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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

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.
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pone-0004241-g001: 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.

Mentions: Sialic acids can be bound to underlying glycans by α2-3, α2-6, or α2-8 glycosidic linkages. Presumably because the binding pocket of antibodies can accommodate >4 monosaccharides [34]–[37], all mouse and chicken monoclonal Neu5Gc-dependent antibodies reported to date show specificity for both the sialic acid linkage and the precise structure of the underlying glycan chain [19], [20]. Despite the fact that the primary immunogen used to immunize chickens (GM3-Neu5Gc) contained Neu5Gc only in the α2-3linkage, the final antibody preparation reacted with all linkages and underlying glycan structures tested. A likely explanation is that the BSA that is traditionally used as a carrier is contaminated with Neu5Gc-containing bovine serum sialoglycoproteins (our unpublished observations). Regardless of the reason, this broad specificity was found both with synthetically prepared compounds (Figure 1A) and with naturally-occurring molecules containing Neu5Gc (Figure 1B). Even O-acetylation of the glycerol-like side chain of sialic acid, such as that found in Bovine submaxillary mucin (BSM) did not block recognition, as reactivity was not altered by prior de-O-acetylation of BSM by base treatment (Figure 1B). These results are typical of many ELISAs done with Neu5Gc-containing molecules. We have so far not encountered any instance where the antibody did not detect such molecules.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626223&req=5

pone-0004241-g001: 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.
Mentions: Sialic acids can be bound to underlying glycans by α2-3, α2-6, or α2-8 glycosidic linkages. Presumably because the binding pocket of antibodies can accommodate >4 monosaccharides [34]–[37], all mouse and chicken monoclonal Neu5Gc-dependent antibodies reported to date show specificity for both the sialic acid linkage and the precise structure of the underlying glycan chain [19], [20]. Despite the fact that the primary immunogen used to immunize chickens (GM3-Neu5Gc) contained Neu5Gc only in the α2-3linkage, the final antibody preparation reacted with all linkages and underlying glycan structures tested. A likely explanation is that the BSA that is traditionally used as a carrier is contaminated with Neu5Gc-containing bovine serum sialoglycoproteins (our unpublished observations). Regardless of the reason, this broad specificity was found both with synthetically prepared compounds (Figure 1A) and with naturally-occurring molecules containing Neu5Gc (Figure 1B). Even O-acetylation of the glycerol-like side chain of sialic acid, such as that found in Bovine submaxillary mucin (BSM) did not block recognition, as reactivity was not altered by prior de-O-acetylation of BSM by base treatment (Figure 1B). These results are typical of many ELISAs done with Neu5Gc-containing molecules. We have so far not encountered any instance where the antibody did not detect such molecules.

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