CHARACTERIZATION OF 2 DOG IGE-SPECIFIC ANTIBODIES ELICITED BY DIFFERENT RECOMBINANT FRAGMENTS OF THE EPSILON-CHAIN IN HENS

Two recombinant [His](6)-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domain s: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [H is](6)-tagged proteins, and affinity-purified over nickel-nitrilotriac etic acid columns. The recombinant proteins were used to immunize hens . The raised and affinity-purified chicken antibodies (Ab) isolated fr om egg yolk exhibited specific binding to the respective recombinant c anine IgE fragment (IgEf) on immunoblots and displayed high titers aga inst the IgEf in ELISA. Immunoblotting of canine serum separated by PA GE under native conditions with the IgEf2- and IgEf3/4-specific Ab res ulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4 -specific Ab affinity-enriched dog serum separated under denaturing co nditions. In an ELISA for the detection of Two recombinant [His](6)-ta gged fragments of the canine immunoglobulin E (IgE) heavy chain (secon d domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, ex pressed in Escherichia coli (E. coli) as [His](6)-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The reco mbinant proteins were used to immunize hens. The raised and affinity-p urified chicken antibodies (Ab) isolated from egg yolk exhibited speci fic binding to the respective recombinant canine IgE fragment (IgEf) o n immunoblots and displayed high titers against the IgEf in ELISA. Imm unoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a prot ein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab fur ther recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enric hed dog serum separated under denaturing conditions. In an ELISA for t he detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog seru m. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]B-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recogni zed cells in paraffin-embedded tissue sections of lymph nodes. Further more, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In con clusion, both lgEf-specific Ab recognize native dog IgE with the advan tages that they are directed against different and known constant doma ins of the IgE molecule, and that they can be used for immunohistochem istry on paraffin-embedded tissue. The two dog IEE-specific Ab could i nitiate clinical research on the involvement of immediate-type hyperse nsitivity reactions in dogs. (C) 1998 Elsevier Science B.V. All rights reserved.