Abstract
Sensitive, specific, reproducible ELISAs were developed and validated for cow's milk beta-lactoglobulin and alpha-casein specific IgG and IgA antibodies and beta-lactoglobulin antigen. A novel method for the quantitation of cow's milk beta-lactoglobulin and alpha-casein specific IgG sera standards was developed. This involved affinity purification and nephelometry. These assays were applied to a number of studies investigating the physiological and pathological levels of these antibodies and antigens in various body fluids from adults and infants. There was a large individual variation in the beta-lactoglobulin and alpha-casein specific antibody levels and the beta-lactoglobulin antigen levels in sera, breast milk and saliva samples in both atopic and non-atopic individuals. beta-lactoglobulin IgG antibody levels were higher than the alpha-casein IgG antibody levels in all of the sera samples assayed. Explanations are offered for these differences. A prospective study on 39 infants was performed to investigate the effect of parental atopy and of a maternal milk-free diet during late pregnancy and lactation, on the immune response to milk specific antigens and the allergy incidence in their infants. The presence of atopy had a significant influence on the allergy incidence of infants, with infants born to atopic parents having a significantly higher allergy incidence than infants born to non-atopic parents (p<0.001). Atopic mothers also showed a tendency to transfer higher quantities of antigenically intact beta-lactoglobulin via the placenta and breast milk compared to non-atopic women, and in addition had a lower milk specific IgA antibody concentration in their breast milk. These factors could add to an underlying predisposition of allergy in infants, increasing the risk of allergy development later. The strict removal of all milk and milk products from the diet significantly reduced the circulating beta-lactoglobulin IgG antibody levels after a period of 7 weeks (p<0.05). In "at risk" infants it was demonstrated that a strict maternal milk-free diet during late pregnancy and lactation significantly reduced the allergy incidence up to 18 months postpartum (p<0.0005). A significant increase in IgG antibodies against beta-lactoglobulin and alpha-casein was demonstrated in all infants on their first milk ingestion: this response was found to be higher in infants who developed allergy, but not significantly. The clinical implications of such a dietary regimen are discussed. In an adult study undertaken to examine allergic disorders, both beta-lactoglobulin and alpha-casein specific IgG4 antibody levels were significantly higher (p<0.001) compared with IgGl, IgG2 and IgG3 subclasses. A significantly higher alpha-casein IgG4 antibody level (p<0.05) was found in individuals suffering from food allergy compared with controls and atopy, not related to food. A positive correlation was found between the total IgE antibody levels and the beta-lactoglobulin and alpha-casein specific IgG4 antibody levels. In a study investigating absorption of antigens in normal adult subjects antigenically intact beta-lactoglobulin was absorbed across the gut wall in a biphasic pattern in 7/8 healthy adult subjects after a milk load. Levels of beta-lactoglobulin were found to peak at 30-90 mins and 165-240 mins with a significant increase in antigenically intact beta-lactogIobulin with time (p<0.01). The levels of beta-lactoglobulin and alpha-casein specific IgG antibody levels were not influenced by the milk load. The significance of the transfer of milk antigens was discussed. The data reported in this thesis offers more information regarding the immunological handling of cow's milk protein by atopic and non-atopic adults and infants born to such women.