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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

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.
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pone-0004241-g002: 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.

Mentions: Human serum and bovine serum proteins were electrophoresed on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes as described in the Methods. While human serum did not react at all with the antibody, the lane for bovine serum shows a full spectrum of positive bands (Figure 2, lane A). Sialidase pretreatment of the bovine serum abolished almost all reactivity confirming the specificity for sialic acids (Figure 2, lane B). There was no background staining seen when probing the blot with the control IgY antibody, nor with secondary antibody alone (data not shown). Thus, this antibody preparation can detect a complex mixture of Neu5Gc-containing proteins in a specific manner. Bovine fetuin was also used for a sensitivity check, as it carries a variety of different sialic acid linkages and but only a low amount of Neu5Gc (∼2% of total sialic acids). Low µg amounts of fetuin and asialofetuin were electrophoresed into a 10% SDS-PAGE gel. Only the fetuin gave positive reactions, giving detection of as little as 4 pmoles Neu5Gc contained in 1 µg of protein (Figure 2). This is more sensitive than conventional detection of Neu5Gc by DMB-derivatization and HPLC analysis with fluorescent detection [38] (previously the most sensitive method, with a detection limit of ∼10–20 pmoles, depending on the background).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626223&req=5

pone-0004241-g002: 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.
Mentions: Human serum and bovine serum proteins were electrophoresed on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes as described in the Methods. While human serum did not react at all with the antibody, the lane for bovine serum shows a full spectrum of positive bands (Figure 2, lane A). Sialidase pretreatment of the bovine serum abolished almost all reactivity confirming the specificity for sialic acids (Figure 2, lane B). There was no background staining seen when probing the blot with the control IgY antibody, nor with secondary antibody alone (data not shown). Thus, this antibody preparation can detect a complex mixture of Neu5Gc-containing proteins in a specific manner. Bovine fetuin was also used for a sensitivity check, as it carries a variety of different sialic acid linkages and but only a low amount of Neu5Gc (∼2% of total sialic acids). Low µg amounts of fetuin and asialofetuin were electrophoresed into a 10% SDS-PAGE gel. Only the fetuin gave positive reactions, giving detection of as little as 4 pmoles Neu5Gc contained in 1 µg of protein (Figure 2). This is more sensitive than conventional detection of Neu5Gc by DMB-derivatization and HPLC analysis with fluorescent detection [38] (previously the most sensitive method, with a detection limit of ∼10–20 pmoles, depending on the background).

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