in TBST with 0.5% gelatin from cold water fish skin or with a control non-specific Chicken IgY antibody pool (Jackson ImmunoResearch) at the same protein concentration. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST with 0.5% gelatin from cold water fish skin and 1% human serum at room temperature for 1 hr. The membranes were washed again and incubated with Pierce SuperSignal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, human IgG 1 µg; B, blank lane; C–G, 10 µg each of various FDA-approved biotherapeutic IgG molecules; H and I, blank lanes; J, human serum, 1 µg; K, blank lane; L, bovine serum, 1 µg.