Figure 1.
Affinity purified Chicken IgY antibody recognizes Neu5Gc with various linkages to underlying glycans.
A. Detection of Neu5Gc on synthetic and semi-synthetic molecules. Reactivity of the affinity-purified anti-Neu5Gc antibody was tested in triplicate, by ELISA against synthetic and semi-synthetic Neu5Gc- and Neu5Ac-target pairs, using Neu5Ac-glycans for background subtraction (the Neu5Ac A490 value was subtracted from the corresponding Neu5Gc A490 value). B. Detection of Neu5Gc on natural glycoproteins. The affinity-purified anti-Neu5Gc antibody was tested in triplicates by ELISA, against natural glycoproteins containing Neu5Gc and Neu5Ac, using buffer only for background subtraction. Error bars indicate the standard deviation of triplicates. Gc, alpha-linked Neu5Gc; GM3, GM3 ganglioside; HSA, Human serum albumin; PAA, polyacrylamide; and, SPG, sialylparagloboside.
Figure 2.
Sensitive and specific detection of Neu5Gc-containing glycoproteins by Western Blot Analysis.
Standard proteins were run on a 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked overnight at 4°C with Neu5Gc-free 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 with TBST. The membranes were washed with TBST and then incubated with Donkey anti-chicken HRP (Jackson ImmunoResearch) 1∶50,000 in TBST at room temperature for 1 hr. The membranes were washed and incubated with Pierce Super-Signal West Pico Substrate (Pierce) as per manufacturer's recommendation, exposed to X-ray film and the film developed. A, bovine serum 1 µg; B, bovine serum 1 µg+sialidase; C, human serum; D, blank lane. E–H, bovine fetuin 10, 5, 2, 1 µg. The smallest amount of bovine fetuin contained only 4 pmoles of Neu5Gc per µg of protein as determined by DMB-HPLC.
Figure 3.
Sensitive and specific detection of Neu5Gc on cells by flow cytometry.
CHO-K1 cells were detached from the tissue culture dish using 10 mM EDTA in PBS, pH 7.3. The cells were immediately washed in blocking buffer (0.5% gelatin from cold water fish skin in PBS, pH 7.5) containing 5 mM EDTA and counted by hemocytometer. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll Paque Plus (GE Healthcare), washed in blocking buffer and counted. 1×106 cells were used for each staining which was done at 4°C. The cells were washed with 1 ml cold blocking buffer then pelleted at 500×g for 5 min. The supernatant was carefully removed and discarded. The cell pellet was gently suspended in 100 µl of either affinity purified chicken anti-Neu5Gc antibody or control non-specific IgY antibody diluted 1∶4000 in blocking solution and incubated on ice for 1 hr. The cells were washed by adding 1 ml of blocking buffer, mixed gently, and pelleted as above. The cell pellet was gently suspended in 100 µl Donkey-anti-chicken IgY Cy5-conjugated diluted 1∶4000 in blocking buffer and incubated on ice for 1 hr. The cells were washed as above, resuspended in 400 µl PBS, analyzed on a FACSCalibur (BD Biosciences Immunocytometry Systems, San Jose, CA) and the data analyzed using Flowjo software (Tree Star, Ashlan, OR). Human PBMCs were negative for Gc (bottom), while mouse PBMCs (data not shown) and CHO cells were positive for Gc (top). The gray peak represents cells stained with total IgY of un-immunized chickens, and the black trace represents cells stained with anti-Neu5Gc.
Figure 4.
Sensitive and specific detection of Neu5Gc in mouse and human tissues by immunohistochemistry.
A. Direct scan of glass slides with frozen sections of mouse embryos d16.5 or paraffin sections of adult mouse tissues, and detected using biotinylated anti-chicken antibody followed by HRP-Streptavidin. B. Frozen sections of Human Placenta immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (top) or CY3-Streptavidin (bottom). (400× magnification) C. Frozen sections of normal human tissues immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin. (400× magnification) D. Frozen sections of examples of human tumors immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (400× magnification). E. Frozen sections of human ovarian carcinoma immunostained with primary reagents at 1∶1000 each (5 ug/ml each) and detected using biotinylated anti-chicken antibody, followed by HRP-Streptavidin (top) or CY3-Streptavidin (bottom) (200× magnification).
Figure 5.
Detection of Neu5Gc on Some FDA-Approved Biotherapeutic Antibodies.
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.
Figure 6.
Lack of Neu5Gc in a biotherapeutic agent prepared in the human PER.C6® cell line under Neu5Gc-free/serum-free conditions.
PER.C6® cells were stably transfected with cDNA encoding human EPO and co-transfected with ST3GalIV for full sialylation of the glycans of EPO. EPO-expressing PER.C6® cells were cultured under serum-free conditions (VPRO medium) and EPO was purified from the medium. The sialic acid content of PER.C6®-rEPO and of CHO-rEPO (Eprex) was similar as determined by IEF (data not shown). A. The presence of Neu5Gc on CHO-rEPO (Eprex) or PER.C6®-rEPO was examined by DMB-HPLC according to [38]. B. The presence of Neu5Gc on both CHO-rEPO (Eprex) and PER.C6®-rEPO was examined by the highly sensitive Western blot as described in the Methods. 1 and 2 µg of EPO and 5 µg of positive control (bovine fetuin) were run on a 4–12% SDS-PAGE gel and blotted onto nitrocellulose membrane. Immunostaining for Neu5Gc was performed as described in the Methods and as above under figure 2.