Antibody-based enzyme-linked lectin assay (ABELLA) for the sialylated recombinant human erythropoietin present in culture supernatant.
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.
Kim
Hyoung Jin
HJ
College of Pharmacy, Chung-Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea.
Lee
Seung Jae
SJ
Kim
Hong-Jin
HJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001681:Biological Assay / Q000379:methods; D055592:Biophysical Phenomena; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004921:Erythropoietin / Q000097:blood* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D037102:Lectins / Q000032:analysis*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000494:pharmacology; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology
D000911:Antibodies, Monoclonal; D003470:Culture Media; D037102:Lectins; D011994:Recombinant Proteins; D010504:Periodic Acid; D004921:Erythropoietin; D012710:Serum Albumin, Bovine; C009288:metaperiodate; D019158:N-Acetylneuraminic Acid