A highly sensitive nanobody-based ELISA for bovine β-lactoglobulin to classified donated human milk destined to susceptible newborns

The β-lactoglobulin protein from cow's milk is a common allergen present in human milk that leads to serious allergic reactions in susceptible infants. This creates a serious safety issue in Human Milk Banks, and the best practice to assure the safety of susceptible newborns would be to classify the donated human milk according to its content of β-lactoglobulin. To provide a simple and fast assay to detect this molecule in human milk we developed a nanobody-based sandwich ELISA. The capture and detection nanobodies were isolated by phage display from a llama immunized with the antigen using a sequential selection strategy for antibody pair identification. This allowed to attain a highly sensitive test with a limit of quantitation of 40 pg/mL and extended lineal range of up to 3000 pg/mL, showing an excellent performance with human milk. For these characteristics, the ELISA developed might be useful for the classification of donated human milk according to their β-lactoglobulin content, improving the quality of milk destined to susceptible newborns. [Display omitted] •Highly specific nanobodies against β-lactoglobulin were generated.•The nanobody pair allowed detection of up to 40 pg/mL of β-lactoglobulin.•Oriented immobilization of the capture nanobody boosted the assay sensitivity.•The nanobody-based β-lactoglobulin ELISA was validated with human milk samples.•The method can be reproduced in any laboratory using the provided nanobody sequences.