Clinical relevance of the Hevea brasiliensis lipid transfer protein Hev b 12
2015; Elsevier BV; Volume: 135; Issue: 6 Linguagem: Inglês
10.1016/j.jaci.2014.12.1919
ISSN1097-6825
AutoresMargaretha A. Faber, Vito Sabato, Chris H. Bridts, Ajay P. Nayak, Donald H. Beezhold, Didier G. Ebo,
Tópico(s)Occupational exposure and asthma
ResumoNatural rubber latex (NRL) from Hevea brasiliensis (Euphorbiaceae) constitutes an important cause of allergy with distinct phenotypes depending on the sensitization route and profile of the patient.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google Scholar In health care workers NRL allergy is dominated by sensitization to the proteins Hev b 5, 6.01, 6.02, and 6.03.2Ebo D.G. Hagendorens M.M. De Knop K.J. Verweij M.M. Bridts C.H. De Clerck L.S. et al.Component-resolved diagnosis from latex allergy by microarray.Clin Exp Allergy. 2010; 40: 348-358Crossref PubMed Scopus (96) Google Scholar Because of the ubiquity of these proteins in the plant kingdom, patients frequently display cross-reactivity to various fruits and vegetables. In patients who have undergone multiple operations, such as those with spina bifida, cross-reactivity is rare because NRL allergy mainly results from sensitization to components without homologues in edible plants, namely Hev b 1 and Hev b 3.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google ScholarHowever, NRL allergy has recently been confirmed in patients without clear sensitization to the components Hev b 1, 3, 5, 6, 7, 8, 9, 10, and 11.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google Scholar The exact explanation for this observation probably relates to the fact that the panel of recombinant proteins currently available for diagnostics does not cover the entire NRL IgE response. Although Beezhold et al3Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Google Scholar identified the nonspecific lipid transfer protein (nsLTP) Hev b 12 from NRL as a potential cross-reactive panallergen more than a decade ago, literature about the clinical significance of Hev b 12 sensitization is limited, and no Hev b 12 sensitization was demonstrable in 25 children with latex allergy.4Pamies R. Oliver F. Raulf-Heimsoth M. Rihs H.P. Barber D. No rHev b 12-specific IgE-response in children sensitized to natural rubber latex.Allergy. 2005; 60: 709-710Crossref PubMed Scopus (7) Google Scholar Rihs et al5Rihs H.P. Rueff F. Lundberg M. Rozynek P. Barber D. Scheurer S. et al.Relevance of the recombinant lipid transfer protein of Hevea brasiliensis: IgE-binding reactivity in fruit-allergic adults.Ann Allergy Asthma Immunol. 2006; 97: 643-649Abstract Full Text PDF PubMed Scopus (25) Google Scholar observed Hev b 12–specific IgE (sIgE) reactivity in only 6 (12.5%) of 48 patients with fruit allergy but without symptoms on latex exposure. In these patients Hev b 12 sensitization seemed attributable to cross-reactive nsLTPs from different fruits and apparently of limited role in NRL allergy.Because of limited data about the clinical relevance of Hev b 12, this study aims to re-evaluate the role of Hev b 12 in patients with NRL allergy, and in addition, particular focus is put on the position of NRL allergy in patients with the nsLTP syndrome.Five patients with a definite history of immediate allergic symptoms on exposure to NRL and an objective allergy to different plant foods were selected. All patients with latex allergy had a positive sIgE level to NRL extract, and 2 patients showed no sIgE reactivity to any of the available recombinant latex components. Demographic and detailed clinical data of the entire study population are shown in Table I. Patients provided informed consent in accordance with the Declaration of Helsinki.Table IDemographic and clinical data for the study population and results of skin prick tests with NRL and sIgE determination (ImmunoCAP or CBA)Patient no.Sex/age (y)Clinical symptoms on NRLAllergic symptoms to:SPT (NRL)Total IgE (kU/L), ImmunoCAPsIgE (kUA/L), NRL extract (ImmunoCAP)Sensitization profile to commercially available NRL components (ImmunoCAP)sIgE (MFI)sIgE (kUA/L), rPru p 3 (ImmunoCAP)rHev b 12 (CBA)nCan s 3 (CBA)1F/26U, AECannabis sativa, cherry, tree nuts peanut, apple, tobacco, peach, tomato, pineapple, raspberry, cucumber, fennel, red wine, wheat+2123.23None2.1912.5734.402M/11UPeanut, tree nuts, beans, carrot, pumpkinNT6040.64None0.97<0.102.883F/39D, WhSoy, apple, kiwifruit, tree nuts, potato, tomato, melon, paprika, peanutNT263.92Hev b 8<0.10<0.10<0.104F/53U, RhChestnut, passion fruit, kiwifruit+416.36Hev b 5/Hev b 6.01/Hev b 6.02<0.10<0.10<0.105F/52AEKiwifruit, peanut, almond+60.75Hev b 5<0.10<0.10<0.10AE, Angioedema; D, dyspnea; F, female; M, male; NT, not tested; Rh, rhinitis; SPT, skin prick test; U, urticaria; Wh, wheezing. Open table in a new tab NRL extract (Stallergenes, Fresnes, France) was used for skin prick tests. Results are shown in Table I.Levels of total IgE and sIgE to NRL extract; rHev b 1, 3, 5, 6.01, 6.02, 8, 9, and 11; and the peach nsLTP (Prunus persica, rPru p 3) were quantified by using a single-plexed technique (ImmunoCAP; Thermo Fisher Scientific, Uppsala, Sweden), and results of 0.35 kUA/L or greater were considered positive.A flow cytometric bead array (CBA) (BD Biosciences, Franklin Lakes, NJ) was developed to detect sIgE to the nsLTP allergens rHev b 12 and nCan s 3 of Cannabis sativa.6Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (11) Google Scholar The recombinant nsLTP from H brasiliensis latex and the native nsLTP extract from C sativa were purified, as previously reported.7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar Mean fluorescence intensity (MFI) results of 0.10 or greater were considered positive (detailed methodology is described in the Online Repository at www.jacionline.org).The basophil activation test (BAT) was performed with NRL extract, rHev b 12, nCan s 3, and nPru p 3 and prepared as previously described.3Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Google Scholar, 7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar Flow-assisted BAT was performed with 2 concentrations of NRL extract (0.01 μg/mL or 1 μg/mL) and 5 concentrations of rHev b 12, nCan s 3, and nPru p 3 (0.0001, 0.001, 0.01, 0.1, and 1 μg/mL). Three healthy subjects served as the control group.As displayed in Table I, both patients without sIgE to any of the available latex components had sIgE antibodies to rHev b 12. In addition, both patients showed sIgE reactivity to rPru p 3, and one of the patients showed a positive sIgE result to nCan s 3. In the other allergic patients sensitized to Hev b 5, 6.01, 6.02, and/or 8, no sIgE antibodies were found to rHev b 12, nCan s 3, or rPru p 3.Fig 1 shows a clear upregulation of the activation marker CD63 for rHev b 12 in both patients with latex allergy and rHev b 12 sensitization. In the other patients with latex allergy and the healthy control subjects, rHev b 12 did not trigger basophil activation. Both Hev b 12–sensitized patients had a positive BAT result to the nsLTP extract of peach. As demonstrated in Fig 2, the patient with sIgE antibodies to nCan s 3 also had a clear upregulation of CD63 with the nsLTP extract of cannabis. In the other Hev b 12– and Pru p 3–sensitized patient, no basophil activation to nCan s 3 was observed (data not shown).Fig 2Basophil selection and activation: BAT plot in a patient with latex allergy and sensitization to rHev b 12 and nCan s 3. A and B, Detection of basophils by using side scatter (SSC) and gating out IgEhigh and CD203c+ cells. C, Within this gate, numbers of activated basophils expressing CD63 were measured. Stimulation with activation buffer served as a negative control for spontaneous CD63 expression. D, Stimulation with anti-human IgE (10 μg/mL, BD Biosciences) was used as a positive control. E-H, Basophil stimulation with NRL extract (1 μg/mL), rHev b 12 (0.1 μg/mL), nCan s 3 (0.1 μg/mL), and nPru p 3 (0.1 μg/mL), respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These data indicate that sensitization to Hev b 12, the nsLTP from H brasiliensis latex, can be clinically relevant on exposure to products containing NRL.Although the exact routes of exposure to Hev b 12 remain elusive, we observed that 1 patient had symptoms of latex allergy after having nsLTP-related allergic symptoms to C sativa and the other patient had NRL allergy after being sensitized to multiple nsLTP-containing fruits. For both patients, it seems less probable that Hev b 12 is the primary sensitizer. The female patient with positive sIgE results to rHev b 12 and rCan s 3 was notable in that her first allergic symptoms occurred while smoking marijuana. It is noteworthy that this patient did not mention latex-induced allergic symptoms at first consultation and appeared to have the new sensitization to NRL in a timeframe of 1 year, with seroconversion of sIgE results to NRL and skin prick test responses with latex extract becoming positive.8Faber MA, Van Gasse A, Sabato V, Hagendorens MM, Bridts CH, De Clerck LC, et al. Marihuana allergy: beyond the joint. J Investig Allergol Clin Immunol 2015 [in press].Google Scholar Additional studies are needed to obtain better insight into the allergenicity of Hev b 12 and to confirm the hypothesis that sensitization to Can s 3 can cause cross-reactive allergies extending beyond fruits and vegetables.7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar In these studies BATs, along with IgE-binding studies, might be adopted to elucidate the clinical relevance of different nsLTPs. We should note that we did not determine sIgE levels to Hev b 2, an allergen of NRL also associated with the latex-fruit syndrome.9Palomares O. Villalba M. Quiralte J. Polo F. Rodriguez R. 1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.Clin Exp Allergy. 2005; 35: 345-351Crossref PubMed Scopus (87) Google Scholar Therefore we cannot exclude the possibility that our Hev b 12–sensitized patients are cosensitized to Hev b 2. In conclusion, Hev b 12 seems to be a clinically relevant allergen in patients with an nsLTP allergy, although further studies are needed in larger study populations and additional geographic areas. Natural rubber latex (NRL) from Hevea brasiliensis (Euphorbiaceae) constitutes an important cause of allergy with distinct phenotypes depending on the sensitization route and profile of the patient.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google Scholar In health care workers NRL allergy is dominated by sensitization to the proteins Hev b 5, 6.01, 6.02, and 6.03.2Ebo D.G. Hagendorens M.M. De Knop K.J. Verweij M.M. Bridts C.H. De Clerck L.S. et al.Component-resolved diagnosis from latex allergy by microarray.Clin Exp Allergy. 2010; 40: 348-358Crossref PubMed Scopus (96) Google Scholar Because of the ubiquity of these proteins in the plant kingdom, patients frequently display cross-reactivity to various fruits and vegetables. In patients who have undergone multiple operations, such as those with spina bifida, cross-reactivity is rare because NRL allergy mainly results from sensitization to components without homologues in edible plants, namely Hev b 1 and Hev b 3.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google Scholar However, NRL allergy has recently been confirmed in patients without clear sensitization to the components Hev b 1, 3, 5, 6, 7, 8, 9, 10, and 11.1Schuler S. Ferrari G. Schmid-Grendelmeier P. Harr T. Microarray-based component-resolved diagnosis of latex allergy: isolated IgE-mediated sensitization to latex profilin Hev b8 may act as confounder.Clin Transl Allergy. 2013; 3: 11Crossref PubMed Scopus (37) Google Scholar The exact explanation for this observation probably relates to the fact that the panel of recombinant proteins currently available for diagnostics does not cover the entire NRL IgE response. Although Beezhold et al3Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Google Scholar identified the nonspecific lipid transfer protein (nsLTP) Hev b 12 from NRL as a potential cross-reactive panallergen more than a decade ago, literature about the clinical significance of Hev b 12 sensitization is limited, and no Hev b 12 sensitization was demonstrable in 25 children with latex allergy.4Pamies R. Oliver F. Raulf-Heimsoth M. Rihs H.P. Barber D. No rHev b 12-specific IgE-response in children sensitized to natural rubber latex.Allergy. 2005; 60: 709-710Crossref PubMed Scopus (7) Google Scholar Rihs et al5Rihs H.P. Rueff F. Lundberg M. Rozynek P. Barber D. Scheurer S. et al.Relevance of the recombinant lipid transfer protein of Hevea brasiliensis: IgE-binding reactivity in fruit-allergic adults.Ann Allergy Asthma Immunol. 2006; 97: 643-649Abstract Full Text PDF PubMed Scopus (25) Google Scholar observed Hev b 12–specific IgE (sIgE) reactivity in only 6 (12.5%) of 48 patients with fruit allergy but without symptoms on latex exposure. In these patients Hev b 12 sensitization seemed attributable to cross-reactive nsLTPs from different fruits and apparently of limited role in NRL allergy. Because of limited data about the clinical relevance of Hev b 12, this study aims to re-evaluate the role of Hev b 12 in patients with NRL allergy, and in addition, particular focus is put on the position of NRL allergy in patients with the nsLTP syndrome. Five patients with a definite history of immediate allergic symptoms on exposure to NRL and an objective allergy to different plant foods were selected. All patients with latex allergy had a positive sIgE level to NRL extract, and 2 patients showed no sIgE reactivity to any of the available recombinant latex components. Demographic and detailed clinical data of the entire study population are shown in Table I. Patients provided informed consent in accordance with the Declaration of Helsinki. AE, Angioedema; D, dyspnea; F, female; M, male; NT, not tested; Rh, rhinitis; SPT, skin prick test; U, urticaria; Wh, wheezing. NRL extract (Stallergenes, Fresnes, France) was used for skin prick tests. Results are shown in Table I. Levels of total IgE and sIgE to NRL extract; rHev b 1, 3, 5, 6.01, 6.02, 8, 9, and 11; and the peach nsLTP (Prunus persica, rPru p 3) were quantified by using a single-plexed technique (ImmunoCAP; Thermo Fisher Scientific, Uppsala, Sweden), and results of 0.35 kUA/L or greater were considered positive. A flow cytometric bead array (CBA) (BD Biosciences, Franklin Lakes, NJ) was developed to detect sIgE to the nsLTP allergens rHev b 12 and nCan s 3 of Cannabis sativa.6Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (11) Google Scholar The recombinant nsLTP from H brasiliensis latex and the native nsLTP extract from C sativa were purified, as previously reported.7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar Mean fluorescence intensity (MFI) results of 0.10 or greater were considered positive (detailed methodology is described in the Online Repository at www.jacionline.org). The basophil activation test (BAT) was performed with NRL extract, rHev b 12, nCan s 3, and nPru p 3 and prepared as previously described.3Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Google Scholar, 7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar Flow-assisted BAT was performed with 2 concentrations of NRL extract (0.01 μg/mL or 1 μg/mL) and 5 concentrations of rHev b 12, nCan s 3, and nPru p 3 (0.0001, 0.001, 0.01, 0.1, and 1 μg/mL). Three healthy subjects served as the control group. As displayed in Table I, both patients without sIgE to any of the available latex components had sIgE antibodies to rHev b 12. In addition, both patients showed sIgE reactivity to rPru p 3, and one of the patients showed a positive sIgE result to nCan s 3. In the other allergic patients sensitized to Hev b 5, 6.01, 6.02, and/or 8, no sIgE antibodies were found to rHev b 12, nCan s 3, or rPru p 3. Fig 1 shows a clear upregulation of the activation marker CD63 for rHev b 12 in both patients with latex allergy and rHev b 12 sensitization. In the other patients with latex allergy and the healthy control subjects, rHev b 12 did not trigger basophil activation. Both Hev b 12–sensitized patients had a positive BAT result to the nsLTP extract of peach. As demonstrated in Fig 2, the patient with sIgE antibodies to nCan s 3 also had a clear upregulation of CD63 with the nsLTP extract of cannabis. In the other Hev b 12– and Pru p 3–sensitized patient, no basophil activation to nCan s 3 was observed (data not shown). These data indicate that sensitization to Hev b 12, the nsLTP from H brasiliensis latex, can be clinically relevant on exposure to products containing NRL. Although the exact routes of exposure to Hev b 12 remain elusive, we observed that 1 patient had symptoms of latex allergy after having nsLTP-related allergic symptoms to C sativa and the other patient had NRL allergy after being sensitized to multiple nsLTP-containing fruits. For both patients, it seems less probable that Hev b 12 is the primary sensitizer. The female patient with positive sIgE results to rHev b 12 and rCan s 3 was notable in that her first allergic symptoms occurred while smoking marijuana. It is noteworthy that this patient did not mention latex-induced allergic symptoms at first consultation and appeared to have the new sensitization to NRL in a timeframe of 1 year, with seroconversion of sIgE results to NRL and skin prick test responses with latex extract becoming positive.8Faber MA, Van Gasse A, Sabato V, Hagendorens MM, Bridts CH, De Clerck LC, et al. Marihuana allergy: beyond the joint. J Investig Allergol Clin Immunol 2015 [in press].Google Scholar Additional studies are needed to obtain better insight into the allergenicity of Hev b 12 and to confirm the hypothesis that sensitization to Can s 3 can cause cross-reactive allergies extending beyond fruits and vegetables.7Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (12) Google Scholar In these studies BATs, along with IgE-binding studies, might be adopted to elucidate the clinical relevance of different nsLTPs. We should note that we did not determine sIgE levels to Hev b 2, an allergen of NRL also associated with the latex-fruit syndrome.9Palomares O. Villalba M. Quiralte J. Polo F. Rodriguez R. 1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity.Clin Exp Allergy. 2005; 35: 345-351Crossref PubMed Scopus (87) Google Scholar Therefore we cannot exclude the possibility that our Hev b 12–sensitized patients are cosensitized to Hev b 2. In conclusion, Hev b 12 seems to be a clinically relevant allergen in patients with an nsLTP allergy, although further studies are needed in larger study populations and additional geographic areas. MethodsMethodology: CBAA CBA was developed to detect sIgE to nsLTP allergens of H brasiliensis latex (Hev b 12) and C sativa (Can s 3).E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar The recombinant nsLTP from H brasiliensis latex was produced by Beezhold et al,E2Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Scopus (48) Google Scholar and the native nsLTP extract from C sativa was purified as previously reported.E3Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (55) Google Scholar The method was validated and standardized by using rBet v 1 (Stallergenes) from birch pollen (Betula verrucosa).Conjugation procedureFunctional beads (BD CBA functional beads; BD Biosciences, Franklin Lakes, NJ) were conjugated according to the manufacturer's procedures with selected allergens, as described by Pomponi et al.E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar Briefly, 75 μL of microbeads were incubated with 1.9 μL of 1 mol/L dithiothreitol for 1 hour in the dark at room temperature, vortexing the tubes every 15 minutes. Afterward, the microbeads were washed with coupling buffer (BD Biosciences) and centrifuged at 900g for 3 minutes, and the supernatant was resuspended in 20 μL of coupling buffer. Ninety microliters of the selected allergens at a concentration of 1 mg/mL were incubated with 2 μL of fresh prepared sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) solution (2 mg/mL; Thermo Scientific, Pierce, Ill) for 1 hour at room temperature in the dark vortexing every 15 minutes. The Bio Spin columns (Bio-Rad, Milan, Italy) were prewashed with coupling buffer, and the protein/sulfo-SMCC mix was transferred to the column, followed by spinning at 1000g for 2 minutes. The modified proteins in the eluate were transferred to functional microbeads and incubated for 1 hour at room temperature and protected from light. The tubes were vortexed every 15 minutes. Afterward, 2 μL of N-ethylmaleimide (2 mg/mL in dimethyl sulfoxide, Thermo Scientific) was added for 15 minutes, with vortexing every 5 minutes. After incubation, 1 mL of storage buffer was added, and the tubes were spun for 3 minutes at 900g. Washing with storage buffer was performed 3 times before the microbead pellet was resuspended in 500 μL of storage buffer at a concentration of 6 × 106 microbeads/mL and stored at 2°C to 8°C and protected from light before use.Testing procedureOne microliter of each allergen-conjugated microbead was used for each test. The microbeads were washed with 1 mL of PBS with 0.05% Tween 20 and 0.05% NaN3 (PBS-T) and centrifuged for 5 minutes at 500g. After removing the supernatant, the microbeads were resuspended in 50 μL of PBS-T with 1% BSA (PBS-T–BSA).Fifty microliters of diluted serum samples (1:5 in PBS-T–BSA) was added to 50 μL of the mixed capture beads and incubated for 1 hour at room temperature in the dark. Tubes were then washed with PBS-T and resuspended in 100 μL of PBS-T–BSA after removing the supernatant. Then 50 μL of diluted phycoerythrin-conjugated anti-human IgE (1:100 in PBS 0.05% NaN3; BioLegend, San Diego, Calif) was added and incubated for 1 hour at room temperature in the dark. The samples were washed with 1 mL of PBS-T, centrifuged for 5 minutes at 200g, and resuspended in 200 μL of PBS-T. Afterward, the samples were measured on a FACSCanto II Flow Cytometer (BD Biosciences), and results were expressed as MFI.Validation of the CBAStandardization of the flow cytometric immunobead array was performed with rBet v 1–positive samples compared with the ImmunoCAP technique. At least 5 samples with known rBet v 1 IgE concentrations (ImmunoCAP; in kilounits of allergen per liter) were pooled and tested. A 5-parameter logistic nonlinear standard curve was constructed to normalize the MFI. Reproducibility was tested, and a coefficient of variation of 10% was found. Methodology: CBAA CBA was developed to detect sIgE to nsLTP allergens of H brasiliensis latex (Hev b 12) and C sativa (Can s 3).E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar The recombinant nsLTP from H brasiliensis latex was produced by Beezhold et al,E2Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Scopus (48) Google Scholar and the native nsLTP extract from C sativa was purified as previously reported.E3Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (55) Google Scholar The method was validated and standardized by using rBet v 1 (Stallergenes) from birch pollen (Betula verrucosa). A CBA was developed to detect sIgE to nsLTP allergens of H brasiliensis latex (Hev b 12) and C sativa (Can s 3).E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar The recombinant nsLTP from H brasiliensis latex was produced by Beezhold et al,E2Beezhold D.H. Hickey V.L. Kostyal D.A. Puhl H. Zuidmeer L. van Ree R. et al.Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein.Ann Allergy Asthma Immunol. 2003; 90: 439-445Abstract Full Text PDF PubMed Scopus (48) Google Scholar and the native nsLTP extract from C sativa was purified as previously reported.E3Ebo D.G. Swerts S. Sabato V. Hagendorens M.M. Bridts C.H. Jorens P.G. et al.New food allergies in a European non-Mediterranean region: is Cannabis sativa to blame?.Int Arch Allergy Immunol. 2013; 161: 220-228Crossref PubMed Scopus (55) Google Scholar The method was validated and standardized by using rBet v 1 (Stallergenes) from birch pollen (Betula verrucosa). Conjugation procedureFunctional beads (BD CBA functional beads; BD Biosciences, Franklin Lakes, NJ) were conjugated according to the manufacturer's procedures with selected allergens, as described by Pomponi et al.E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar Briefly, 75 μL of microbeads were incubated with 1.9 μL of 1 mol/L dithiothreitol for 1 hour in the dark at room temperature, vortexing the tubes every 15 minutes. Afterward, the microbeads were washed with coupling buffer (BD Biosciences) and centrifuged at 900g for 3 minutes, and the supernatant was resuspended in 20 μL of coupling buffer. Ninety microliters of the selected allergens at a concentration of 1 mg/mL were incubated with 2 μL of fresh prepared sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) solution (2 mg/mL; Thermo Scientific, Pierce, Ill) for 1 hour at room temperature in the dark vortexing every 15 minutes. The Bio Spin columns (Bio-Rad, Milan, Italy) were prewashed with coupling buffer, and the protein/sulfo-SMCC mix was transferred to the column, followed by spinning at 1000g for 2 minutes. The modified proteins in the eluate were transferred to functional microbeads and incubated for 1 hour at room temperature and protected from light. The tubes were vortexed every 15 minutes. Afterward, 2 μL of N-ethylmaleimide (2 mg/mL in dimethyl sulfoxide, Thermo Scientific) was added for 15 minutes, with vortexing every 5 minutes. After incubation, 1 mL of storage buffer was added, and the tubes were spun for 3 minutes at 900g. Washing with storage buffer was performed 3 times before the microbead pellet was resuspended in 500 μL of storage buffer at a concentration of 6 × 106 microbeads/mL and stored at 2°C to 8°C and protected from light before use. Functional beads (BD CBA functional beads; BD Biosciences, Franklin Lakes, NJ) were conjugated according to the manufacturer's procedures with selected allergens, as described by Pomponi et al.E1Pomponi D. Bernardi M.L. Liso M. Palazzo P. Tuppo L. Rafaiani C. et al.Allergen micro-bead array for IgE detection: a feasibility study using allergenic molecules tested on a flexible multiplex flow cytometric immunoassay.PLoS One. 2012; 7: e35697Crossref PubMed Scopus (36) Google Scholar Briefly, 75 μL of microbeads were incubated with 1.9 μL of 1 mol/L dithiothreitol for 1 hour in the dark at room temperature, vortexing the tubes every 15 minutes. Afterward, the microbeads were washed with coupling buffer (BD Biosciences) and centrifuged at 900g for 3 minutes, and the supernatant was resuspended in 20 μL of coupling buffer. Ninety microliters of the selected allergens at a concentration of 1 mg/mL were incubated with 2 μL of fresh prepared sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) solution (2 mg/mL; Thermo Scientific, Pierce, Ill) for 1 hour at room temperature in the dark vortexing every 15 minutes. The Bio Spin columns (Bio-Rad, Milan, Italy) were prewashed with coupling buffer, and the protein/sulfo-SMCC mix was transferred to the column, followed by spinning at 1000g for 2 minutes. The modified proteins in the eluate were transferred to functional microbeads and incubated for 1 hour at room temperature and protected from light. The tubes were vortexed every 15 minutes. Afterward, 2 μL of N-ethylmaleimide (2 mg/mL in dimethyl sulfoxide, Thermo Scientific) was added for 15 minutes, with vortexing every 5 minutes. After incubation, 1 mL of storage buffer was added, and the tubes were spun for 3 minutes at 900g. Washing with storage buffer was performed 3 times before the microbead pellet was resuspended in 500 μL of storage buffer at a concentration of 6 × 106 microbeads/mL and stored at 2°C to 8°C and protected from light before use. Testing procedureOne microliter of each allergen-conjugated microbead was used for each test. The microbeads were washed with 1 mL of PBS with 0.05% Tween 20 and 0.05% NaN3 (PBS-T) and centrifuged for 5 minutes at 500g. After removing the supernatant, the microbeads were resuspended in 50 μL of PBS-T with 1% BSA (PBS-T–BSA).Fifty microliters of diluted serum samples (1:5 in PBS-T–BSA) was added to 50 μL of the mixed capture beads and incubated for 1 hour at room temperature in the dark. Tubes were then washed with PBS-T and resuspended in 100 μL of PBS-T–BSA after removing the supernatant. Then 50 μL of diluted phycoerythrin-conjugated anti-human IgE (1:100 in PBS 0.05% NaN3; BioLegend, San Diego, Calif) was added and incubated for 1 hour at room temperature in the dark. The samples were washed with 1 mL of PBS-T, centrifuged for 5 minutes at 200g, and resuspended in 200 μL of PBS-T. Afterward, the samples were measured on a FACSCanto II Flow Cytometer (BD Biosciences), and results were expressed as MFI. One microliter of each allergen-conjugated microbead was used for each test. The microbeads were washed with 1 mL of PBS with 0.05% Tween 20 and 0.05% NaN3 (PBS-T) and centrifuged for 5 minutes at 500g. After removing the supernatant, the microbeads were resuspended in 50 μL of PBS-T with 1% BSA (PBS-T–BSA). Fifty microliters of diluted serum samples (1:5 in PBS-T–BSA) was added to 50 μL of the mixed capture beads and incubated for 1 hour at room temperature in the dark. Tubes were then washed with PBS-T and resuspended in 100 μL of PBS-T–BSA after removing the supernatant. Then 50 μL of diluted phycoerythrin-conjugated anti-human IgE (1:100 in PBS 0.05% NaN3; BioLegend, San Diego, Calif) was added and incubated for 1 hour at room temperature in the dark. The samples were washed with 1 mL of PBS-T, centrifuged for 5 minutes at 200g, and resuspended in 200 μL of PBS-T. Afterward, the samples were measured on a FACSCanto II Flow Cytometer (BD Biosciences), and results were expressed as MFI. Validation of the CBAStandardization of the flow cytometric immunobead array was performed with rBet v 1–positive samples compared with the ImmunoCAP technique. At least 5 samples with known rBet v 1 IgE concentrations (ImmunoCAP; in kilounits of allergen per liter) were pooled and tested. A 5-parameter logistic nonlinear standard curve was constructed to normalize the MFI. Reproducibility was tested, and a coefficient of variation of 10% was found. Standardization of the flow cytometric immunobead array was performed with rBet v 1–positive samples compared with the ImmunoCAP technique. At least 5 samples with known rBet v 1 IgE concentrations (ImmunoCAP; in kilounits of allergen per liter) were pooled and tested. A 5-parameter logistic nonlinear standard curve was constructed to normalize the MFI. Reproducibility was tested, and a coefficient of variation of 10% was found.
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