A Protein Allergen Microarray Detects Specific IgE to Pollen Surface, Cytoplasmic, and Commercial Allergen Extracts

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

FGFR2 amplification in colorectal adenocarcinoma

FGFR2 is recurrently amplified in 5% of gastric cancers and 1%–4% of breast cancers; however, this molecular alteration has never been reported in a primary colorectal cancer specimen. Preclinical studies indicate that several FGFR tyrosine-kinase inhibitors (TKIs), such as AZD4547, have in vitro activity against the FGFR2-amplified colorectal cell line, NCI-H716. The efficacy of these inhibitors is currently under investigation in clinical trials for breast and gastric cancer. Thus, better characterizing colorectal tumors for FGFR2 amplification could identify a subset of patients who may benefit from FGFR TKI therapies. Here, we describe a novel FGFR2 amplification identified by clinical next-generation sequencing in a primary colorectal cancer. Further characterization of the tumor by immunohistochemistry showed neuroendocrine differentiation, similar to the reported properties of the NCI-H716 cell line. These findings demonstrate that the spectrum of potentially clinically actionable mutations detected by targeted clinical sequencing panels is not limited to only single-nucleotide polymorphisms and insertions/deletions but also to copy-number alterations.</jats:p

A Protein Allergen Microarray Detects Specific IgE to Pollen Surface, Cytoplasmic, and Commercial Allergen Extracts

Background: 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.Methodology/Principal Findings: 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 Conclusions/Significance: 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.</p

Reproducibility of the allergy microarray.

<p>Six individuals were serially tested 18 times for 80 allergens on the allergen microarray. <b>A</b>) Representative correlation plot of corrected fluorescence intensity values (circles) of one serum sample binding to 80 allergens on two replicate arrays. <b>B</b>) Histogram of slopes of regression curves calculated for 918 pair-wise comparisons of replicates.</p

CV of replicates.

<p>Median coefficient of variation (CV) of fluorescence intensity reported for pollen surface fractions (n = 21), pollen cytoplasmic fractions (n = 21), pollen commercial extracts (n = 21), non-pollen commercial extracts (n = 9), and recombinant allergens (n = 5). Only values where the mean of the replicates was >0.5 were analyzed.</p

Allergen microarray testing vs clinical diagnosis of patients.

<p>Heatmap depiction of microarray results of six patients who were diagnosed as either not having pollen allergy, but having some other aeroallergy (P−/O+) or as having both pollen and other aeroallergy (P+/O+). Diagnoses were made at the <i>in vitro</i> allergy lab by ImmunoCAP RAST on 12 of the same (*) allergen species as on the array, four similar (+) allergen species as on the array, and five other weeds not present of the array. All other allergens on the allergen array were not tested by ImmunoCAP RAST.</p

Allergen list.

<p>Abbr. – abbreviations.</p

IgE profiling using protein microarrays.

<p><b>A</b>) General layout of the allergen microarray, <b>B</b>) scanned image of an array probed only with secondary antibody to show autofluorescence, <b>C–F</b>) and scanned images of arrays probed with sera from four individuals showing different allergen sensitization profiles. Images were pseudocolored with a color spectrum adjusted so that blue indicates low signal and red indicates high signal.</p

Clustering of allergen-specific IgE levels.

<p>Hierarchical clustering of allergen-specific IgE to <b>A</b>) recombinant, <b>B</b>) non-pollen, and <b>C</b>) pollen allergens. Within the pollens, grasses are indicated in green, trees in black, weeds in blue, and cedar (a gymnosperm) in red.</p