Sandwich immunoassay for lactoferrin detection in milk powder

Abstract

Lactoferrin (LF) content in infant milk powder has been strictly regulated by many governments and there is a need for convenient and reliable assays. Using hybridoma techniques, fourteen monoclonal antibodies (mAbs) active against LF were prepared. Two antibodies (mAb2 and mAb3), recognizing spatially distant epitopes of LF, were selected to establish a sandwich enzyme-linked immunosorbent assay (ELISA). A solution of mAb3 (1 μg mL⁻¹) was coated onto micro-titer plates for LF capture while mAb2 labeled with horseradish peroxidase (2.2 μg mL⁻¹) was used as a detection antibody. Under optimized conditions, the proposed sandwich ELISA was evaluated, and linearly responded to LF standards in a range of 5–600 ng mL⁻¹ and the limit of detection was 3.23 ng mL⁻¹. Lactoferrin samples were able to be determined after simple dilution, and the recovery in fortified milk powder averaged between 98% and 109%. The developed assay showed both high specificity (with no obvious cross-reactivity with related proteins) and reproducibility (the coefficient of variation ranged from 4.5% to 7.1%), indicating the utility of this sandwich ELISA in LF monitoring.