Dataset Information

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

ABSTRACT: German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

SUBMITTER: Khurana T

PROVIDER: S-EPMC4596881 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

Khurana Taruna T Dobrovolskaia Ekaterina E Shartouny Jessica R JR Slater Jay E JE

PloS one 20151007 10

<h4>Background</h4>German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.<h4>Objective</h4>Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.<h4>Methods</h4>Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocyte ...[more]

PMID: 26444288

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Project description:Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.

Dataset Information

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

Publications

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

Similar Datasets

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