Individual Sensitization Pattern Recognition to Cow's Milk and Human Milk Differs for Various Clinical Manifestations of Milk Allergy

Abstract

Cow's milk allergy (CMA) belongs to one of the most common food allergies in early childhood affecting 2-3% of children under 3 years of age. However, approximately 1% of adults remain allergic to cow's milk, often showing severe reactions even to traces of milk. In our study, we recruited patients with different clinical manifestations of CMA, including patients with anaphylaxis and less severe symptoms. We assessed the sensitization patterns and allergic responses of these subgroups through different immunological and cell-based methods. Sera of patients were investigated for IgE against whole cow's milk and its single allergens by CAP- FEIA. In a newly developed in-house multiplex dot assay and a basophil activation test (BAT), cow's milk allergens, in addition to human breast milk and single allergens from cow's and human milk were analyzed for IgE recognition and severity of CMA in the included patients. Both the CAP-FEIA routine diagnostic and the multiplex dot test could differentiate CMA with severe from milder allergic reactions by means of the patients' casein sensitization. The BAT, which mirrors the clinical response in vitro, confirmed that basophils from patients with severe reactions were more reactive to caseins in contrast to the basophils from more moderate CMA patients. By means of this improved component-resolved diagnosis of CMA, individual sensitization patterns could be assessed, also taking sensitization against human milk into consideration.

Keywords: CAP-FEIA (Fluorescence Enzyme Immunoassay); Cow’s milk allergy (CMA), anaphylaxis; basophil activation test (BAT), human breast milk; cow’s milk allergens; multiplex dot test; sensitization pattern.

Figure 1

Synopsis of data of the sensitization patterns of ID1, ID2 and ID3 with severe reactions in the ImmunoCAP, multiplex dot test and basophil activation test (BAT). Caseins were associated with this clinical response. CAP data of the cow’s milk allergy (CMA) patients are depicted in a bar graph. The filled bars show the data of the first blood sample, the hatched bars those of the second blood sample. Determination of IgE recognition to whole and single allergens of cow’s milk and human milk in the multiplex dot test. The concentration of the dot-blotted analytes as indicated was 1.0 µg/µL and 2.5 µg/µL, respectively. The IgE recognition of the first blood sample is given in a box with a solid line, of the second blood sample with a box with a dotted line. The BAT was performed with blood of the patients using whole cow’s milk and human milk as well as single bovine and human milk allergens. Percentages represent CD63 positive basophils determined by flow cytometric analysis. Blood sample stimulation was conducted with the analytes as indicated. fMLP, anti-IgE were run as positive controls, PBS as negative control.

Figure 2

Synopsis of data of the sensitization pattern of ID4, ID5 and ID6 with milder allergic reactions in the ImmunoCAP, multiplex dot test and BAT. Whey proteins were more pronounced with this clinical response. CAP data of the CMA patients were depicted in a bar graph. The filled bars show the data of the first blood sample, the hatched bars are those of the second blood sample. Determination of IgE recognition to whole and single allergens of cow’s milk and human milk in the multiplex dot test. The concentration of the dot-blotted analytes as indicated was 1.0 µg/µL and 2.5 µg/µL, respectively. The IgE recognition of the first blood sample was given in a box with a solid line, of the second blood sample with a box with a dotted line. The BAT was performed with blood of the patients using whole cow’s milk and human milk and single bovine and human milk allergens. Percentages represent CD63 positive basophils determined by flow cytometry. Blood sample stimulation was conducted with the analytes as indicated. fMLP, anti-IgE were run as positive controls, PBS as negative control.