Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic ProductsReportar como inadecuado




Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

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.



Autor: Sandra L. Diaz, Vered Padler-Karavani, Darius Ghaderi, Nancy Hurtado-Ziola, Hai Yu, Xi Chen, Els C. M. Brinkman-Van der Linden, A

Fuente: http://plos.srce.hr/



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