maintenance dosing for sublingual immunotherapy …...have shown conclusive effective doses for...

Maintenance dosing for sublingual immunotherapyby prominent European allergen manufacturersexpressed in bioequivalent allergy unitsDésirée Larenas-Linnemann, MD*; Robert Esch, PhD†; Greg Plunkett, PhD‡; Shannon Brown, MS§;Daniel Maddox, MD¶; Charles Barnes, PhD�; and Derek Constable, PhD§

Background: Sublingual immunotherapy (SLIT) has become established in Europe, and its efficacy is being evaluated in theUnited States. The doses used for SLIT in Europe today are difficult to evaluate, because each manufacturer expresses thepotency of its extracts differently.

Objectives: To compare in vitro European SLIT maintenance solutions against US licensed standardized allergenic extractconcentrates and to determine the monthly SLIT doses delivered expressed in bioequivalent allergy units ([B]AU).

Methods: We studied Dermatophagoides pteronyssinus, timothy grass pollen, cat (hair) and short ragweed pollen allergenextracts. The SLIT maintenance solutions of 4 leading European manufacturers and standardized concentrate extracts of 3 USmanufacturers were analyzed with the following assays: protein content, relative potency (immunoglobulin E [IgE]-bindingenzyme-linked immunosorbent assay [ELISA] inhibition) and major allergen content. The relative monthly allergen dose in(B)AU was calculated for each recommended SLIT schedule.

Results: Relative potency was approximately 10 times higher for US concentrate standardized extracts—which are meant tobe diluted—than for European SLIT maintenance solutions of D pteronyssinus and timothy grass pollen. For cat (hair) and shortragweed pollen, the difference was less. Measurements of relative potency and major allergen content correlated well. In ourassays, European mite extracts contain a very low quantity of Der p 2 compared with US mites.

Conclusion: Recommended SLIT doses in Europe vary widely among the manufacturers, but are consistently lower (Eur1)or higher (Eur4) over all four allergens tested. SLIT efficacy probably depends on additional factors apart from the exact dose.SLIT dose finding studies should be done for each product.

Ann Allergy Asthma Immunol. 2011;xx:xxx.

INTRODUCTIONSubcutaneous immunotherapy (SCIT) in the United Statesconsists of the administration of aqueous allergen solutions,derived from glycerinated concentrate extracts, standardizedaccording to the Center for Biologics Evaluation and Re-search (CBER) of the Food and Drug Administration (FDA)guidelines. Dosing recommendations in the United States forSCIT1 are inferred from efficacy data mostly obtained fromtrials with European extracts,2-7 with the exception of thedosing recommendations for cat and short ragweed pollen,

for which the dosing is based on trials with US extracts. Theonly study published on SLIT using a US extract has beencarried out with a sublingual short ragweed pollen solution,based on the same glycerinated allergen extract used for thepreparation of SCIT.8

For immunotherapy in Europe, glycerinated products areadministered, just as in the United States, although extractsare standardized according to in-house-references of eachmanufacturer.9 In contrast to in the United States, however,for SCIT almost all extracts are adsorbed to a depot. This isnot the case for SLIT extracts, and as such European SLITsolutions bear a close resemblance to US concentrate ex-tracts, both containing a lyophilized allergen dissolved in50% glycerin. Many clinical trials have been conducted withEuropean SLIT solutions,10 and each manufacturer has partlybased the dosing of its SLIT products on these results.11,12

Several of these trials have been included in a recent updatedCochrane meta-analysis on SLIT.13

However, a universal potency measure14 to quantify theallergen quantity administered by each manufacturer’s solu-tion is lacking, and different designs of the many studiesobfuscate the drawing of straight dosing conclusions thatmight apply to all solutions.15 Some international guidelineshave given recommendations for SLIT doses in relation toSCIT doses.16 Lately, clinical trials with grass SLIT tablets

Affiliations: * Hospital Médica Sur, Mexico City, Mexico; † Greer Lab-oratories Inc. Lenoir, North Carolina; ‡ ALK-Abelló, Rockville, Maryland;§ Hollister-Stier Laboratories, Spokane, Washington; ¶ Mayo Clinic, Roch-ester, Minnesota; � Children’s Mercy Hospital, Kansas City, Kansas.

Disclosures: Drs. Esch, Plunkett, Brown, and Constable are employed byallergen manufacturers in the US. Dr. Larenas Linnemann has received travelgrants from ALK-Abelló, Alerquim and Stallergènes and speakers honorariafrom Schering Plough. Dr Maddox and Dr. Barnes have made no declara-tions.

Funding Sources: This project was supported by a grant from theAmerican Academy of Allergy Asthma and Immunology.

Received for publication March 8, 2011; Received in revised form June11, 2011; Accepted for publication July 6, 2011.

© 2011 American College of Allergy, Asthma & Immunology.Published by Elsevier Inc. All rights reserved.

doi:10.1016/j.anai.2011.07.001

VOLUME xx, MONTH, 2011
1

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have shown conclusive effective doses for adults17,18 andchildren,19,20 but how and whether these data can be extrap-olated to SLIT solutions is not clear.

In an ideal world, all laboratories would use the sameassays and reagents, generating reliable data on the concen-tration of major allergens in extracts. This would provide theinternational intellectual community with accurate informa-tion on dose efficacy for European SLIT products, and withthe many European SLIT trials conducted, making a clearstatement on how much US allergen extract should begiven daily to attain a probably effective SLIT dose shouldbe possible. However, reality is far from this ideal sce-nario.

Allergens are complex biological substances to which eachindividual can react in a different manner. Thus, not onelaboratory assay has been able to fully express all aspects ofextracts’ potency. As a consequence, the average result of theoutcomes of several in vitro tests might give the most com-plete picture. The CBER/FDA dictates which assays andreagents are suitable to assign (Bioequivalent) allergy units(BAU and AU) to the commercial batches released onto theAmerican market. For this study, various qualitative andquantitative assays were carried out, including the CBER/FDA tests.

The objective of the current study is to estimate themonthly dose given at SLIT maintenance with the products ofprominent European manufacturers, as expressed in US dos-ing units. As such, we decided to analyze European SLITproducts from several major European allergen manufactur-ers, with assays usually employed to determine the potency ofUS extracts, including those dictated by the CBER/FDA. Toestablish this direct potency comparison with the US stan-dardized extracts, analyzing laboratories were asked to runthe potency tests they normally run for batch release, simul-taneously, on all US and European extracts under investiga-tion.

METHODSStandardized sublingual maintenance extracts were pur-chased from 4 prominent European allergen manufacturers.Commercial standardized extracts including dust mite (Der-matophagoides pteronyssinus), timothy grass pollen (Phleumpratense), cat (hair) (Felis sp), and (short) ragweed pollen(Ambrosia artemisiifolia) were obtained from 3 US manufac-turers, and reference extracts of these 4 allergens were ac-quired from CBER/FDA (Table 1). Analysis took place in thelaboratories of the Mayo Clinic, the Children’s Mercy Hos-pital, CBER/FDA, ALK-Abelló Inc., Greer Laboratories, andHollister-Stier Laboratories. Each laboratory performed onlythose analyses that were already run routinely. Because theimprecision of the analyses goes beyond the minor lot-to-lotvariability of standardized extracts,21 only 1 lot of each wastested. Conversely, each assay was performed at least twice;
values reported in the tables and figures are mean results. c
2

rotein content (qualitative and quantitative)wo-dimensional gels were used to analyze the quality of thextracts showing the different proteins in 1 of the laborato-ies. The protein bands were visualized with Silver Stain. Theonditions for running these gels were optimized to demon-trate the entire range of protein content in the samples, ratherhan being optimized for the best separation of specific aller-ens. To adjust for the protein content, some extracts wereirst diluted. A 3,000-kDa cutoff dialysis membrane was usedo prepare the samples before electrophoresis. For detailedescription of the methods, see eMethods online.Methods to determine the total protein content of the

llergen extracts were: Coomassie Plus, a modified Bradfordssay (Thermo Fisher Scientific, Rockford, Illinois; labora-ory 1), Bradford assay (Thermo Fisher Scientific; laboratory), ninhydrin assay (laboratory 3), and Lowry assay (BCAssay Sigma Chemicals, Sigma-Aldrich, St. Louis, Missouri;

aboratory 4). All results are expressed in absolute (micro-rams per milliliter) and relative (% of highest) values. Bo-ine serum albumin was used as the standard. See e-Methodsor a detailed description of the methods.

elative Potency Testinghe relative potency was determined for D pteronyssinus and

imothy grass pollen extracts. Laboratories 1, 2, 3, and 5 usedompetition enzyme-linked immunosorbent assays (ELISAs)o measure the inhibition of allergen-specific immunoglobu-in E (IgE) binding with respect to current FDA references,sing a parallel line assay. Laboratory 4 performed ELISAnhibition with a kit from Indoor Biotechnologies (Charlot-esville, Virginia). The results were multiplied by 10,000 toield AU/mL for the D pteronyssinus extracts and by 100,000o give BAU/mL for the timothy grass pollen extracts.

ajor Allergen Contentor cat and short ragweed pollen extracts, only the amount ofajor allergen was examined. The major allergen contentas determined by a radial immunodiffusion assay in almost

ll laboratories, with the exception of laboratory 1, whichsed for cat the ALK-Abelló Fel d 1 ELISA and convertedhe micrograms per milliliter obtained into Fel d 1 units withhe following calculation: 1 �g Fel d 1 � 0.4 Fel d 1 units.

The major allergen content of D pteronyssinus and timothyrass pollen extracts were studied in 2 laboratories. Labora-ory 1 uses reagents from ALK (ALK-Abelló, Madrid,pain), and Laboratory 3 determined all microgram majorllergen data with the sandwich ELISA kits from Indooriotechnologies. For D pteronyssinus, standard curves used

n the CREATE project (full project name: Development ofertified Reference Materials for Allergenic Products andalidation of Methods for their Quantification) were appliedy laboratory 1. Laboratory 3 used reference standards in-luded with the kits from Indoor Biotechnologies (Der p 1tandard for mite, group 1 allergen, Phl p 5 standard forimothy grass extract, and a Universal Allergen Standard for
at and mite group 2 allergen). The concentration of Phl p 5
ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY

Table 1. Manufacturer, Allergen, Trade Name, and Concentration of the Allergen Extracts Analyzed in the Presented Assays

Extract D. pteronyssinus Timothy Cat Ragweed

ManufacturerTrade name,

allergenPotency

Trade name,allergen

PotencyTrade name,

allergenPotency

Trade name,allergen

Potency

CBER/FDAa ReferenceD. pteronyssinus

5,000 AU/mL Reference timothy 100,000 BAU/mL Reference cat hair 5,000 BAU/mL Referenceshort rag-weed

315 U/mL

ALK-USA Diagnostic con-centrate

D. pteronyssinus

5,000 AU/mL Diagnostic con-centrate timothy

10,000 BAU/mL Diagnostic con-centrate cat hair

5,000 BAU/mL Diagnosticconcentrateshort rag-weed

234 U/mL

Greer Diagnostic con-centrate

D. pteronyssinus

10,000 AU/mL Diagnostic con-centrate timothy

100,000 BAU/mL Diagnostic con-centrate cat hair


220 U/mL

Hollister-Stier Diagnostic con-centrate

D. pteronyssinus

10,000 AU/mL X X Diagnostic con-centrate

AP-Cat


264 U/mL

ALK-Euro SLIToneD. pteronyssinus

10,000 STU/mL SLITone timothy 1,000 STU/mL SLITone cat 200 STU/Dose SLITone shortragweed

1,000 ST/mL

Bencardc OralvacD. pteronyssinus

320,000 TU/mL Oralvac,B2-grass**

768,000 TU/mL Oralvac,Katze

32,000 TU/mL Oralvac, rag-weed

768,000 TU/mL

HALb SublivacD. pteronyssinus

10,000 AU/mL Sublivac grass 10,000 AU/mL SublivacKatzenepi,

10,000 AU/mL Sublivac shortragweed

10,000 NE/mL

Stallergènesc StaloralD. pteronyssinus

300 IR/mL Staloral timothy 300 IR/mL StaloralCat

100 IR/mL Staloral rag-weed

300 IR/mL

a Reference extracts of the FDA were E8-Dp for Dermatophagoides pteronyssinus, E8-Ti for timothy, E5 cat hair for cat, and E15-Ras for short ragweed.b B2-grass of Bencard is a mixture of several grasses.c According to product characteristic leaflet these extracts do not contain phenol as preservative.

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was calculated with the CREATE-Allergopharma referenceby laboratory 1 and the Indoor Biotechnologies reference bylaboratory 3.

Pearson correlations between the different assays werecalculated (R2) for each allergen, using the mean values of theresults per test.

Monthly SLIT Maintenance Dose Expressed in PotencyMeasurements Used in the United StatesThe relative monthly SLIT maintenance dose for each SLITextract was calculated based on 2 variables: the relativepotency found for each extract in our study (AU, BAU, Fel d1 U, and Amb a 1 U/mL for D pteronyssinus, timothy grass

Figure 1. SDS-PAGE of European SLIT maintenance extracts and US coand D, ragweed pollen.

pollen, cat, and short ragweed pollen extracts, respectively) s

4

nd the dosing recommendations of each manufacturer con-erning frequency and volume of extract given in each dose.e used the following calculation:‘relative potency (BAU/mL)’ � ‘volume of each dose

mL)’ � ‘number of doses per month’ � ‘monthly doseBAU)’

ESULTS

rotein Analysesn sodium dodecyl sulfate polyacrylamide gel electrophore-

is (SDS-PAGE) gels with silver staining, European and USxtracts show high similarity for timothy grass pollen and

es of A, Dermatophagoides pteronyssinus; B, Timothy grass pollen; C, cat;
ncentrat
hort ragweed pollen (Fig 1). The SDS-PAGE analysis for


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1. (Co

US extracts of D pteronyssinus and cat revealed a greaternumber of individual peptide bands, with the exception of theFDA cat hair extract, which showed fewer.

The protein content of US standardized extract concen-trates is generally higher than that of European SLIT main-tenance extracts. Strong variation exists among the differentmethods. However, the relative values for protein content(data not shown) when determined by the same methodcorrelate quite well with the other potency data (see below).The Lowry assay results were excluded, because they ap-peared to be affected by the phenol content present in most of

Figure

the samples. n


elative Potency Testing and Content of Major Allergensn Figure 2, the relative potency results of the extracts of the

allergens are shown. The recommended monthly subcuta-eous immunotherapy doses for the US standardized extractsre depicted as small bars to the right of the concentrates: forouse dust mite, 500 to 2,000 AU; timothy grass pollen,,000 to 4,000 BAU; cat, 1,000 to 4,000 BAU (�1.9–7.6 Fel1 U); and short ragweed pollen, 1,000 to 4,000 AU (6–12mb a 1 U).1 The relative potencies of the US D pteronys-

inus and timothy grass pollen extracts were more than 10imes higher than those of the corresponding SLIT mainte-

nt’d)

ance extracts from the 4 European manufacturers tested.

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Moreover, the slopes of the regression lines generated by theEuropean D pteronyssinus extracts were not parallel to thoseof the US D pteronyssinus extracts, suggesting that they werequalitatively different as well.

The radial immunodiffusion results for cat and short rag-weed pollen extract demonstrated higher potency for someEuropean extracts in comparison with others. The more po-tent European extracts of these 2 allergens were comparablein potency to the US standardized extract concentrates. For Dpteronyssinus and cat extracts, approximately 50% variabilitywas found between the US extracts.

Figure 3 shows the Der p 1 and Der p 2 content ofEuropean and US extracts, as analyzed by the 3 differentlaboratories. The mean content of the major allergens Der p

Figure

1, Der p 2, Phl p 5, and Fel d 1 can be found in Table 2. c

6

elative Monthly SLIT Maintenance Dose (Expressed asAU)

able 3 describes the total quantity of allergen given at SLITaintenance during 1 month of treatment, based on the

utcomes of the relative potency tests and the Europeananufacturers’ package insert recommendations on dosing.

orrelations between Potency Measurementsow the different methods of measuring allergen extractotency correlate with one another is shown in Table 4. In thepper part of the table, the results of protein content areorrelated with the relative potency measurements and theontent of major allergens. In the lower part of the table,

nt’d)

orrelations between the latter 2 are shown.


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DISCUSSIONTo our knowledge, this is the first report of US allergenextracts directly compared with European immunotherapysolutions in laboratory testing, carried out with the samemethods in the same laboratories. For the European extracts,we could not use SCIT solutions, because these containmodified depot extracts, so we chose the SLIT maintenancesolutions, because these contain natural allergens dissolved inglycerin, just as the US commercial extracts. This allowed usto compare their quality and potency and make a roughestimate of how much allergen is given at SLIT maintenanceby the different European manufacturers in terms of (bio-equivalent) allergy units. In one of our previous studies, USand European diagnostic extracts were compared, showing in

Figure

general that US diagnostic extracts of house dust mite, cat, a


nd Bermuda grass pollen are at least twice as potent asuropean extracts when tested in vitro.22 Another group of

nvestigators studied laboratory characteristics and potency ofuropean timothy pollen extracts, demonstrating that potencyaried considerably among products from different allergenanufacturers.23 Mösges et al.24 compared SLIT maintenance

olutions of 2 major European allergen manufacturers inuantitative skin prick testing, again showing significant po-ency differences.24

elative Dosing of SLIT by the Four Europeananufacturers Studiedhe SLIT maintenance extracts are less potent than the USoncentrates, because the latter are meant to be diluted before

nt’d)

dministration. When extracts are compared, what really is of

7

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ose, 1/2

interest is the dose that is actually delivered to the patient. Assuch, in Table 3 we compare the maintenance monthly SCITdose recommended in the latest update of the Practice Pa-rameters on Immunotherapy1 with the maintenance monthlySLIT dose that is given with the different products, followingthe manufacturers’ recommendations. A major difference canbe seen between the maintenance SLIT doses given by the 4European manufacturers studied here. This difference ishighly consistent over the 4 allergens we analyzed: D ptero-nyssinus, timothy grass pollen, cat hair, and short ragweedpollen, with Europe 1 giving low dose SLIT and Europe 4

Figure 2. A, Relative potency (AU/mL) of European SLIT maintenance eas measured with ELISA inhibition in several laboratories (FDA/CBER10,000AU/mL US extracts, is marked in dark gray. B, Relative potency (BBAU/mL) of timothy grass pollen, as measured with ELISA inhibition inmaintenance dose, 1/50th of the 100,000BAU/mL US extracts, is marked inextracts and US concentrates (10,000BAU/mL) of cat, as measured with radsubcutaneous monthly maintenance dose, 1/5th of the 10,000BAU/mL US exSLIT maintenance extracts and US concentrates (10,000BAU/mL) of short(FDA/CBER method). Recommended subcutaneous monthly maintenance d

high dose SLIT. m

8

Sublingual immunotherapy dosing has been addressed ineveral previous publications, and in the past it has been 1 ofhe points raised against this form of immunotherapy, be-ause no clear dosing guidelines existed.10,25 However, that aelatively high dose is needed for SLIT to be effective isenerally accepted. In the first Allergic Rhinitis and its im-act on Asthma (ARIA) guidelines, dosing of 50 to 100 timeshe SCIT dose was recommended for SLIT,16 although in theatest ARIA update,26 no absolute numbers are given any-ore. The World Allergy Organization consensus paper on

ublingual immunotherapy mentions the probable effective

and US concentrates (10,000AU/mL) of Dermatophagoides pteronyssinus,). Recommended subcutaneous monthly maintenance dose, 1/10th of theL) of European SLIT maintenance extracts and US concentrates (100,000laboratories (FDA/CBER method). Recommended subcutaneous monthly

ay. C, Relative potency (Fel d 1 Units/mL) of European SLIT maintenanceunodiffusion in several laboratories (FDA/CBER method). Recommended

s marked in dark gray. D, Relative potency (Amb a 1 Units/mL) of Europeand pollen, as measured with radial immunodiffusion in several laboratories8th of the 255 Units/mL US extracts, is marked in dark gray.

xtractsmethod

AU/mseveraldark grial imm

tracts, iragwee

onthly dose for timothy grass pollen SLIT to be approxi-


fraAg11ao

PTmpELdilreto

REaradrmtor

CApttal

Ti(

mately 600 �g of the major allergen Phl p 5.27 However, theoptimal dose has not yet been firmly established for allextracts. Since 2006, convincing data have been generated forsome pollen extracts that leave no doubt that the greatestimprovement is seen only in the high-dose groups. Almost allof these latter studies are done with tablets.17–20,28,29 One of thefew exceptions was the dose-finding SLIT study in childrenof Valovirta et al,30 which used a liquid solution of tree pollenextract. Recently, a US sublingual standardized glycerinatedshort ragweed pollen allergenic extract containing 4.8, or 48�g of Amb a 18 (approximately 10 and 100 times, respec-tively, of the recommended SCIT monthly dose) adminis-tered daily in rhinitis patients showed only in the high-dosegroup both a 15% reduction in nasal symptom score and astatistically significant reduction of 51% in the medicationscore during the peak pollen season. The final analysis of ourstudy indicates that high dosing in SLIT is not universallyapplied in Europe at this moment.

Quality of the ExtractsThe data derived from the analysis of the quality of theextracts is also of importance for subcutaneous immunother-apy, as has been previously discussed by Esch.31 To give only1 example, for the US house dust mite (HDM), dosing rec-ommendations are based on studies with European extracts,as discussed in the introduction. Extrapolating doses forHDM like this is problematic, because our study demon-strates notable qualitative differences.

We analyzed US concentrate and SLIT maintenance ex-tracts with various specific and nonspecific laboratory assays.For HDM, European extracts seem to be different in compo-sition, as indicated by the SDS-PAGE gels (Fig 1) and by thenonparallelism in the relative potency tests (ELISAs). This isconfirmed by the specific tests that determine the content ofthe 2 major allergens, Der p 1 and Der p 2, with the latterlacking almost completely in most European extracts. For cat

Figure 3. Content of major allergens Der p 1 and Der p 2 (�g/mL) ofEuropean SLIT maintenance extracts and US concentrates (10,000AU/mL)of Dermatophagoides pteronyssinus, as assayed with several techniques.* The FDA extract was only analyzed by laboratories 2 and 3.

extracts, differences exist between solutions that are extracted


rom cat hair or cat epithelium. For timothy grass and shortagweed pollen, the European and US extracts are more alike,ccording to the gels and the parallelism in the ELISA lines.lso, for these 2 extracts, the different analyzing laboratoriesave potency test results within a smaller range. Even thoughof the European solutions was extracted from the pollen of

2 grasses and not just from timothy grass pollen, it showedremarkably similar SDS-PAGE protein band pattern to the

ther extracts.

rotein Analysishe absolute protein content of extracts is a poor measure-ent of potency. Moreover, we came across some specific

roblems: phenol, used as a preservative in all US and someuropean extracts (Table 1), gives falsely high readings in theowry assay, and ninhydrin is such a sensitive method that itetects even protein molecules, too small to be immunolog-cally active, resulting in falsely high readings when nondia-yzed extracts are compared with dialyzed ones. However, theelative protein content—expressed as percentage of a leadxtract assigned a 100% value—bears a quite close relationo the specific potency measurements, especially for the tim-thy grass pollen and cat extracts (Table 4).

elative Potency Tests and Content of Major Allergensven though the reference sera and curves varied between thenalyzing laboratories, quite consistent data are shown by theelative potency tests (Fig 2) and the determination of majorllergens (Table 3). Evaluation of the performance of theifferent assay methods (Table 4) indicates that a good cor-elation can be found between the relative potency and theajor allergen contents. For the D pteronyssinus extracts,

he best correlation coefficient is generated when the sumf the Der p 1 and Der p 2 content is plotted against theelative potency (AU/mL).

ONCLUSIONSs can be deduced from these published results of the relativeotency testing of several European SLIT maintenance solu-ions, the quantity of allergen given during maintenance SLITreatment in Europe varies considerably among the differentllergen manufacturers. However, apart from the precise al-ergen quantity delivered to the patient, other details may

able 2. Major allergen content of European sublingualmmunotherapy maintenance extracts and US concentrate extracts�g/mL)

Manufacturers Der p 1 Der p 2 Phl p 5 Fel d 1

Eur1 2.8 0.0 0.04 4.5Eur2 29.3 0.5 14.7 0.3Eur3 8.1 1.2 48.1 16.9Eur4 24.0 7.7 65.4 21.0US1 53.1 51.8 398.5 34.7US2 66.2 36.6 513.7 55.3US3 25.2 12.1 NP 40.6
FDA 34.9 35.0 645.7 22.0
9

bi

AJWy

SSi

R

1

short ra

influence the efficacy of SLIT: the volume in which the doseis given (concentration), the stickiness of the solution (muco-adhesive substances are under investigation32), and other ad-ditives that might influence the reaction of the immune sys-tem. Furthermore, higher doses of SLIT lead also to a higherfrequency of side effects, which might be different in differ-ent populations,18,33–35 and the compositions of US and Euro-pean extracts vary, as we showed here specifically for housedust mite extracts. Therefore, the only way to truly prove theefficacy–safety balance of a sublingual allergen extract is totest each individual product in a clinical trial. Some SLITtrials are being undertaken in the United States, and majorchanges are underway in Europe, where the regulatory agen-cies are starting to require the manufacturers to show efficacyof immunotherapy extracts in clinical trials.36,37 Most of theEuropean SLIT products had been registered until now aspatient-named products. All these developments will proba-

Table 4. Correlations between different methods of measuringpotency of an allergen extract in vitro

Protein (mean %) vs relative potencymeasurementa

Protein (mean %) vs Dpt AU/mL R2 � 0.763 (0.647)Protein (mean %) vs Timothy BAU/mL R2 � 0.971 (0.953)Protein (mean %) vs Fel d 1 Units/mL R2 � 0.772 (0.562)Protein (mean %) vs Amb a 1 Units/mL R2 � 0.697 (0.695)

Protein (mean %) vs major allergencontenta

Protein (mean %) vs Der p 1 �g/mL R2 � 0.676 (0.540)Protein (mean %) vs Der p 2 �g/mL R2 � 0.483 (0.392)Protein (mean %) vs Der p 1 � 2 �g/mL R2 � 0.629 (0.506)Protein (mean %) vs Phl p 5 �g/mL R2 � 0.881 (0.856)Protein (mean %) vs Fel d 1 �g/mL R2 � 0.836 (0.66)

Major allergen content vs relative potencytest values

Der p 1 vs Dpt AU/mL R2 � 0.751Der p 2 vs Dpt AU/mL R2 � 0.899Der p 1�2 vs Dpt AU/mL R2 � 0.895Phl p 5 �g/mL vs BAU R2 � 0.864Fel d 1 �g/mL vs Units/mL R2 � 0.979

a For the calculation of correlations protein results from laboratory 3for Eur3 were not taken into account, as they represented strong

Table 3. Cumulative monthly doses of SLIT maintenance therapy of

Manu-facturers D pteronyssinus (AU)a Tim

Eur1 Daily: 35Monthly: 1,050

DailyMon


DailyMon


DailyMon


DailyMon

a As a reference: monthly probably effective doses recommended in1,000–4,000BAU, cat 1,000–4,000 BAU (� 1.9–7.6 Fel d 1 U), and

outliers (uncorrected values are given in brackets).

10

ly lead to more specific dosing indications for allergenmmunotherapy products.

CKNOWLEDGMENTSay E. Slater, MD, reviewed the first draft of this manuscript.

e also thank other reviewers, who preferred to stay anon-mous.

UPPLEMENTARY DATAupplementary data associated with this article can be found,

n the online version, at doi: 10.1016/j.anai.2011.07.001.

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3. Ewan PW, Alexander MM, Snape C, Ind PW, Agrell B, Dreborg S.Effective hyposensitization in allergic rhinitis using a potent partiallypurified extract of house dust mite. Clin Allergy. 1988;18:501–508.

4. Haugaard L, Dahl R, Jacobsen L. A controlled dose-response study ofimmunotherapy with standardized, partially purified extract of housedust mite: clinical efficacy and side effects. J Allergy Clin Immunol.1993;91:709–722.

5. Olsen OT, Larsen KR, Jacobsan L, Svendsen UG. A 1-year, placebo-controlled, double-blind house-dust-mite immunotherapy study in asth-matic adults. Allergy. 1997;52:853–859.

6. Frew AJ, Powell RJ, Corrigan CJ, Durham SR. Efficacy and safety ofspecific immunotherapy with SQ allergen extract in treatment-resistantseasonal allergic rhinoconjunctivitis. J Allergy Clin Immunol. 2006;117:319–325.

7. Walker SM, Pajno GB, Lima MT, Wilson DR, Durham SR. Grass pollenimmunotherapy for seasonal rhinitis and asthma: a randomized, con-trolled trial. J Allergy Clin Immunol. 2001;107:87–93.

8. Leynadier F, Banoun L, Dollois B, et al. Immunotherapy with a calciumphosphate-adsorbed five-grass-pollen extract in seasonalrhinoconjunctivitis: a double-blind, placebo-controlled study. Clin Exp Al-lergy. 2001 Jul;31(7):988–96.

9. Skoner D, Gentile D, Bush R, Fasano MB, McLaughlin A, Esch RE.Sublingual immunotherapy in patients with allergic rhinoconjunctivitiscaused by ragweed pollen. J Allergy Clin Immunol. 2010;125:660–666,6 e1-6 e4.

0. Larenas-Linnemann D, Cox LS. European allergen extract units andpotency: review of available information. Ann Allergy Asthma Immunol.

ominent European manufacturers

BAU) Cat (BAU) Short ragweed (AU)

900Daily: 170Monthly: 5,100

Daily: 420Monthly: 12,600

,000Daily: 85Monthly: 2,550

Daily: 3,550Monthly: 106,500

,000Daily: 1,100Monthly: 33,000


9,000Daily: 2,600Monthly: 78,000


r SCIT are for house dust mite 500–2,000AU, timothy grass pollengweed pollen 1,000–4,000AU (6–12 Amb a 1 U).(1)

four pr

othy (

: 130thly: 3,: 1,400thly: 42: 3,800thly: 54: 6,300thly: 18

US fo

2008;100:137–145.


2

2

2

2

3

3

3

3

3

3

3

3

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RDHPCD1

11. Cox LS, Larenas Linnemann D, Nolte H, Weldon D, Finegold I, NelsonHS. Sublingual immunotherapy: a comprehensive review. J Allergy ClinImmunol. 2006;117:1021–1035.

12. Pfaar O, Klimek L. Efficacy and safety of specific immunotherapy witha high-dose sublingual grass pollen preparation: a double-blind, placebo-controlled trial. Ann Allergy Asthma Immunol. 2008;100:256–263.

13. Pham-Thi N, Scheinmann P, Fadel R, Combebias A, Andre C. Assess-ment of sublingual immunotherapy efficacy in children with house dustmite-induced allergic asthma optimally controlled by pharmacologictreatment and mite-avoidance measures. Pediatr Allergy Immunol. 2007;18:47–57.

14. Radulovic S, Calderon MA, Wilson D, Durham S. Sublingual immuno-therapy for allergic rhinitis. Cochrane Database Syst Rev. 2010;12:CD002893.

15. van Ree R, Chapman MD, Ferreira F, Vieths S, Bryan D, Cromwell O,et al. The CREATE project: development of certified reference materialsfor allergenic products and validation of methods for their quantification.Allergy. 2008;63:310–326.

16. Bousquet J, Van Cauwenberge P, Khaltaev N. Allergic rhinitis and itsimpact on asthma. J Allergy Clin Immunol. 2001;108(5 Suppl):S147–334.

17. Durham SR, Yang WH, Pedersen MR, Johansen N, Rak S. Sublingualimmunotherapy with once-daily grass allergen tablets: a randomizedcontrolled trial in seasonal allergic rhinoconjunctivitis. J Allergy ClinImmunol. 2006;117:802–809.

18. Didier A, Malling HJ, Worm M, Horak F, Jager S, Montagut A, et al.Optimal dose, efficacy, and safety of once-daily sublingual immunother-apy with a 5-grass pollen tablet for seasonal allergic rhinitis. J AllergyClin Immunol. 2007;120:1338–1345.

19. Wahn U, Tabar A, Kuna P, Halken S, Montagut A, de Beaumont O, etal. Efficacy and safety of 5-grass-pollen sublingual immunotherapytablets in pediatric allergic rhinoconjunctivitis. J Allergy Clin Immunol.2009;123:160–6 e3.

20. Bufe A, Eberle P, Franke-Beckmann E, et al. Safety and efficacy inchildren of an SQ-standardized grass allergen tablet for sublingualimmunotherapy. J Allergy Clin Immunol. 2009;123:167–73 e7.

21. Pagani M, Antico A, Cilia M, et al. Comparison of different diagnosticproducts for skin prick testing. Eur Ann Allergy Clin Immunol. 2009;41:23–31.

22. Larenas-Linnemann D, Esch RE, Guidos-Fogelbach G, Rodriguez-PerezN. A comparison of in vitro potency between European and Mexicanallergen extracts and US (CBER/FDA) reference extracts. Allergol Im-munopathol (Madr). 2010;Mar 23.

23. Sander I, Fleischer C, Meurer U, Bruning T, Raulf-Heimsoth M. Aller-gen content of grass pollen preparations for skin prick testing andsublingual immunotherapy. Allergy. 2009; Apr 6.

24. Mosges R, Pasch N, Schlierenkamper U, Lehmacher W. Comparison ofthe biological activity of the most common sublingual allergen solutionsmade by two European manufacturers. Int Arch Allergy Immunol. 2006;139:325–329.

25. Roder E, Berger MY, de Groot H, van Wijk RG. Immunotherapy in


6. Bousquet J, Khaltaev N, Cruz AA, et al. Allergic Rhinitis and its Impacton Asthma (ARIA) 2008 update (in collaboration with the World HealthOrganization, GA(2)LEN and AllerGen). Allergy. 2008;63(Suppl 86):8–160.

7. Canonica GW, Bousquet J, Casale T, Lockey RF, Baena-Cagnani CE,Pawanker R, et al. Sub-lingual immunotherapy: World Allergy Organi-zation position paper 2009. Allergy. 2009;64(suppl 91):1–59.

8. Nelson HS, Nolte H, Creticos P, Maloney J, Wu J, Bernstein DI.Efficacy and safety of timothy grass allergy immunotherapy tablettreatment in North American adults. J Allergy Clin Immunol. 2011;127:72–80, e1-2.

9. Blaiss M, Maloney J, Nolte H, Gawchik S, Yao R, Skoner DP. Efficacyand safety of timothy grass allergy immunotherapy tablets in NorthAmerican children and adolescents. J Allergy Clin Immunol. 2011;127:64–71, e1-4.

0. Valovirta E, Jacobsen L, Ljorring C, Koivikko A, Savolainen J. Clinicalefficacy and safety of sublingual immunotherapy with tree pollen extractin children. Allergy. 2006;61:1177–1183.

1. Esch RE. Evaluation of allergen vaccine potency. Curr Allergy AsthmaRep. 2006;6:402–406.

2. Saint-Lu N, Tourdot S, Razafindratsita A, Mascarell L, Berjont N,Chabre H, et al. Targeting the allergen to oral dendritic cells withmucoadhesive chitosan particles enhances tolerance induction. Allergy.2009;64:1003–1013.

3. Kleine-Tebbe J, Ribel M, Herold DA. Safety of a SQ-standardised grassallergen tablet for sublingual immunotherapy: a randomized, placebo-controlled trial. Allergy. 2006;61:181–184.

4. Larsen TH, Poulsen LK, Melac M, Combebias A, Andre C, Malling HJ.Safety and tolerability of grass pollen tablets in sublingualimmunotherapy: a phase-1 study. Allergy. 2006;61:1173–1176.

5. Esch RE, Bush RK, Peden D, Lockey RF. Sublingual-oral administra-tion of standardized allergenic extracts: phase 1 safety and dosingresults. Ann Allergy Asthma Immunol. 2008;100:475–481.

6. Bousquet J, Schünemann HJ, Bousquet PJ, et al. How to design andevaluate randomized controlled trials in immunotherapy for allergicrhinitis: an ARIA-GA(2) LEN statement. Allergy. 2011; Apr 18. doi:10.1111/j.1398-9995.2011.02590.x. [Epub ahead of print]

7. Eichler I, Sala Soriano E. Close collaboration between academia, indus-try and drug regulators is required in the development of allergenproducts for specific immunotherapy in children. Allergy. 2011; Mar 23.DOI: 10.1111/j.1398-9995.2011.02582.x [Epub ahead of print]

8. Blum LJ, Gautier SM, Coulet PR. Design of luminescence photobiosen-sors. J Biolumin Chemilumin. 1989;4:543–550.

equests for reprints should be sent to:ésirée Larenas-Linnemann, MD, FAAAAI, Dist. Intl. FACAAIospital Médica Sur, Torre 2, cons.602uente de Piedra 150ol. Toriello Guerrael. Tlalpan4050 México D.F., México
children and adolescents with allergic rhinoconjunctivitis: a systematic
review. Pediatr Allergy Immunol. 2008;19:197–207. E-mail: [email protected]

11

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PROTEIN CONTENT: SDS-PAGE PROTEINPROFILESA volume equivalent to 50 �g (or all of the sample if proteinlevel was undetectable by assay) of buffer-exchanged samplewas dried down by speed vac, and resolubilized in 200 �L of2D sample buffer (9M urea, 4% CHAPS detergent, 30 mMdithiothreitol [DTT], 1.25% ampholytes). An 11-cm 3–10 pHimmobilized pH gradient isoelectric focusing strip (Bio-RadLaboratories, Hercules, California) was rehydrated for 11hours with each sample solution, and proteins were focusedfor 32,000 Vh. The strips were frozen at -80°C until ready forSDS-PAGE.

After thawing, the strips were equilibrated in 20% glycerol,36% urea, 2% SDS, 62.5 mM Tris/HCl pH 8.8 with 1% DTTfor 10 minutes (to be sure proteins are reduced), followedwith 2% iodoacetamide (for alkylation of reduced proteins toprevent reoxidation) in the same buffer for 15 minutes. Stripswere then positioned on top of 10.5–14% Criterion Tris/HClprecast SDS-PAGE gels (Bio-Rad Labs) and sealed in placewith 1% melted agarose in running buffer. Precision proteinstandards (Bio-Rad Labs; 0.5 �L) was added to the singlewell on the acidic side of the strips. Gels were electropho-resed for 1 hour at 200 V, 15°C. After fixing overnight in50% methanol/ 10% acetic acid, gels were washed and silverstained according to a modified method of Blum et al.38 Staindevelopment time was the same within each allergen group,except that those samples with low protein load (lower thandetection limit of protein assay) were allowed to develop untilbackground started to darken to see as many spots as possi-ble. Gels were scanned using PDQuest software and a GS800Densitometer (Bio-Rad Labs). The pH gradient of the imagedgels is from left (acidic; pH 3) to right (basic; pH 10).

PROTEIN CONTENT, QUANTITATIVELY:DETAILS OF METHODSLaboratory 1: Coomassie Plus (Pierce modified Bradfordassay)

Laboratory 2: Bradford assay (Thermo Fisher Scientific)with bovine serum albumin as the standard

Laboratory 3: Ninhydrin Assay for Total Protein. Testsamples and diluted bovine serum albumin standards thatcontain proteins are hydrolyzed to amino acids using 10 Msodium hydroxide and heat. Excess base is neutralized bythe addition of concentrated hydrochloric acid. With theapplication of heat and a Ninhydrin solution (combinationof methylcellusolve, ninhydrin, citric acid, and stannouschloride), the amino acids react with Ninhydrin in a redoxreaction to form a purple compound, which is quantifiedcolorimetrically at 570 nm.

Laboratory 4: Lowry (BCA Assay Sigma Chemicals)

RELATIVE POTENCY TESTING: DETAILS OF THEMETHODS FOR ELISA INHIBITIONLaboratory 1: Same Methods Used as in Laboratory 2. For
the coating of the plates, FDA reference extracts were used of
11.e1

pteronyssinus and Timothy grass pollen for the respectivenalyses.

Laboratory 2: For specific IgE competition ELISAs, ref-rence D pteronyssinus or Timothy grass pollen extract wassed to coat microplate wells (Costar, Lowell, MA) at ap-roximately 2 �g/mL in carbonate coating buffer pH 9.6.hreefold serial dilutions of the test and reference extractsere prepared in phosphate buffer containing 0.05% Tween0 (PBS-T), mixed with an equal volume of a 1:15 dilution ofhe reference human allergic serum pool, and added in du-licate to the respective allergen-coated microplate wells.fter incubation at room temperature for 4 to 6 hours andashing the wells with PBS-T, the specific IgE bound wasetected by overnight incubation of the wells with biotinyl-ted anti-human IgE (Kirkegaard & Perry Laboratories,aithersburg, Maryland), followed by washing in PBS-T and

lkaline-phosphatase–labeled avidin (Zymed Laboratories,an Francisco, California). After washing the plate and theells in PBS-T, 1 mg/mL para-nitrophenyl phosphate sub-

trate (Amresco, Solon, Ohio) was added to each well, andhe absorbance was measured at 405 nm using a plate reader.he percent inhibition values were calculated for each sampleilution using the formula (Ap–As/Ap) � 100, where Ap ishe average positive control (no inhibitor) and As is theverage sample absorbance value. The best-fit dose–responseines containing at least 4 dilutions between 10% and 90%nhibition and bracketing 50% inhibition are constructed us-ng linear regression analysis: y � a � bx, where y is theercent inhibition value, a is the y-intercept, b is the slope,nd x is the logarithm to the base 3 of the dose. The best-fitarallel lines are computed using the combined data for theest and respective reference samples. For a valid assay, theorrelation coefficient for both the reference and test extractsust have been 0.95 or greater. Parallelism of the reference

nd test regression lines was checked using Student’s t-test,nd the lines had to be parallel at P � .01. The log3 relativeotency (W) was calculated using the following formula:

� (It–Ir)/B, where It and Ir are the test and reference-intercepts calculated from the parallel lines, and B is theooled slope. Three independent valid assays on the testntigen were used to calculate the relative potency (3w). Theeference extract for D pteronyssinus had a potency of 10,000U/mL, and for Timothy grass pollen, 100,000 BAU/ml.Laboratory 3: ELISA relative potency (Based on a CBER

ethod). Extracts at various dilutions compete with immobi-ized allergens on microtiter plates for specific IgE obtainedrom several allergic donors. Bound IgE is determined usingoat anti-IgE coupled to horseradish peroxidase and the en-yme substrate system TMB–hydrogen peroxidase. Uninhib-ted IgE produces the greatest color, and as more allergennhibitor is mixed with the specific IgE, less color is gener-ted. Potency of a sample is determined by comparing theegression line of the dose–response produced by the sampleith that of a reference extract (ie, parallel-line bioassayethod). Reference extract and sera are supplied by CBER.
e-Table 1 lists the key parameters for each ELISA.

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LLm

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e-Table 1.

Timothy grass Mite, D pter

Plate coating E8-Ti (100,000 BAU/mL), 1:600

E8-Dp (10,000 AU/mL)1:500

Reference E8-Ti (100,000 BAU/mL) E8-Dp (10,000 AU/mL)Serum S5-GR S5-Dpf

Laboratory 4: did not perform.Laboratory 5: ELISA based on a CBER method for D ptero-nyssinus and timothy.

RELATIVE POTENCY TESTING: DETAILS OF THEMETHODS FOR RADIAL IMMUNODIFFUSIONLaboratory 1: For SRagweed, the current FDA Amb a 1 RIDwas used. For cat, the major allergen content was determinedusing ALK-Abelló Fel d 1 ELISA, and the Fel d 1 Units werecalculated: 1 Unit � 2.5 �g/mL Fel d 1. Laboratory 2: Forradial immunodiffusion assays, dilutions of cat and SRag-weed allergenic extracts were added to 3-mm-diameter wells,in quadruplicate, cut into 1% agarose plates containing ref-erence anti-Fel d 1 serum. After incubating the plates forapproximately 48 hours in a humidified chamber, the preci-pitin circles were visualized by dipping the plates in a 10%acetic acid solution for approximately 2 minutes. Alterna-tively, the precipitins were stained after exhaustive rinsing ofthe plates in distilled water followed by silver staining. Thediameters of the precipitin circles were measured to thenearest 0.1 mm. The standard curves were generated usingthe labeled values of the Fel d 1 reference dilutions and theaverage diameter from quadruplicate wells. From these val-ues, the best-fit regression line was calculated using theformula y � a [log(x)] � b, where y is average diameter inmm, x is the labeled value of the specific antigen for thatpreparation in Fel d 1 units/mL, a is the slope, and b is they-intercept. The correlation coefficient of the regression linemust be greater than or equal to 0.9 for a valid assay.The average diameters of the of the test extract samples werecalculated, and the Fel d 1 content was computed from thebest fit regression line. Laboratory 3: Cat: (Based on a CBERmethod) This assay measures the quantity of Fel d 1 and Amba 1 in allergenic extracts using radial immunodiffusion. Forcat, a dose–response curve is determined from the diametersof precipitated circles in agar. Four dilutions of a referencepreparation and a specific antiserum, which are provided byCBER, are used to generate the reference curve. From thesedata, a best-fit regression line is calculated. From the diam-eter of the circle produced by a test preparation, the concen-tration of Fel d 1 is calculated using the best-fit regression


ine of the standard dose–response curve. SRagweed poly-lonal antisera, specific for Amb a 1, is mixed with dissolvedgar and poured on a glass microscope slide or RID plate toolidify. The wells are then made in the agar, and the samplextract is added. The Amb a 1 will diffuse from the wells andnteract with the polyclonal antibodies to form a precipitateing. The diameter of this ring is directly related to theoncentration of the Amb a 1 in the applied sample extracthen compared with reference standards applied in the sameanner. e-Table 2 lists the key parameters for the assay.

e-Table 2.

Lot number Supporting information

Reference-Cat C11-Cat Provided by CBER; 5, 10,15, 20 units/mL.

Antisera-Cat S-2b Provided by CBER.Reference-SRW* C15-Ras Provided by CBER; 5, 10,

20, 30 units/mL.Antisera-SRW RS9478 Internal preparation of burro

serum.

RW, short ragweed.

aboratory 4: not performed.aboratory 5: radial immunodiffusion with the CBER recom-ended method for cat and short ragweed pollen.

ETERMINATION OF MAJOR ALLERGENONTENT: DETAILS OF THE METHODSaboratory 1: For the determination of de content of Der p 1nd Der p 2, laboratory 1 used the ALK-Abelló ELISA;imilarly, for Timothy and cat, the ALK-Abelló Phl p 5 andel d 1 ELISAs were used.Laboratory 3: Assay kits for the determination of major

llergens were purchased from Indoor Biotechnologies, Inc.Charlottesville, VA). The assays are all direct sandwichLISAs. In brief, monoclonal antibodies (mAb), which haveeen raised against the allergen of interest, are immobilizedn microtiter plates. Antigen (in the reference or in samples)s added and binds to the captured mAb. A secondary, bio-inylated mAb is added and binds to the immobilized antigen.treptavidin-peroxidase and ABTS (with hydrogen peroxide)re used as the detection system. The responses of the sam-les are compared with a standard curve to determine theajor allergen content of the sample. Four assay kits were

sed to determine the major allergen content of three extractypes (Dpt, Timothy, and cat). e-Table 3 lists the key param-ters for each ELISA. Neat samples were diluted so that thebserved major allergen value was within the working rangef the reference standards.

11.e2

e-Table 3.

ExtractAllergen

Mite, D pter Timothy grasspollenPhl p 5

CatFel d 1Der p 1 Der p 2

ELISA kit item no. &batch no.

EL-DP12905

EL-DP230032

EL-PP530016

EL-FD130007

Coating (capture)antibody

Anti-Der p 1 mAbItem No. MA-5H8Lot No. 2760

Anti-Group 2 mAbItem No. MA-1D8Lot No. 2760

Anti-Phl p 5 mAbItem No. MA-1D11Lot No. 6001916

Anti-Fel d 1 mAbItem No. MA-6F9Lot No. 29021

Reference Standard Der p 1 Standard2,500 ng/mLItem No. ST-DP1Lot No. 2901

Universal Allergen Standard2,500 ng/mLItem No. ST-UASLot No. 30006

Phl p 5 Standard5,000 ng/mLItem No. ST-PP5Lot No. 29083

Universal Allergen Standard1,300 ng/mLItem No. ST-UASLot No. 30006

Reference range 0.5–250 ng/mL 0.5–250 ng/mL 1–500 ng/mL 0.25–1300 ng/mLSecondary antibody Biotinylated anti-Mite

Group 1 mAbItem No. B1-4C1Lot No. 2761

Biotinylated anti-Mite Group2 mAb

Item No. B1-7A1Lot No. 2837

Biotinylated anti-Phlp 5 mAb

Item No. B1-B01Lot No. 29082

Biotinylated anti-Fel d 1mAb

Item No. B1-3E4Lot No. 29059

Detection system Streptavidin-peroxidase and ABTS with H2O2

11.e3 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY

maintenance dosing for sublingual immunotherapy …...have shown conclusive effective doses for...

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