Editors: Donna E. Hansel, MD, PhD, chief, Division of Anatomic Pathology, and professor, Department of Pathology, University of California, San Diego; John A. Thorson, MD, PhD, associate professor of pathology, director of the Clinical Genomics Laboratory, Center for Advanced Laboratory Medicine, UCSD; Sarah S. Murray, PhD, associate professor, Department of Pathology, and director of genomic technologies, Center for Advanced Laboratory Medicine, UCSD; and James Solomon, MD, PhD, resident, Department of Pathology, UCSD.
Generation of allergen-specific human IgEs as standards in diagnostic allergy testing
Diagnostic allergy testing for patients with moderate to severe allergies is usually performed by an allergy specialist and can include skin prick and patch tests or blood tests. The skin tests are performed by intradermally injecting a patient with a small amount of purified allergen or by placing an allergen patch on the skin and assessing the skin’s response to each of the antigens tested. Blood tests are performed by quantifying the levels of the patient’s individual antigen-specific IgE antibodies via an enzyme-linked immunosorbent assay. The benefits of the blood tests are numerous: They require only a single venipuncture as opposed to many painful intradermal injections; they can be performed in patients with skin abnormalities; and they are significantly safer, especially for patients at risk for anaphylaxis from skin exposure to the tested antigens. Drawbacks of the blood test include higher cost, as well as high interassay variance and low reproducibility, likely due to a lack of purified antigen-specific human IgE antibodies to serve as standards and calibrators. The authors developed a methodology to produce allergen-specific human IgE antibodies for use in the immunoassays required for blood testing. They created a knock-in mouse strain in which the gene segment encoding mouse γ1 constant region is replaced by human ε constant region and the gene segment encoding mouse κ constant region is replaced by human κ constant region. After being immunized with the antigen papain, the serum levels of antibodies in the knock-in mouse strain were compared. The authors found that the knock-in mice expressed human IgE at levels 10 to 30 times greater than those of mouse IgE. In addition, because of the high concentration of mouse cells expressing human IgE, the authors were able to use B cells from the spleens of the mice to prepare and culture hybridomas to produce monoclonal antigen-specific human IgE antibodies. The authors also demonstrated that the purified antigen-specific monoclonal human IgEs were able to activate and degranulate a rat basophil cell line in the presence of the antigen, confirming the activity of the generated IgE antibodies. The authors concluded that the methodology described herein can be used to generate large quantities of human IgE antibodies that are specific to a wide array of allergens, thereby creating standards to improve the blood tests used in diagnostic allergy testing.
Lu C-S, Hung AF, Lin C-J, et al. Generating allergen-specific human IgEs for immunoassays by employing human ε gene knock-in mice [published online ahead of print January 23, 2015]. Allergy. doi:10.1111/all.12572.
Correspondence: Dr. Tse Wen Chang at [email protected]