Detecting Allergens From Black Tiger ShrimpPenaeus monodonThat Can Bind and Cross-link IgE by ELISA, Western Blot, and a Humanized Rat Basophilic Leukemia Reporter Cell Line RS-ATL8

Abstract

BackgroundBlack tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8.MethodsSera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens.ResultsAllergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32–39 kDa and 91–230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens.ConclusionsThe RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.

Overcoming Shellfish Allergy: How Far Have We Come?

“…The latter assay made use of a rat basophilic leukemia cell line transfected with human IgE receptor FcεRI and luciferase reporter gene (RS-ATL8) [54], which obviates the need for complicated procedures involving blood stimulation and flow cytometry-based analyses while keeping the benefits of measuring specific allergen-IgE crosslinking. A very recent study by Jarupalee et al [55] presented remarkable findings, where only the 38 kDa-and 115 kDa-shrimp protein fractions induced higher reporter signals in EXiLE test while fractions containing shrimp proteins of 19 kDa, 38 kDa, 41 kDa and 115 kDa displayed IgE reactivity in Western blot. The 115-kDa proteins indeed exhibited weak IgE reactivity but were capable of inducing sufficient IgE crosslinking.…”

Emerging approaches in the diagnosis and therapy in shellfish allergy

“…The measurement of luciferase signal is thus theoretically reflecting the degree of IgE crosslinking. The utility of EXiLE was exemplified by the remarkable findings by Jarupalee et al [30] whom illustrated that only the 38 ]. Thirty-five shrimp allergic and tolerant subjects defined by DBPCFC were assessed in this study to compare the diagnostic accuracy of conventional SPT, shrimp extract and rPen a 1 sIgE measurement with BAT and EXiLE.…”

“…The measurement of luciferase signal is thus theoretically reflecting the degree of IgE crosslinking. The utility of EXiLE was exemplified by the remarkable findings by Jarupalee et al [30] whom illustrated that only the 38 kDa- and 115 kDa-shrimp protein fractions induced higher reporter signals by EXiLE test whereas fractions containing 19 kDa, 38 kDa, 41 kDa and 115KDa shrimp proteins fractions were IgE reactive in western blotting. The study data also showed that 115-kDa proteins were only weakly IgE positive by Western blot but could induce sufficient IgE crosslinking detectable by EXiLE test.…”

Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies

“…All the reagents for RS-ATL8 cell culture were procured from Gibco; ThermoFisher Scientific, USA. Measurement of luciferase activity was performed following the procedures described previously (Nakamura et al, 2012;Gericke et al, 2015;Jarupalee et al, 2018) with some modifications. Briefly, the RS-ATL8 cells were seeded at 5×10 4 cells per well in 96 wellplate (50 mL per well) and incubated for 3 h. Cells were then sensitized overnight with the pre-mixture of 5 mg/mL of human-IgE (Abcam, USA) and various concentrations (0.5-512 mg/mL) of plant-produced anti-human IgE (stock 3.4 mg/mL) or Omalizumab (stock 75 mg/mL) diluted in PBS, for 1 h at 37°C.…”