A sensitive reverse ELISA for the measurement of specific IgE to Der p 2, a major Dermatophagoides pteronyssinus allergen

A sensitive reverse ELISA for the measurement of specific IgE to Der p 2, a major Dermatophagoides pteronyssinus allergen
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  A sensitive reverse ELISA for the measurementof specific IgE to Der p 2, a major  Dermatophagoides pteronyssinus  allergen Deise A. O. Silva, DMV*; Aure´lia M. Gerva´sio, DDS, MS*; Moˆnica C. Sopelete, DMV*;Erika Arruda-Chaves, MD, PhD*; L. Karla Arruda, MD, PhD†; Martin D. Chapman, PhD‡;Sun-Sang J. Sung, PhD§; and Ernesto A. Taketomi, MD, PhD* Background:  Epidemiologic studies have shown that the presence of IgE anti-bodies to house dust mite and other indoor allergens is an important risk factor forasthma. Objective:  The aim of this study was to develop a reverse ELISA (rELISA) formeasuring specific IgE to Der p 2, a major  Dermatophagoides pteronyssinus  (Dpt)allergen, as a potential tool for followup of allergen immunotherapy. Methods:  Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 wasused to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Serafrom 48 asthmatic patients with positive skin prick test (SPT  ) to  D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results werecompared with a group of 41 SPT  asthmatic and 30 SPT  control subjects. Results:  The sensitivity of the two assays for Der p 2-specific IgE was 3.9EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISAand rELISA ( r   0.74;  P  0.0001). However, rELISA was more sensitive than wascELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Derp 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT  asthmatic patients. Conclusions:  rELISA has shown to be a sensitive and alternative method formeasuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cellepitopes in allergens will provide valuable information for the design of allergenanalogs and peptides for immunotherapy. Ann Allergy Asthma Immunol 2001;86:545–550. INTRODUCTION The importance of house dust mites(HDMs) as a source of indoor aller-gens and their role in the developmentof allergic diseases have been recog-nized for many years. 1 Epidemiologicstudies from tropical countries includ-ing Brazil have shown that the pres-ence of IgE antibodies to HDM andother indoor allergens is an importantrisk factor for asthma. 2,3 Mites of thegenus  Dermatophagoides  (  D. pteron- yssinus ,  D. farinae , and  D. microceras )account for   90% of the mites foundin house dust samples and are the prin-cipal cause of mite allergy. 4 The ma- jority of HDM allergic patients havehigh levels of IgE antibodies to group1 (  80%) and group 2 (  90%) aller-gens of   D. pteronyssinus  (Dpt) and  D. farinae  (Df).Crude mite extracts are currentlyused in assays for measuring HDM-specific IgE, in part because of diffi-culties in obtaining large amounts of purified group 1 and 2 allergens. 5 Thesensitive RAST has still been used formeasuring specific IgE to  Dermato- phagoides  sp. However, radioactivematerials are relatively unstable, ex-pensive, and potentially hazardous, re-quiring special facilities. 6 These disad-vantages have prompted investigatorsto develop alternative methods for IgEmeasurements.Kemeny et al 7 have used allergen-specific monoclonal and polyclonalantibodies to measure IgE by ELISA.These authors reported that sandwichELISA using polyspecific rabbit anti-body was more sensitive than was con-ventional ELISA (cELISA) and alsoslightly more sensitive than RAST.Mastrandrea et al 8 reported that IgEantibodies to Dpt whole extract and tothe natural major allergens Der p 1 andDer p 2, could also be measured byELISA. A new automated fluoroim-munoassay system using reagents suit-able for increased speed and accuracy(UniCAP; Pharmacia & Upjohn, Upp-sala, Sweden) was evaluated by Pagan-elli et al. 9 They showed that the UniCAPsystem for IgE measurement is efficientfor routine diagnostic testing of allergy.Recently, Peng et al 5 have described amonoclonal antibody-based captureELISA for measuring Der f 1-specificIgE. This method has shown to be moresensitive and specific than the conven-tional Der f 1-capture ELISA to diag-nose dust mite allergy.We have developed a sensitive re-verse ELISA for measuring Der p2-specific IgE in serum. In this article,we report the results of measurementof IgE antibodies to Der p 2 by acELISA, in which purified antigen wasbound directly to microtiter plates, andby a reverse ELISA (rELISA), in * Department of Immunology, Microbiology,and Parasitology, Federal University of Uber-laˆndia, Uberlaˆndia, MG, †Ribeirao Preto, SP,Brazil; † Department of Immunology, School of Medicine of Ribeira˜o Preto-USP, Brazil; andDivision of ‡ Allergy, Asthma, and Immunologyand § Rheumatology, Health Sciences Center,University of Virginia, Charlottesville, Virginia.Received for publication July 10, 2000.Accepted for publication in revised form De-cember 22, 2000.VOLUME 86, MAY, 2001 545  which antigen was captured by anti-Der p 2 monoclonal antibody bound tothe plates. In addition, we comparedthe sensitivity and specificity of thetwo ELISAs for measurement of Der p2-specific IgE in asthmatic patientswith positive or negative skin prick test(SPT) to dust mite extract, and in non-allergic healthy control subjects. METHODS Subjects Eighty-nine patients (19 males), ages18 to 60 years, with mild intermittentto severe persistent asthma, seen at theAllergy and Infectious Diseases Unitof the Immunology Division of FederalUniversity of Uberlaˆndia, Brazil, wereincluded. The study was approved bythe human investigation committee atthe Federal University of Uberlaˆndiaand informed written consent was ob-tained from patients. Diagnosis of asthma was based on medical history,physical examination, and spirome-try. 10,11 Patients were submitted to SPTwith HDM extract (  Dermatophagoides pteronyssinus ; Bayer Corporation,Spokane, WA) and venipuncture. Skintest reactions were evaluated based onthe mean wheal diameter at 15 minutesafter application of the extract, consid-ering values  4 mm as a positive SPT.Among the 89 patients, 48 had pos-itive SPT to Dpt (asthma SPT  group),and 41 patients had negative SPT(asthma SPT  group). Thirty healthysubjects without asthma or clinical his-tory of atopic disease and with nega-tive skin tests to Dpt were included ascontrols (control group).  Mite Extract  Crude Dpt extract was prepared fromdried material kindly provided by Dr.Larry Arlian (Wright State University,Dayton, OH) and contained 285  g/mL of Der p 1 and 121   g/mL of Der p 2. Recombinant Der p 2 (rDer p2) was produced by means of a bacte-rial expression system. 12 Dpt extract,50% glycerin, 10,000 allergen units(AU)/mL, was purchased from BayerCorporation.  Der p 2-Specific IgE by rELISA High-binding microtiter plates (Corn-ing Laboratories Inc, New York, NY)were coated (50   L/well) with mousemonoclonal antibody to Der p 2 (clone1D8) at 1   g/well in 0.06 M carbonatebuffer (pH 9.6) overnight at 4° C. Ascontrol of the mouse monoclonal anti-body, plates were coated in parallelwith purified mouse IgG at 10   g/mL.Plates were washed three times with0.01 M phosphate buffered saline(PBS; pH 7.2) containing 0.05%Tween 20 (PBS-T) and active siteswere blocked (200   L/well) withPBS-T plus 1% bovine serum albumin(BSA; Sigma, St. Louis, MO) for 1hour at room temperature. Subsequentsteps were carried out using 1% BSA-PBS-T as diluent (ELISA buffer). Af-ter blocking, wells were washed andsubsequently incubated (50   L/well)with Dpt extract (40   g/mL) for 1 hourat room temperature. Plates werewashed five times and incubated (50  L/well) with serum samples dilutedat 1:2.5 and 1:5 for 2 hours at roomtemperature. After washing as above,biotinylated goat anti-human IgE(Kirkegaard & Perry Laboratories,Gaithersburg, MD) diluted at 1:4,000was added to the wells (50   L/well)and plates were incubated for 1 hour atroom temperature. After washing, 50  L/well of streptavidin-peroxidaseconjugate (Sigma) diluted at 1:1,000were added and incubated for 30 min-utes at room temperature. After wash-ing, the assay was developed by add-ing 50  L/well of the enzyme substrate0.01 M 2,2  -azino-bis-(3-ethyl-benz-thiazoline sulfonic acid; Sigma) in0.07 M citrate-phosphate buffer (pH4.2) containing 0.03% H 2 O 2.  The reac-tion was read at 405 nm with a Titertek Multiskan Plus MK II plate reader(Flow Laboratories, McLean, VA).Results were expressed as ELISA units(EU)/mL and compared with a controlcurve obtained by measuring HDM-specific IgE levels in parallel, using astandard mite allergic serum pool(UVA 89/01; University of Virginia,Charlottesville, VA). The UVA serumpool contained 1,000 RAST units(RU)/mL of specific IgE to  Dermato- phagoides farinae  (1 RU is equivalentto 0.1 ng of IgE). 13 The control curvewas included in each plate with dupli-cate samples, and values ranged from500 to 0.5 RU/mL.Levels of Der p 2-specific IgE werearbitrarily classified in ELISA reactiv-ity classes based on the sensitivity of the assay. Thus, the following classeswere determined using 5-fold serialfactor: class 0:  3.9 EU/mL (negativespecific IgE); class 1: 3.9 to   19.5EU/mL (low positive specific IgE);class 2: 19.5 to   97.5 EU/mL (mod-erate positive specific IgE); class 3:97.5 to   487.5 EU/mL (high positivespecific IgE); class 4:  487.5 EU/mL(very high positive specific IgE).  Der p 2-Specific IgE by cELISA cELISA for Der p 2-specific IgE wascarried out according to Mastrandrea etal, 8 with modifications. High-bindingmicrotiter plates (Corning LaboratoriesInc) were coated overnight at 4° C (50  L/well) with 10   g/mL of recombi-nant rDer p 2 allergen in 0.06 M car-bonate buffer (pH 9.6). Plates werewashed three times with PBS-T andblocked with 1% BSA-PBS-T for 1hour at room temperature. Subsequentsteps were carried out using ELISAbuffer. After washing, 50   L/well of serum samples diluted at 1:2 wereadded in duplicate to the wells andincubated for 2 hours at 37° C. Plateswere washed five times and incubatedwith 50   L/well of biotinylated goatanti-human IgE (Kirkegaard & PerryLaboratories) diluted at 1:1,000 for 1hour at 37° C. Subsequent steps (addi-tion of streptavidin-peroxidase conju-gate and enzyme substrate) were sim-ilar to those described for rELISA.Results were expressed as EU/mL,compared with a control curve ob-tained as previously mentioned. Levelsof Der p 2-specific IgE were arbitrarilyclassified in ELISA reactivity classesas described for rELISA. Specificity of cELISA and rELISA Specificity of cELISA and rELISAwas evaluated using inhibition tests.Dpt extract (Bayer Corporation) was 546 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY  10-fold serially diluted, from 15,000 to15 AU/mL in ELISA buffer. Each di-lution of the extract was mixed 1:1with a 1/50 dilution of serum that wasdesignated as containing 1,000 EU/mLof Der p 2-specific IgE, and furtherincubated at 37° C for 2 hours. Thereference serum incubated with bufferonly was used as positive control. Derp 2-specific IgE was determined bymeasuring the absorbance in twoELISAs as described above. Percent-age of inhibition was calculated as fol-lows: [1.0    (test sample absorbance/ positive control absorbance)]  100. Total Serum IgE  Total serum IgE was measured by amonoclonal antibody-based ELISAmodified from a previously describedradioimmunoassay. 13 Briefly, polysty-rene plates (Immulon II; DynatechLaboratories, Chantilly, VA) werecoated with mouse monoclonal anti-human IgE (Sigma) diluted 1:5,000 in0.06 M carbonate buffer (pH 9.6) over-night at 4° C. Plates were washed andblocked as previously described. Se-rum samples diluted 1:5, 1:25, and1:125 in 1% BSA-PBS-T were incu-bated for 1 hour at room temperature.After washing, biotinylated goat anti-human IgE (Kirkegaard & Perry Lab-oratories) diluted 1:4,000 was addedand plates were incubated for 1 hour atroom temperature. Subsequent steps(streptavidin-peroxidase and enzymesubstrate) were similar to those de-scribed for rELISA. Results were ex-pressed as international units (IU)/mLof serum (1 IU/mL    2.4 ng/mL of IgE) and were calculated based on acontrol curve obtained by serial 2-folddilutions of a serum that was desig-nated as containing 3,000 IU/mL of total IgE. The control curve valuesranged from 0.3 to 300 IU/mL. Statistical Analysis Unpaired  t   tests were used to comparespecific IgE values between groupsand obtained by different techniques.The    2 test was used to compare per-centages of positives within thegroups. Levels of specific IgE mea-sured by cELISA and rELISA wereanalyzed by Spearman’s correlationtest.  P  values  0.05 were consideredas statistically significant. RESULTS Sensitivity and Specificity of ELISAs Control curves of cELISA and rELISAobtained with the standard serum(UVA 89/01) were analyzed for intra-and interassay variations. The sensitiv-ity of the two ELISAs for Der p 2-spe-cific IgE was 3.9 EU/mL (Fig 1). Theaverage coefficients of variation foreach standard serum dilution assayedin duplicate were 12.7% and 5.8% incELISA and rELISA, respectively. Forrepeated assays, the average coeffi-cients of variation were 16.5% and9.6% in cELISA and rELISA, respec-tively.Specificities of cELISA and rELISAfor the Der p 2 allergen were con-firmed by inhibition tests (Fig 2). BothELISAs were inhibited in a dose-de-pendent manner, when reference se-rum was mixed with increasing con-centrations of crude Dpt extract.Inhibition was up to 80% with thehighest concentration of mite extract(15,000 AU/mL).  Levels of Total IgE and Der p 2-Specific IgE  Total serum IgE was significantlyhigher in the SPT  asthmatic patients(geometric mean; GM: 375.2 IU/mL)compared with either the SPT  asth-matic patients (GM: 141.3 IU/mL;  P  0.01) or the control subjects (GM: 17.8IU/mL;  P  0.0001).The relationship between levels of Der p 2-specific IgE (EU/mL) obtainedby the two assays in SPT  asthmaticpatients is shown in Figure 3. Therewas a significant positive correlationbetween cELISA and rELISA ( r    0.74;  P  0.0001).As presented in Figure 4, the GM of Der p 2-specific IgE obtained byrELISA in SPT  asthmatic patients(28.4 EU/mL) was significantly higherthan in SPT  asthmatic patients (0.6EU/mL) or control subjects (0.6 EU/ mL;  P    0.0001). Using cELISA, themean levels of Der p 2-specific IgEwere also significantly higher in theSPT  asthmatic patients (4.5 EU/mL),compared with SPT  asthmatic pa-tients and control subjects (0.2 EU/mLand 0.3 EU/mL, respectively;  P   0.0001). In addition, levels of Der p2-specific IgE measured by rELISA inSPT  asthmatic patients were signifi-cantly higher than those obtained bycELISA ( P  0.01). Serodiagnostic Performanceof ELISAs The serodiagnostic performance of thecELISA and rELISA was evaluated forsensitivity and specificity in relation toskin prick testing. Positivity rate of Der p 2-specific IgE measured bycELISA and rELISA was calculatedbased on the sensitivity of both assays(3.9 EU/mL), in asthmatic patients(SPT  and SPT  groups) and controlsubjects. Thus, rELISA for Der p 2showed significantly higher sensitivity(70.8%) than did cELISA (52.1%;  P  Figure 1. Standard curves of cELISA and rELISA for the measurement of Der p 2-specific IgE. *Thesensitivity of the two assays was 3.9 EU/mL.VOLUME 86, MAY, 2001 547  0.05). Specificity was 100% for bothELISAs (Fig 5).  Der p 2-Specific IgE by cELISAand rELISA at Different  Reactivity Classes Der p 2-specific IgE levels were ana-lyzed according to reactivity classes(classes 0, 1, 2, and   3) obtained bycELISA and rELISA in the SPT  asthma, SPT  asthma and controlgroups, and the results are shown inTable 1.Among the 34 positive samples(70.8%) for Der p 2-specific IgE byrELISA in the SPT  asthma group, themajority (41.7%) displayed high orvery high ELISA reactivity (class  3),whereas a lower proportion of samples(20.8%) showed ELISA reactivityclass   3 when using cELISA ( P   0.05). In contrast, in the SPT  asthmaand control groups, neither ELISAsdemonstrated any reactivity (class 0),resulting in total concordance withSPT and no difference in their speci-ficity. DISCUSSION ELISA has shown to be a sensitive andalternative method for measuring spe-cific IgE antibodies to mite allergenswithout using radioactive techniques.cELISA is usually performed usingcrude extracts or purified antigens/aller-gens to coat microtiter plates. However,purified antigen or allergens are difficultto obtain in large amounts. Mite aller-gen-specific monoclonal antibodies arebroadly used to measure environmentalallergens in two-site monoclonal anti-body-based ELISA. 14,15 Few reportshave described the use of monoclonalantibodies in ELISAs to detect specificIgE antibodies, and these assays are usu-ally named mAb-capture ELISA 5 orsandwich ELISA. 7 In the present study,we have developed a rELISA for mea-suring specific IgE antibodies to Der p 2,a major  D. pteronyssinus  allergen. Be-cause the terminology of classical cap-ture ELISA is commonly used whenmonoclonal antibodies bound to micro-titer plates are used to capture IgM, IgA,orIgEantibodies,wepreferredtousetheterm rELISA because the plates were Figure 3. Correlation between rELISA and cELISA for measurement of Der p 2-specific IgE in SPT  asthmatic patients (N  48).Figure 4. Levels of Der p 2-specific IgE (EU/mL) measured by rELISA and cELISA in SPT  andSPT  asthmatic patients and control subjects. The horizontal bars indicate the GM.Figure 2. Inhibition tests for the measurements of Der p 2-specific IgE by cELISA and rELISA. Dptallergenic extract was 10-fold serially diluted from 15,000 to 15 AU/mL in ELISA buffer. Each dilutionof the extract was mixed 1:1 with a reference serum (20 EU/mL) and incubated at 37° C for 2 hours. Aspositive control, the reference serum was incubated with ELISA buffer only. Der p 2-specific IgE wasdetermined by measuring the absorbance obtained by two ELISAs. Percentage of inhibition wascalculated as follows: [1.0  (test sample absorbance/positive control absorbance)]  100.548 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY  coated with monoclonal antibody to cap-ture the antigen/allergen, and then to de-tect allergen-specific IgE antibodies.Although the sensitivity of the twoassays (rELISA and cELISA) was 3.9EU/mL according to the reference se-rum used (UVA 89/01), the rELISAstandard curve showed slightly higherabsorbance values than did cELISA. Inaddition, the inter- and intraassay vari-ations found for rELISA were lowerthan were those observed for cELISA.The specificity of both assays for Der p2-specific IgE was confirmed by inhi-bition with crude Dpt extract in a dose-dependent manner. We believed thatIgE-Der p 2 complexes in this inhibitionassay for rELISA did not compete forbinding to the plate-coated monoclonalantibody because it was obtained a dose-response curve of inhibition.Specificity of rELISA was furtherconfirmed in all sera from SPT  asth-matic patients, which showed no reac-tivity in wells coated with mouse IgG(used as control) in contrast to the re-activity observed when using themouse monoclonal antibody to Der p 2(data not shown). Thus, the possibilitythat these sera could contain IgE iso-type rheumatoid factor (IgE antibodiesagainst Ig    -chain) and then reactingwith the plate-coated mouse IgGmonoclonal antibody was eliminated.The probability that an IgE epitopeis blocked by the plate-coated mono-clonal antibody in rELISA was ruledout because of the fact that the resultsobtained in rELISA were higher thanthose obtained in cELISA using Dptcrude extract instead of rDer p 2 forcoating the microtiter plates (data notshown).In contrast, a direct comparison be-tween cELISA and rELISA using thesame allergen preparation (rDer p 2)was not possible because the monoclo-nal antibody (clone 1D8) used inrELISA does not react to a relevantisoform of Der p 2, which has an as-partic acid at position 114 and this isalso the isoform most frequently usedas recombinant Der p 2. 16 Serodiagnostic performance of thetwo ELISAs for measuring the levelsof Der p 2-specific IgE was evaluatedin asthmatic patients (SPT  and SPT  )and control subjects. Thus, rELISAwas found to be more sensitive thancELISA with respect to both the posi-tivity rate (70.8% vs 52.1%) and GMlevels (28.4 vs 4.5 EU/mL) in theSPT  asthmatic patients. In contrast,the specificities of both ELISAs were100%, and total concordance withSPT-negative results was found both inasthmatic patients and control subjects.A likely explanation for the highersensitivity of rELISA found in thepresent study is provided by Kemenyet al. 7 These authors reported that theincreased sensitivity of sandwichELISA compared with RAST and con-ventional ELISA is not attributable tolarger amounts of IgE antibody beingbound in ELISA than in RAST, butbecause a very high concentration of nonradioisotopic anti-IgE can saturatethe small amounts of bound IgE. Afurther advantage of sandwich orrELISA for detection of specific IgEantibodies is that the use of a consid-erable excess of anti-IgE results in amuch steeper binding curve and this inturn contributes to a lower interassayvariation coefficient, 7 compared withRAST, which has been associated withhigh interassay variation. 17 This fea-ture was also observed in our studywhen comparing the interassay varia-tion coefficients between rELISA andcELISA.Levels of total serum IgE in ourSPT  asthmatic patients (375.2 IU/ mL) were slightly higher than thoseobserved by Gelber et al 18 among asth-matic patients (160 IU/mL) and controlsubjects (44 IU/mL), living in Wil-mington, Delaware. In contrast, verysimilar results were seen in the SPT  (141.3 IU/ml) and control (17.8 IU/ mL) groups.When analyzing the different reactiv-ity levels of the two ELISAs amongSPT  asthmatic patients, rELISAshowed a significantly higher percentage Figure 5. Positivity rate of Der p 2-specific IgE by rELISA and cELISA in asthmatic patients (SPT  and SPT  ) and control subjects. * P  0.05. Table 1. Analysis of Different Reactivity Classes Obtained by rELISA and cELISA for Der p2-Specific IgE in Asthmatic Patients (SPT  and SPT   ) and Control Subjects GroupELISA reactivity class*Der p 2-specific IgE subjects(%)rELISA cELISA  SPT  asthma (N  48) 0 14 (29.2)† 23 (47.9)1 7 (14.6) 5 (10.4)2 7 (14.6) 10 (20.8)  3 20 (41.7)† 10 (20.8)SPT  asthma (N  41) 0 41 (100) 41 (100)Control (N  30) 0 30 (100) 30 (100)* Class 0 :   3.9 EU/mL (negative specific IgE); class 1 : 3.9 to   19.5 EU/mL (low positivespecific IgE); class 2 : 19.5 to   97.5 EU/mL (mild positive specific IgE); class    3 :    97.5EU/mL (high or very high positive specific IgE).†  P  0.05. VOLUME 86, MAY, 2001 549
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