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


METHODS
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 approximately 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.


CONCLUSIONS
Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.
Shin
K
K
Nutritional Science Laboratory, Morinaga Milk Industry Co Ltd, Zama, Kanagawa, Japan.
Hayasawa
H
H
Lönnerdal
B
B
D000906:Antibodies; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques; D007774:Lactation; D007784:Lactoperoxidase / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D008895:Milk, Human / Q000201:enzymology*; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007784:Lactoperoxidase