Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

Abstract

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA, for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.

Heat Denaturation of Egg‐White Proteins Abrogates the Induction of Oral Tolerance of Specific Th2 Immune Responses in Mice

“…Using polyclonal antibodies specific for native or chemically denatured OVA, we have also shown that native OVA, but not chemically denatured OVA, is the unique molecular species in OVA-fed mouse serum [17]. Similarly, OVA was not detected in the mouse serum after ingestion of EW boiled for 60 min.…”

“…Absorption of minute amounts of fed proteins has been described repeatedly [17,30]. Although the effects of uptake of oral antigens are unclear, it has been postulated that the induction of oral tolerance is related to the intestinal permeation of monomeric forms of oral antigens from immuogenic aggregates [2,15].…”

“…Mice were bled on day 14 and serum-specific IgG and IgG l antibody responses were assayed by ELISA as described previously [17,18]. Briefly, 96-well vinyl plates were coated with 100 mg/ml OVA, DNP-HSA, lysozyme, conalbumin or ovomucoid in 0.05 M carbonate buffer, pH 9.6 (100 ml/ well) overnight at 4ЊC.…”

“…Mice were bled 1 h after feeding 50 mg raw EW or EW boiled for 5 or 60 min. Serum levels of immunoreactive OVA were examined by a sandwich ELISA as described previously [17]. Briefly, 96-well vinyl plates were coated with 5 mg/ml rabbit anti-OVA IgG at 4ЊC.…”

“…Chicken eggs are the most common food and contain several food allergens such as ovalbumin (OVA), ovomucoid, conalbumin and lysozyme [16]. We have recently shown that chemical denaturation of OVA can prevent the induction of oral tolerance of Th1 and Th2 immune responses in mice [17,18]. Therefore, the effects of heat denaturation of egg-white (EW) proteins on the induction of oral tolerance are investigated further.…”