Measurement of allergen-specific secretory IgA in stool of neonates, infants and toddlers by protection against degradation of immunoglobulins and allergens

Background and aims : Measurement of secretory immunoglobulin A (SIgA) level is important to monitor various disease conditions. However SIgA in gut mucosa is degraded by pancreatic proteases and proteolytic enzymes from enteric microbiota. Currently, there is no reliable quantitation method that measures allergen-specific SIgA levels in stool of neonates, infants and toddlers. Methods : Allergen-specific SIgA levels in stool of 10 healthy neonates, infants and toddlers aged 0 to 36 months were measured by our new allergen microarray with densely carboxylated arms on a glass slide chip. Results : Protease activities in stool of 3-day-old neonates were low and no degradation of SIgA, IgA and allergens was detected. However, immunofluorescence signals of SIgA, IgA and allergen on the chip were markedly reduced by stool extracts obtained from infants and toddlers aged more than one month in dose- and time-dependent manners. Such reduction was almost completely inhibited by serine protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) and partly by leupeptin, but not by a variety of other protease inhibitors tested. Conclusion : Allergen-specific SIgA levels in stool of neonates, infants and toddlers under 36 months of age could be analyzed using protease inhibitors, including PMSF and leupeptin.