A mouse model for food allergy using intraperitoneal sensitization(1)
There exists a correlation between the relative resistance of a protein to digestion by pepsin in a simulated gastric fluid and allergenic activity, this relationship is not absolute, with reports of both pepsin resistant non-allergens, and pepsin sensitive allergens. The BALB/c strain mouse was selected as such mice are high IgE responders, equivalent to an atopic phenotype. SpecifIc IgG antibody production is measured by enzyme-linked immunosorbent assay (ELISA) and specific IgE antibody responses assessed by homologous passive cutaneous anaphylaxis (PCA) assay. One important issue is contamination of the protein preparations with bacterial endotoxins as there is evidence that low doses of endotoxin can enhance IgE antibody responses. In practice, levels of endotoxin of 100EU/mg of protein appear to be without marked effect on IgE anti-protein responses. Groups of mice receive 0.25 ml of protein in PBS by intraperitoneal injection. Seven days later treatment is repeated.
Antibody responses induced by two proteins, peanut agglutinin and potato agglutinin, are used as examples of proteins that stimulate vigorous IgE production or little detectable IgE, respectively. IgG antibody titres achieved following treatment with peanut agglutinin ranged from 1 in 256 to 1 in 8192 whereas those derived following exposure to potato agglutinin varied from 1 in 256 to 1 in 1024.
For serum derived from peanut agglutinin immunized mice, the majority of cutaneous reactions elicited in recipient mice (8/10) are maximal responses (10 mm; the approximate diameter of a mouse ear and thus the maximum size of skin response achievable) and that very similar sized responses are generally induced in each of the two recipient sites. Serum derived from all five mice immunized with peanut agglutinin caused average skin reactions of 3 mm or greater. Three of the potato agglutinin immunized mice are IgE responders, with skin reactions varying from 3 to 8 mm, with reactions of similar diameters recorded in each duplicate reading. The remaining two mice are non-IgE responders, with cutaneous reactions of <3 mm recorded (0 mm or 1 mm) in all analyses.
High concentrations of serum (neat and 1 in 2 dilution) will almost certainly be strongly positive. The dilution series selected to determine IgE titre for the pooled sample from the peanut agglutinin sensitized mice comprises serial doubling dilutions of 1 in 4 to 1 in 64.
Failure to generate an IgG antibody response may be a result of the lack of immunogenicity of the protein in the mouse strain of choice, or may relate to prior dietary exposure to a cross-reactive protein. In some circumstances it might be necessary to generate positive control sera (for both IgG and IgE analyses) by immunizing mice with protein in the presence of a known adjuvant such as alum. Even gavage exposure appears to be considerably less sensitive than is parenteral administration with respect to eliciting IgE antibody responses in BALB/c mice. The use of adjuvant may compromise the ability to discriminate between proteins with respect to allergenic potential, possibly generating false positives and conferring the appearance of sensitizing potential on non-allergens.