Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens.

Cockroach allergens can lead to serious allergy and asthma symptoms. Termites are evolutionarily related to cockroaches, cohabitate in human dwellings, and represent an increasing pest problem in the United States. The Formosan subterranean termite (Coptotermes formosanus) is one of the most common species in the southern United States. Several assays were used to determine if C. formosanus termite proteins cross-react with cockroach allergens. Expressed sequence tag and genomic sequencing results were searched for homology to cockroach allergens using BLAST 2.2.21 software. Whole termite extracts were analyzed by mass-spectrometry, immunoassay with IgG and scFv antibodies to cockroach allergens, and human IgE from serum samples of cockroach allergic patients. Expressed sequence tag and genomic sequencing results indicate greater than 60% similarity between predicted termite proteins and German and American cockroach allergens, including Bla g 2/Per a 2, Bla g 3/Per a 3, Bla g 5, Bla g 6/Per a 6, Bla g 7/Per a 7, Bla g 8, Per a 9, and Per a 10. Peptides from whole termite extract were matched to those of the tropomyosin (Bla g 7), arginine kinase (Per a 9), and myosin (Bla g 8) cockroach allergens by mass-spectrometry. Immunoblot and ELISA testing revealed cross-reaction between several proteins with IgG and IgE antibodies to cockroach allergens. Several termite proteins, including the hemocyanin and tropomyosin orthologs of Blag 3 and Bla g 7, were shown to crossreact with cockroach allergens. This work presents support for the hypothesis that termite proteins may act as allergens and the findings could be applied to future allergen characterization, epitope analysis, and clinical studies

Immunoblots of termite extract with anti-cockroach allergen antibodies.

<p>(A) GCr (left lane of each panel) and Cf termite extract (right lane of each panel) (1 μg) were resolved by SDS-PAGE and stained to visualize protein or transferred to PVDF and probed with rabbit anti-cockroach allergen antibodies (1:500) followed by IRdye 800 labeled anti-rabbit secondary antibody (1:10000). Molecular weight markers are shown to the left of the SDS-PAGE gel. (B) The GCr (left lane of each panel) and Cf extracts (right lane of each panel) were probed with a monoclonal anti-Bla g 1 antibody and an IRdye800 labeled goat anti-mouse secondary antibody. Molecular weight markers are shown to the left of the PVDF membrane. (C) Western blots of Cf termite whole body extract. Purified scFvs were used as primary antibodies (1.0 μg/mL) and horseradish peroxidase-conjugated anti-hemagglutinin (1:1000) was used for detection using a chemiluminescent substrate.</p

Termite peptide matches to cockroach allergen protein sequences.

<p>Termite peptide matches to cockroach allergen protein sequences.</p

Termite proteins cross-react with anti-cockroach allergen scFvs in ELISA assays.

<p>(A) This assay was performed in sandwich format using two separate collections of detection agents. Serially diluted Cf termite extract was first added to a mixture of anti-cockroach scFvs either generated non-specifically using whole body GCr extract or specifically targeted against recombinant Bla g 1, 2, or 4, and one scFv from a naïve human library screened against whole GCr extract that were coupled to unique bead sets as capture agents in 96-well filter bottom plates. A mixture of rabbit anti-E6Cg cockroach extract polyclonal antibodies, anti Bla g 1, anti Bla g 2, and anti Bla g 4 IgGs (1:500) were used as detection antibodies, followed by biotinylated anti-rabbit (1:1000) and streptavidin-RPE (1:500). MFI detected for selected scFvs is on the y-axis. (B) Ninety six well plates were coated with serially diluted Cf termite extract overnight at 4°C. ScFvs (1.0 ug/mL) were added to each well after blocking. The binding was determined after addition of HRP-conjugated anti-HA antibodies. Samples were tested 2 times and mean absorbance values are reported. Serially diluted extract (log scale) is on the x-axis.</p

Termite proteins cross-react with IgE from human cockroach allergic sera.

<p>(A) Western blot of GCr (left lane of each panel) and Cf termite (right lane of each panel) extracts. The proteins were separated on SDS-PAGE gel followed by transfer onto PVDF membrane. After blocking for 2 hr the blot was incubated with four GCr allergic serum pools S1Cr and P1-4 (1:10). Specific IgE binding was determined following sequential addition of biotinylated goat anti-human IgE (1:1000), and HRP-conjugated streptavidin (1:10,000). (B). 96-well plates were coated with termite extract (100 μg/mL) overnight at 4°C. After blocking for 2 hr, GCr allergic serum pools (1:10) were added to each well. Specific IgE binding was determined following sequential addition of biotinylated goat anti-human IgE (1:1000), HRP-conjugated streptavidin (1:10,000), and TMB substrate. (C) Dose response binding of human IgE in human serum pool (S1Cr) with the indicated amount of Cf termite extract.</p

Putative termite homologs of cockroach allergen genes.

<p>Putative termite homologs of cockroach allergen genes.</p

Termite proteins cross-react with anti-cockroach allergen antibodies.

<p>Cf termite extract (1 μg) or German cockroach extract (GCr, 1 μg) was added to the wells of a microtiter plate for ELISA and probed with rabbit anti-cockroach allergen antibodies (1:500) followed by IRdye 800 labeled anti-rabbit antibody (1:10000). Black bars represent GCr extract and white bars represent Cf termite extract signals. Samples were tested 4 times and mean values are shown with standard deviation included as error bars. Relative IRdye800 signal is shown on the y-axis and anti-cockroach allergen antibody designation is shown on the x-axis.</p