Isolation and preparation of antisera to ovine IgE.
Elevated levels of ovine reaginic antibody were induced by immunization with Ascaris suum antigens. The PCA activity of this antibody persisted in the skin of sheep for 20 days and was abolished by heating to 56 degrees C, suggesting that it was immunoglobulin E. Attempts to isolate IgE from this hyperimmune serum by gel filtration, ion exchange and affinity chromatography resulted in the preparation of a PCA-positive fraction containing proteins with molecular weights of 70, 56 and 22 kD on SDS-PAGE. This preparation was used to raise an antiserum in a rabbit to IgE which was rendered specific by absorption. An antiserum to the heavy chain of ovine IgE was also raised in a rabbit by immunization with a fraction prepared from the 68- to 80-kD region of SDS-PAGE by excision and electroelution. Both antisera were positive in reverse cutaneous anaphylaxis tests and recognized a single protein with a molecular weight of 70-72 kD on SDS-PAGE immunoblotting. The presence of this protein in reaginic sheep serum, the molecular weight of its heavy chain, its heat lability and long-term skin-sensitizing ability are characteristic of IgE.
Yilmaz
H
H
School of Veterinary Science, University of Bristol, Langford, UK.
Roe
J M
JM
Morgan
K L
KL
D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D007106:Immune Sera / Q000378:metabolism*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000302:isolation & purification; D010323:Passive Cutaneous Anaphylaxis / Q000276:immunology; D011935:Reagins / Q000302:isolation & purification*; D012756:Sheep / Q000276:immunology*; D012967:Sodium Dodecyl Sulfate
D007106:Immune Sera; D007143:Immunoglobulin Heavy Chains; D011935:Reagins; D012967:Sodium Dodecyl Sulfate; D007073:Immunoglobulin E