Analysis Of Tropomyosin SIgE And SIgG4 Antibody Level And Epitope Profile In Shrimp Allergy Patients

Objective:The prevalence of seafood allergy is high which is harmful to health,but the accurate diagnosis is a challenge.Tropomyosin(TM)is the major sensitizing component of crustaceans which could improve the diagnostic accuracy with component-resolved diagnostics.TM also helps the diagnosis of other seafood allergy.However,the difference in geographical,ethnic characteristics and dietary cultures brings complexity to food allergies.According to reports,frequencies of TM-sIgE and sIgG4,the epitopes recognized and the cross-reaction with dust mites(HDM)/cockroaches in shrimp allergic patients are controversial.The clinical significance of IgG4 in shrimp allergy is not clear.The aim of this study was to investigate TM-IgE and IgG4 antibody level and its peptides in patients from northern China.Method:1.Study objects.Serum samples were collected from Tianjin Port Hospital and Academy of Traditional Chinese Medicine Affiliated Hospital.The diagnostic of allergies were based on medical history and serum sIgE levels.2.Analyse TM sIgE and sIgG4 antibody level.Prepare recombinant shrimp TM(rTM).TM was labeled with biotin and coated with anti-human IgE/IgG4 to construct the light-initiated chemiluminescent assay(LiCA)system.Conditions of serum dilution and reaction concentration were optimized.Measure TM-sIgE and sIgG4 antibody level and determine the cut-off value.The frequency of TM-sIgE and sIgG4 and the their correlation was calculated.The cross reaction rate to TM-sIgE in dust mite/cockroach allergy patients was counted.3.Analyse TM sIgE and IgG4 epitope profile.Screened TM-sIgE and sIgG4 positive samples.Using biotin label synthesized peptide and luminescent microspheres were coated with anti-human IgE/IgG4 to establish the LiCA system.Conditions of serum dilution and reaction concentration were optimized.The 9 major linear epitopes of TM reported in the literature were synthesized.Analyse sIgE and sIgG4 epitope profile in shrimp sensitized serum and perform epitope inhibition test.Result:1.Among 92 subjects included,there were 35 shrimp allergic patients,29HDM/cockroach allergic patients and 28 healthy volunteers.2.Analyse TM sIgE and sIgG antibody level.After optimization,add 25 ?L 1:20diluted serum,50 ?L 1:200 diluted luminescent microspheres and 1:100 diluted biotinylated TM,incubated at 37 ? for 30 min.Add 150 ?L photosensitive microspheres labeled with streptavidin and incubated at 37 ? for 10 min.Among 35 shrimp allergy patients,25(71.4%)had positive TM-IgE antibodies,22(62.9%)contained increased TM-sIgG4 antibodies.There was a significant positive correlation between TM-sIgE and shrimp sIgE(r = 0.668,P < 0.0001),and between sIgE of TM and sIgG4(r = 0.485,P = 0.003).None of patients with HDM/cockroach allergy showed IgE reactivity to TM.3.Analyse TM sIgE and IgG4 epitope profile.After optimization,add 25 ?L1:20 diluted serum,50 ?L 1:200 diluted luminescent microspheres and 1:100 diluted biotinylated peptides,incubated at 37 ? for 30 min.Add 150 ?L photosensitive microspheres labeled with streptavidin and incubated at 37 ? for 10 min.Two IgE binding epitopes,amino acid(AA)85-105 and 265-284,and nine IgG4 binding epitopes covering the primary sequences of TM were identified.TM-sIgE level was positively correlated with the number of TM-sIgE positive epitopes(r = 0.650,P =0.026).Epitope inhibition test showed no cross-reaction between different epitopes.The TM-IgE and IgG4 recognition patterns showed high interpatient heterogeneity.Conclusion:In this population,TM is the major allergen which has diagnostic value.A strong IgG4 response accompanies the presence of IgE but has no diagnostic value.There was no cross reaction between shrimp allergy and arthropod allergy.May be due to regional differences,frequencies of TM-sIgE and IgG4 epitopes are different from reports.The number of epitopes recognized by IgE was positively correlated with sIgE level.