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Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.

Diaz SL, Padler-Karavani V, Ghaderi D, Hurtado-Ziola N, Yu H, Chen X, Brinkman-Van der Linden EC, Varki A, Varki NM - PLoS ONE (2009)

Bottom Line: Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies.These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies.The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Pathology and Cellular & Molecular Medicine, Glycobiology Research and Training Center, School of Medicine, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/principal findings: We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions: We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.

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Related in: MedlinePlus

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.
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pone-0004241-g005: 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.

Mentions: Biotherapeutic monoclonal antibodies were electrophoresed on 12.5% SDS-PAGE gels and transferred onto nitrocellulose membranes as described in the Methods. Representatives of biotherapeutic monoclonal antibodies studied were all IgG1 molecules: either chimeric or humanized mouse monoclonal antibodies. There was no background staining seen in the blot neither with the control IgY antibody, nor with secondary antibody alone (data not shown). The anti-Neu5Gc antibody gave staining on all these glycoprotein biotherapeutic agents (Figure 5, lane C–G) most likely representing Neu5Gc originating from the mammalian expression system used and/or animal-derived serum or growth medium additives. Human IgG antibodies and human and bovine serum were used as controls (Figure 5; A, J, L). Interestingly, even some human sera gave a faint band, which might be explained by incorporation of exogenous Neu5Gc into a specific human serum glycoprotein.


Sensitive and specific detection of the non-human sialic Acid N-glycolylneuraminic acid in human tissues and biotherapeutic products.

Diaz SL, Padler-Karavani V, Ghaderi D, Hurtado-Ziola N, Yu H, Chen X, Brinkman-Van der Linden EC, Varki A, Varki NM - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626223&req=5

pone-0004241-g005: 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.
Mentions: Biotherapeutic monoclonal antibodies were electrophoresed on 12.5% SDS-PAGE gels and transferred onto nitrocellulose membranes as described in the Methods. Representatives of biotherapeutic monoclonal antibodies studied were all IgG1 molecules: either chimeric or humanized mouse monoclonal antibodies. There was no background staining seen in the blot neither with the control IgY antibody, nor with secondary antibody alone (data not shown). The anti-Neu5Gc antibody gave staining on all these glycoprotein biotherapeutic agents (Figure 5, lane C–G) most likely representing Neu5Gc originating from the mammalian expression system used and/or animal-derived serum or growth medium additives. Human IgG antibodies and human and bovine serum were used as controls (Figure 5; A, J, L). Interestingly, even some human sera gave a faint band, which might be explained by incorporation of exogenous Neu5Gc into a specific human serum glycoprotein.

Bottom Line: Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies.These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies.The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine, Pathology and Cellular & Molecular Medicine, Glycobiology Research and Training Center, School of Medicine, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT

Background: Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/principal findings: We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions: We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.

Show MeSH
Related in: MedlinePlus