Development and validation of a high throughput multiplex immunofluorescence assay to detect all human immunoglobulin isotypes and subclasses in human fluids.

Antibodies in human milk protect infants against infections, but currently no assay is described that is able to simultaneously measure all 9 antibody isotypes and subclasses immunoglobulins in human fluids, such as human milk. Our cohort "Protecting against Respiratory tract Infections through human Milk Analysis" (PRIMA) is focused on the relation between the occurrence of respiratory infections during the first year of life and concentration of maternal antibodies in breastfeeding. We developed and successfully validated a multiplex assay that is able to measure all nine antibody isotypes and subclasses in human plasma and milk (regardeless of the pathogen specificity), using a small sample volume. We used a multiplex immunofluorescence assay (MIA) requiring a minimal sample volume of 25 µl. Commercially available human isotype standards were used in spiking experiments to exclude the presence of cross reactivity. In addition, we prevented signal quenching by milk by determining the optimal dilution of human milk. In conclusion, we have developed a low-volume multiplex assay, that, for the first time, can reliably quantify functionally intact antibodies of all known human isotypes and subclasses and that is able to measure both kappa and lambda heavy chain antibodies. This assay can easily be implemented in other academic labs.