Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase

K. Shin, H. Hayasawa, B. Lönnerdal

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

Background: Two heme-containing peroxidases, secretory lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased. Objective: Our knowledge of lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of lactoperoxidase. Design: Polyclonal antibodies were raised against recombinant human lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively. Results: Two bands with molecular masses of ≈80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate-polyacrylamide gel electophoresis profile of purified lactoperoxidase. The mean (±SD) concentration of lactoperoxidase in 26 whey samples was estimated to be 0.77 ± 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples. Conclusion: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.

Original languageEnglish (US)
Pages (from-to)984-989
Number of pages6
JournalAmerican Journal of Clinical Nutrition
Volume73
Issue number5
StatePublished - 2001

    Fingerprint

Keywords

  • ELISA
  • Enzyme-linked immunosorbent assay
  • Human milk
  • Immunoaffinity chromatography
  • Immunoblotting
  • Lactoperoxidase
  • Myeloperoxidase
  • Recombinant human lactoperoxidase
  • SDS-PAGE

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Food Science

Cite this