losartan-potassium and metaperiodate

The terminal sialic acid of human erythropoietin (hEPO) is essential for in vivo activity. The current resorcinol and HPLC methods for analyzing alpha2,3-linked sialic acid require more than a microgram of purified rhEPO, and purification takes a great deal of time and labor. In this study, we assessed the use of an antibody-based enzyme-linked lectin assay (ABELLA) for analyzing non-purified recombinant hEPO (rhEPO). The major problem of this method was the high background due to terminal sialylation of components of the assay (antibody and bovine serum albumin) other than rhEPO. To solve this problem, we used a monoclonal antibody (Mab 287) to capture the rhEPO, and oxidized the bovine serum albumin used for blocking with meta-periodate. The sialic acid content of non-purified rhEPO measured by ABELLA was similar to that obtained by the resorcinol method on purified rhEPO. ABELLA has advantages such as adaptability and need for minimal amounts of rhEPO (40 ng/ml). Our observations suggest that ABELLA should reduce the time and labor needed to improve culture conditions so as to increase protein sialylation, and also facilitate the study of sialylation mechanisms.

Topics: Animals; Antibodies, Monoclonal; Biological Assay; Biophysical Phenomena; Cattle; CHO Cells; Cricetinae; Cricetulus; Culture Media; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Humans; Lectins; N-Acetylneuraminic Acid; Oxidation-Reduction; Periodic Acid; Recombinant Proteins; Reproducibility of Results; Sensitivity and Specificity; Serum Albumin, Bovine

This paper is a preliminary report on development of a screening method for carbohydrate-specific phage antibodies against recombinant human erythropoietin (rHuEPO), using a phage display antibody library. rHuEPO was oxidized with sodium periodate or treated with 1,4-dithiothreitol and guanidine hydrochloride for detecting the specificity of obtained phage antibodies. Of 100 phage clones, three initially showed higher carbohydrate-related specificity. One of them (No. 62) bound specifically to the carbohydrate chains of rHuEPO, while the other two (Nos. 63 and 83) might recognize the steric conformation related to both the carbohydrate and the polypeptide chain of rHuEPO. These phage antibodies may serve as useful capture ligands for future development of efficient analytical methods for rHuEPO.

Topics: Antibodies; Antibody Specificity; Carbohydrates; Dithiothreitol; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Glycosylation; Guanidine; Humans; Immunoglobulin Fragments; In Vitro Techniques; Oxidation-Reduction; Peptide Library; Periodic Acid; Recombinant Proteins