Table 1.
Putative termite homologs of cockroach allergen genes.
Table 2.
Termite peptide matches to cockroach allergen protein sequences.
Table 3.
Termite peptide matches allowing for a single amino acid substitution to cockroach allergen protein sequences.
Fig 1.
Termite proteins cross-react with anti-cockroach allergen antibodies.
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.
Fig 2.
Immunoblots of termite extract with anti-cockroach allergen antibodies.
(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.
Fig 3.
Termite proteins cross-react with anti-cockroach allergen scFvs in ELISA assays.
(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.
Fig 4.
Termite proteins cross-react with IgE from human cockroach allergic sera.
(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.
Fig 5.
Termite proteins compete for IgE binding to cockroach allergens.
Microtiter plate wells were coated with GCr extract (1 μg) probed with cockroach allergic patient sera (25 μL) that had been pre-incubated for 1 hour with 25 μL of serial half-log dilutions of either GCr or Cf extract in a competitive ELISA. Squares represent GCr extract and circles represent Cf termite extract. IgE binding was visualized by the sequential addition of biotinylated mouse anti-human IgE (1:1000) and IRDye680-conjugated streptavidin (1:5,000). Data points represent the mean of 3 tests and are shown with standard deviation included as error bars. The percentage of IgE binding is indicated on the y-axis and the concentration of GCr or Cf competitor is shown on the x-axis.