Development of a Reliable and Sensitive Peptide Microarray for Large-Scale Epitope Mapping of Milk Allergens

Lin, Jing; Bardina, L.; Bruni, Francesca; Shreffler, Wayne G.; Wang, J.; Sampson, Hugh A.

doi:10.1016/j.jaci.2007.12.974

Cited by 1 publication

(1 citation statement)

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“…Immunolabeling was performed as previously described with some modifications 10,11 . In brief, the slides were blocked with 400μl of 1% human serum albumin (HSA) in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) for 60 minutes at room temperature, followed by incubation with 250μl of patient serum diluted 1:5 in PBS-T/HSA for 24 hours at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens

Vickery

Lin

Kulis

et al. 2013

Journal of Allergy and Clinical Immunology

183

151

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Background Peanut-allergic subjects have highly stable pathologic antibody repertoires to the immunodominant B cell epitopes of the major peanut allergens Ara h 1-3. Objective We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. Methods Measurements of total peanut-specific IgE (psIgE) and psIgG4 were made with CAP-FEIA. We analyzed sera from 22 OIT subjects and 6 controls and measured serum specific IgE and IgG4 binding to epitopes of Ara h 1-3 using a high-throughput peptide microarray technique. Antibody affinity was measured using a competitive peptide microarray as previously described. Results At baseline, psIgE and psIgG4 diversity were similar between subjects and controls, and there was broad variation in epitope recognition. After a median 41 months of OIT, polyclonal psIgG4 increased from a median 0.3 mcg/mL (IQR 0.1-0.43) at baseline to 10.5 mcg/mL (3.95-45.48) (p<0.0001) and included de novo specificities. PsIgE was reduced from a median baseline of 85.45 kUA/L (23.05-101.0) to 7.75 kUA/L (2.58-30.55) (p<0.0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated subjects, several subjects generated new IgE specificities even as the total psIgE decreased. Global epitope-specific shifts from IgE to IgG4 binding occurred, including at an informative epitope of Ara h 2. Conclusion OIT differentially alters Ara h 1-3 binding patterns. These changes are variable between subjects, not observed in controls, and include a progressive polyclonal increase in IgG4, with concurrent reduction in IgE amount and diversity.

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Section: Methodsmentioning

confidence: 99%