Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

Veterinary Immunology and Immunopathology 92 (2003) 45±60

Monoclonal anti-equine IgE antibodies with speci®city for different epitopes on the immunoglobulin heavy chain of native IgE Bettina Wagnera,*, Andreas Radbruchb, Jens Rohwera, Wolfgang Leibolda a

Immunology Unit, Hannover School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hannover, Germany b German Rheumatism Research Center, Schumann Street 21/22, 10117 Berlin, Germany Received 23 October 2002; received in revised form 19 December 2002; accepted 19 December 2002

Abstract In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine VH186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine l1 chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8d, expressing the same heavy chain variable regions and light chains. One of the mAbs (aIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000 Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (aIgE-176) detected a protein of 76,000 Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE speci®c mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no signi®cant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population …1:3  0:5%† of leukocytes from adult Icelandic horses by surface immuno¯uorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Horse IgE; Monoclonal antibodies; IgE heavy chain epitopes; Summer eczema; Icelandic horse; IgE quanti®cation

Abbreviations: IGHC gene; immunoglobulin constant heavy chain gene; IGHE gene; IgE constant heavy chain gene; IGHG gene; IgG constant heavy chain gene; ME; 2-mercaptoethanol; NIP; 4-(hydroxy-3-iodo-5-nitro-phenyl)acetyl; NP; 4-(hydroxy-3-nitro-phenyl)acetyl * Corresponding author. Present address: James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Road, Ithaca, NY 14853, USA. Tel.: ‡1-607-256-5615; fax: ‡1-607-256-5608. E-mail address: [email protected] (B. Wagner). 0165-2427/03/$ ± see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-2427(03)00007-2

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B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

1. Introduction IgE antibodies play an important role in the host defense against parasites (Hagan et al., 1991; Capron et al., 1992) and are involved in type I hypersensitivity responses (Ishizaka et al., 1966). Both the immune reaction against parasites and the hypersensitivity reaction against antigens like pollens are mediated by IgE antibodies, which are bound to immune effector cells via high af®nity IgE receptors (FceRI). In human serum, IgE is detectable at concentrations in the range of nanograms per milliliter, and has a short half-life of approximately 2 days. Due to its high af®nity to the FceRI, IgE is bound to the a chain of the FceRI on mast cells and basophils, resulting in sensitization of these cells (for review: Ravetch and Kinet, 1991). In contrast to the IgG±FcgR interaction, which occurs after formation of IgG-antigen complexes, the mast cell/basophil sensitization requires no previous antigen binding of the IgE. Cross-linking of FceRI-bound IgE by antigen leads to receptor aggregation and induces an intracellular signaling cascade (for review: Turner and Kinet, 1999), resulting in release of preformed mediators of in¯ammation, e.g. histamine, synthesis of lipid mediators, like leukotrienes, and production of cytokines (for review: Wedemeyer et al., 2000). These mediators initiate the allergic symptoms and both, directly and indirectly, stimulate the production of antigen speci®c IgE on the level of T- and B-lymphocytes. In horses, the role of equine IgE has been investigated in a few studies, suggesting that IgE might be involved in parasite speci®c immune reactions (Suter and Fey, 1981) as well as in equine diseases like summer eczema, heaves (recurrent airway obstruction), urticaria or head shaking (Matthews et al., 1983; Larsen et al., 1988; Schmallenbach et al., 1998), which resemble type I hypersensitivities of other species. However, investigations of these studies have been hampered speci®cally by dif®culties in detecting equine IgE. Polyclonal antisera against horse IgE have been described before (Suter and Fey, 1981; Halliwell and Hines, 1985; Watson et al., 1997; Marti et al., 1997), but no equine IgE speci®c monoclonal antibodies (mAbs) have been available thus far. Molecular studies of the number and organization of equine immunoglobulin constant region genes have

identi®ed one IGHE gene per haploid genome (Wagner et al., 1997, 1998). Nucleotide sequencing of equine IGHE genes has identi®ed at least four different IGHE alleles (Navarro et al., 1995; Watson et al., 1997; Wagner et al., 2001). The deduced amino acid sequences of equine IgE allotypes range in homology from 45 to 56% to murine and human IgE, respectively, and up to 66% for canine IgE (Navarro et al., 1995; Wagner et al., 2001). Probably due to the low amino acid homologies shared between different species, cross-reactive antibodies speci®c for human or murine IgE and equine IgE have not been described. The unavailability of puri®ed, monoclonal or recombinant equine IgE is one of the reasons, that no monoclonal antibodies speci®c for equine IgE have been generated. Here, we describe the generation of murine mAbs speci®c for equine IgE, using a recombinant equine IgE (rIgE). This rIgE contains entire equine IgE heavy chain constant regions, murine heavy chain variable regions and murine light chains. This rIgE has been shown to have a high structural and functional homology to native equine IgE and has been immunoaf®nity puri®ed using its antigen speci®city to 4-(hydroxy-3-nitro-phenyl)acetyl (NP) (Wagner et al., 2002a). A new designation of the IgG isotypes of the horse has been proposed at the immunoglobulin and Fcreceptor Workshop of the Sixth International Veterinary Immunology Symposium (IVIS) in Uppsala, Sweden in 2001 (http://www.medicine.uiowa.edu/ CigW). Here, we use this new IgG nomenclature, which is based upon the recently provided nucleotide sequences of the six IGHG genes and their deduced immunoglobulin gamma heavy chains (Wagner et al., 2002b). A comparison of the original nomenclature of the ®ve equine IgGs recognized by earlier serological and biochemical characterizations and the new designation of the six IgG isotypes, according to their molecular analysis is shown in Table 1. 2. Material and methods 2.1. Recombinant equine IgE The equine rIgE contained the entire equine IgE heavy chain constant region, fused to the murine

B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

47

VH186.2 heavy chain variable region and expressed together with murine l1 light chains in J558L cells. It has been expressed as a highly glycosylated immunoglobulin (Ig) monomer with speci®city for 4-(hydroxy-3-nitro-phenyl)acetyl. The antigen speci®city was used for immunoaf®nity puri®cation of the rIgE, using a NP coupled CnBr-activated sepharose 4B column as previously described (Wagner et al., 2002a).

later for anti-IgE antibodies. After 2 weeks of cell culture, HAT-supplement was replaced by HT-supplement (Sigma, Steinheim, FRG). Following an additional 3±4 weeks, the cells were transferred into Hybridoma SFM medium, supplemented with 10% (v/v) Myclone FKS, 100 IU/ml penicillin and 100 mg/ ml streptomycin. Cultures which produced anti-IgE antibodies were cloned twice by limiting dilution to obtain monoclonal cell lines.

2.2. Immunization and cell fusion

2.3. Detection of anti-IgE mAbs by enzyme linked immunosorbent assay (ELISA)

Female BALB/C mice were intraperitoneally immunized with rIgE. The immunization was performed on day 0 (2.5 mg rIgE), day 14 and day 21 (1.25 mg rIgE each) in Gerbu Adjuvanz MM (Gerbu Biotechnik, Gaiberg, FRG) according to manufacturer's instructions. The adjuvant contained cell wall components of Lactobacillus bulgaricus and other immunomodulating substances. Additional boosters of 1.25 mg rIgE (without adjuvant) in phosphate buffered saline (PBS; Biochrom, Berlin, FRG) were given on days 28, 29 and 30. The cell fusion was performed on day 31. The murine spleen cells were carefully isolated, resuspended in Hybridoma SFM medium (Life Technologies, Karlsruhe, FRG) and mixed with murine X63-Ag8.653 myeloma cells (Kearney et al., 1979) in a 1:2 ratio. After centrifugation (100  g, 5 min), the supernatant was removed, the cells were carefully resuspended and slowly mixed with 1.5 ml of prewarmed (37 8C) polyethylene glycol 1500 (Roche, Mannheim, FRG). After 1 min of incubation at 37 8C, a total of 20 ml of Hybridoma SFM medium was added dropwise (1 ml in the ®rst minute, 3 ml in the second minute, 16 ml in third minute). Then, the cells were spun down, the supernatant was removed and 200 ml of Hybridoma SFM medium supplemented with HAT-media supplement (Sigma, Steinheim, FRG), 10% (v/v) Myclone FKS (Life Technologies, Karlsruhe, FRG), 100 IU/ml penicillin, 100 mg/ml streptomycin (PAN Biotech, Aidenbach, FRG) and 4 U/ml human recombinant IL6 (BioConcept, Umkirch, FRG) were carefully added. The cell suspension was then plated out in 24-well cell culture plates (Biochrom, Berlin, FRG). After 7±10 days, single clones were picked and cultivated in 96-well cell culture plates (Biochrom, Berlin, FRG). The supernatants of these clones were tested 2±3 days

Monoclonal antibodies recognizing the rIgE were detected in cell culture supernatants by ELISA: microtiter plates (Nunc, Wiesbaden, FRG) were coated with the NP derivate 4-(hydroxy-3-iodo-5-nitro-phenyl)acetyl (NIP) coupled to BSA (NIP15-BSA; Biosearch Technologies, Navato, CA, USA) in a ®nal concentration of 5 mg/ml in carbonate buffer (15 mmol Na2CO3, 35 mmol NaHCO3, pH 9.6) and incubated overnight at 4 8C. Plates were washed ®ve times with phosphate buffer (2.5 mmol NaH2PO4, 7.5 mmol Na2HPO4, 145 mmol NaCl, 0.1% (v/v) Tween 20, pH 7.2). Cell culture supernatants, containing the NP-speci®c rIgE, were diluted 1:4 in phosphate buffer and incubated Table 1 Original nomenclature and new designationa of the immunoglobulins of the horse, and their corresponding heavy chain constant (IGHC) genesb Original New nomenclature designation

Corresponding GenBank accession IGHC gene number

IgM IgGa

IgM IgG1 IgG2

IGHM IGHG1 IGHG2

L49414 AJ302055 AJ302056

IgG(T) IgGb IgG(T)

IgG3 IgG4 IgG5 IgG6

IGHG3 IGHG4 IGHG5 IGHG6

AJ312379 AJ302057 AJ312380 AJ312381

IgE IgA

IgE IgA

IGHE IGHA

AJ305046 Not yet available

a

IgGc and IgG(B) have not yet been assigned to the new nomenclature, which was proposed at the Immunoglobulin and Fcreceptor Workshop of the Sixth IVIS in Uppsala, Sweden in 2001 (http://www.medicine.uiowa.edu/CigW). b The IGHC nomenclature conforms to the of®cial nomenclature of the international ImMunoGeneTics database (IMGT) (http:// imgt.cines.fr).

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B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

for 1 h on a plate rotator at room temperature. After ®ve washes (see above) plates were ®lled with the cell culture supernatants, containing the anti-IgE mAbs diluted 1:10 in phosphate buffer, and incubated for 1 h. Plates were washed as described above, peroxidase conjugated goat a mouse IgG …H ‡ L† (Dianova, Hamburg, FRG) was added at a dilution of 1:20,000 in phosphate buffer and incubated for 45 min. Plates were washed and ®lled with substrate buffer (33.3 mmol citric acid, 66.7 mmol NaH2PO4, pH 5.0), supplemented with 130 mg/ml 3,30 ,5,50 -tetramethylbenzidine (TMB, Sigma, Steinheim, FRG) and 0.01% (v/v) hydrogen peroxide (Sigma, Steinheim, FRG). Substrate solution was incubated for 20 min in the dark and the reaction was stopped by adding one volume of 0.5 mol H2SO4. Plates were evaluated in an automatic microplate reader (Dynatech, Denkendorf, FRG) at 450 and 630 nm absorbance. In order to identify mAbs that recognize the murine components of the rIgE, the supernatant from the cell line B1-8d, containing NP-speci®c murine IgD (Neuberger and Rajewsky, 1981) was used instead of the NP-speci®c rIgE. The murine NP-speci®c IgD contained the same heavy chain variable regions and light chains as the rIgE. Anti-idiotypic and l light chain speci®c mAbs, which bound to IgD expressed in B1-8d were excluded from this study. 2.4. Isotyping, puri®cation and biotinylation of anti-IgE mAbs The murine isotypes of the anti-IgE mAbs were determined by ELISA using mouse monoclonal antibody isotyping reagents for murine IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA (Sigma, Steinheim, FRG). For puri®cation of aIgE-134, the cell line was cultivated in serum-free Hybridoma SFM medium. The mAb (murine IgG1) was puri®ed from the cell culture supernatant using a protein G sepharose column (Amersham Pharmacia, Uppsala, Sweden). Antibody elution was performed with 0.1 mol glycine±HCl (pH 3.0), which was subsequently collected in 1 mol Tris±HCl (pH 7.0) for neutralization. This solution, containing the puri®ed aIgE-134 mAbs, was dialyzed overnight against PBS (4 8C) and concentratedusing an Ultrafree 15 concentrator, cut off 100.000 Da (Millipore, Eschborn, FRG). The protein concentration of the enriched solution was determined

using the BCA-assay (Pierce, Rockford, IL, USA). For biotinylation, mAbs were diluted to a ®nal concentration of 1 mg/ml and coupled to NHS-biotin (Pierce, Rockford, IL, USA) as described (MuÈller, 1992). 2.5. Competitive ELISA A competitive ELISA was performed using the biotinylated aIgE-134 mAb to identify anti-IgE mAbs recognizing different epitopes on the IgE heavy chain. ELISA plates were coated with NIP15-BSA and NPspeci®c rIgE as described above. Plates were washed and the different anti-IgE supernatants (1:2), the unconjugated puri®ed aIgE-134 mAb (10 mg/ml), as well as phosphate buffer and a supernatant, containing no anti-IgE, for negative controls were incubated for 1 h. After another washing step the plate was ®lled with the biotinylated aIgE-134 mAb diluted 1:4000 in phosphate buffer and incubated for 40 min. Plates were washed and streptavidin-peroxidase (Amersham Pharmacia, Uppsala, Sweden) diluted 1:30,000 in phosphate buffer was added and incubated for 30 min. The following substrate and evaluation steps were as described above. 2.6. Equine isotype speci®c ELISA The ELISA to determine equine Ig isotypes (Wagner et al., 1998) was based on a detection system using different equine isotype speci®c mAbs (Lunn et al., 1998; Sheoran et al., 1998). Here, this assay was modi®ed using the different anti-IgE mAbs. In brief: the ELISA plates were coated with goat a horse IgG …H ‡ L† antibodies (Dianova, Hamburg, FRG). This af®nity matrix then bound the monoclonal equine IgM, IgG1, IgG3, IgG4 (Table 1) or Ig light chains, present in the culture supernatants of equi-murine heterohybridomas (Wagner et al., 1995) or in equine serum (1:10,000). Plates were then incubated with anti-IgE mAbs, which were ®nally detected with peroxidase conjugated goat a mouse IgG …H ‡ L† and hydrogen peroxide/TMB substrate solution as described above. The equine Ig content was con®rmed in the supernatant of heterohybridomas and serum samples in an additional reaction using a peroxidase conjugated polyclonal goat a horse IgG …H ‡ L† detection antibody as described before (Wagner et al., 1995).

B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

2.7. Quantitative detection of equine serum IgE by ELISA For the determination of equine serum IgE two antiIgE mAbs, recognizing different IgE heavy chain epitopes were used. Equine sera were obtained from female Icelandic horses with and without summer eczema, as well as ®ve Icelandic horse foals, 5±6 months in age. To detect equine serum IgE, the ELISA plates were coated using the aIgE-176 mAb (10 mg/ ml). Equine sera were used in a two-fold dilution from 1:200 to 1:25,600. For quanti®cation of the total serum IgE concentrations, puri®ed IgE was used as standard (see below). In the next two steps, the plates were incubated with the biotinylated aIgE-134 mAb and streptavidin coupled peroxidase (Amersham Pharmacia, Uppsala, Sweden). Substrate solution, buffers and washing steps were the same as described above. 2.8. Puri®cation of equine IgE from serum The puri®ed aIgE-134 mAb was coupled to CnBractivated sepharose 4B (Amersham Pharmacia, Uppsala, Sweden) according to manufacturer's instructions. Equine serum was diluted 1:50 in PBS and applied to a protein G column to remove IgG isotypes. The IgG-depleted serum was then puri®ed using the anti-IgE af®nity column. Following passage of the serum, the column was washed twice with PBS. Equine IgE was eluted, dialyzed and concentrated as described above and the protein content was determined by BCA-assay. All serum fractions obtained before and after separations were tested for the amount of different equine Ig isotypes using the equine isotype speci®c ELISA as well as SDS-PAGE. The IgE concentration of the eluted serum fraction was measured by ELISA using the rIgE as a reference. The concentration of the rIgE was determined according to B1-8d expressed IgD of a known concentration by ELISA. Both antibodies were bound to a NIP-BSA af®nity matrix and detected by anti-mouse l light chain antibodies as previously described (Wagner et al., 2002a). 2.9. SDS-PAGE SDS-PAGE, Western blotting and immunoblotting were performed as described (Wagner et al., 1995). In

49

brief, equine sera were separated in 7.5% polyacrylamide gels, with or without 2-mercaptoethanol (ME). Gels were either stained with Coomassie Brilliant Blue or blotted. After Western blotting and one blocking step with 1% (w/v) gelatine for 20 min, the blotting membranes (PVDF, Millipore, Eschborn, FRG) were incubated with anti-IgE for 90 min, using culture supernatants diluted 1:2. Membranes were washed three times with PBS, containing 0.02% (v/v) Tween 20 and incubated with an alkaline phosphatase conjugated goat a mouse-IgG …H ‡ L† antibody (Dianova, Hamburg, FRG). After an additional three washing steps the membranes were stained with alkaline phosphatase substrate solution. 2.10. Surface staining of equine blood leukocytes and ¯ow cytometric analysis IgE surface staining was performed on blood leukocytes of 33 adult Icelandic horses with and without clinical signs of summer eczema and eight healthy foals. The cells were isolated from the leucocyte enriched plasma fraction of spontaneous sedimented heparinized equine blood as described before (Wagner et al., 2002a). A total of 5  106 cells were stained with the various anti-IgE mAbs each, diluted 1:2 in PBS/BSA (PBS, containing 0.5% (w/v) BSA and 0.02% (w/v) NaN3) for 10 min on ice. For isotype controls the cells were incubated with an irrelevant murine monoclonal IgG1 antibody in an additional tube. The cells were washed once with cold PBS/BSA and incubated for 5 min on ice with a phycoerythrin conjugated goat a mouse IgG …H ‡ L† (Dianova, Hamburg, FRG) diluted 1:100 in PBS/BSA. After an additional washing step, dead cells were stained with propidium iodide and analyzed by ¯ow cytometry (FACS-Scan, Becton Dickinson, Heidelberg, FRG). 2.11. Magnetic cell sorting of equine IgE‡ cells Magnetic cell sorting was performed using the MACS technology (Miltenyi Biotec, Bergisch Gladbach, FRG). All staining and cell sorting was performed on ice or at 4 8C, respectively: a total of 5  108 equine leukocytes were stained with aIgE134 using culture supernatant diluted 1:2 in PBS/BSA (®nal volume of 500 ml) and incubated for 15 min.

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B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

Cells were washed once with PBS/BSA and subsequently incubated with 100 ml of rat a-mouse IgG1MicroBeads (Miltenyi Biotec, Bergisch Gladbach, FRG) diluted 1:10 in PBS/BSA. After 10 min of incubation, 400 ml of the phycoerythrin conjugated goat a mouse IgG …H ‡ L† antibody (1:100) were added to the cell suspension, and incubated for additional 5 min. Then, the cells were washed and resuspended in 5 ml PBS/BSA. A LS-column (capacity of 1  108 bound cells) was placed in the magnetic ®eld of a MidiMACS (Miltenyi Biotec, Bergisch Gladbach, FRG) and equilibrated with 5 ml PBS/BSA. To remove cell aggregates and to obtain a single cell suspension, the stained cells ®rst passed a 30 mm nylon preseparation ®lter (Miltenyi Biotec, Bergisch Gladbach, FRG) before entering the column. The ¯ow rate of the stained cell suspension was controlled by a 20 g needle. The ¯owthrough fraction was collected. The LS-column was washed two times with 5 ml of PBS/BSA to remove negative cells. Then, the column was removed from the magnetic ®eld, the 20 g needle was removed, and the IgE‡ cells were eluted with 5 ml PBS/BSA using a plunger. All fractions were tested by ¯ow cytometry to measure the number of IgE‡ cells. The ®rst magnetic cell sorting procedure resulted in a positive fraction of approximately 30% equine IgE‡ leukocytes. This pre-enriched fraction was applied to a MS-column using a MiniMACS and further enriched in IgE‡ cells as described above. Cytospins of the enriched IgE‡ cell aliquots were stained using Accustain, a modi®ed Wright staining reagent (Sigma, Steinheim, FRG). 3. Results 3.1. Equine IgE speci®c monoclonal antibodies recognize various epitopes of the equine rIgE heavy chain region The anti-IgE mAbs were obtained by cell fusion of X63-Ag8.653 cells and spleen cells from a BALB/C mouse immunized with rIgE. A total of 17 cell lines, which produced mAbs speci®c for the rIgE were identi®ed by ELISA. All of these anti-IgE mAbs recognized epitopes on the equine rIgE heavy chain constant region (Table 2), but they failed to react with

murine IgD, containing variable heavy chain domains and l1 light chains, which were identical to those of the rIgE (Fig. 1a). Both, the murine IgD and the rIgE contained identical NP-speci®c antigen binding sites and differed in their heavy chain constant regions, only. To identify anti-IgE mAbs with speci®city for different epitopes on the IgE heavy chain, aIgE-134 was puri®ed from the culture supernatant and biotinylated. Supernatants of all anti-IgE mAbs were then tested for their ability to inhibit the binding of biotinylated aIgE-134 to rIgE. For reference, the binding of biotinylated aIgE-134 was inhibited both by puri®ed unconjugated aIgE-134 (10 mg/ml) and by the supernatant of the aIgE-134 cell line (Fig. 1b). The binding of the biotinylated aIgE-134 mAb was also inhibited by 13 of the other anti-IgE mAbs. In contrast, aIgE-41, aIgE-132 and aIgE-176 did not inhibit the binding of the biotinylated aIgE-134 mAb to rIgE. Thus, these latter mabs de®ne one or more epitopes on rIgE, different from the one recognized by aIgE-134. 3.2. The anti-IgE mAbs do not recognize equine IgM, IgG or IgA antibodies The anti-IgE mAbs were tested for reactivity with other equine Ig isotypes by ELISA. Equine antibodies representing various isotypes (IgM, IgG1 (for previous designation see Table 1), IgG3, IgG4 or equine light chains) were obtained from heterohybridomas and bound to the plate, which was coated before with a polyclonal goat a horse antibody. The anti-IgE mAbs were tested for their ability to bind the different equine Igs. To con®rm the presence of equine Ig in the heterohybridoma supernatants, a polyclonal goat a horse antibody was used. Although all equine isotypes were readily detectable with the polyclonal goat a horse IgG …H ‡ L† antibody, equine IgM, IgG1, IgG3, IgG4 or equine light chains were not detected by any of the anti-IgE mAbs. This is shown in Fig. 2a for the mAbs aIgE-134 and aIgE-176 and is summarized in Table 2 for all of the anti-IgE mAbs. In addition to testing the supernatants of the heterohybridomas which contained a single equine isotype each, we also tested the ability of the anti-IgE mAbs to recognize equine serum antibodies. In the ELISA used here, the coating antibody (goat a horse IgG …H ‡ L†)

B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

bound different serum Igs, depending upon their isotype and their relative concentration in the serum. According to the equine isotype speci®c ELISA described previously which based on the same coating antibody (Wagner et al., 1998), the highest detection signals were obtained for IgG antibodies, which were bound by both, heavy and light chain speci®c antibodies (Wagner et al., 1995). In particular, IgG1 and IgG4, which represent the main IgG serum isotypes of the horse, were bound to the goat a horse IgG …H ‡ L† af®nity matrix. Other serum IgG's, like IgG3 and IgGc, or IgM and IgA, were bound to the af®nity matrix via light chain epitopes (Fig. 2b). Serum antibodies of any of these isotypes were not detected in this assay by the various anti-IgE mAbs (Fig. 2a), con®rming that they also show no cross-reactivity with equine IgGc or IgA, which are not yet available as pure reference proteins. The completely negative signal which we obtained by the use of anti-IgE mAbs for detection of Ig from various equine sera, also indicated that IgE is present at low concentration in serum if at all. In the assay used, the af®nity matrix could have been blocked by antibodies from other classes, if those had been present in excess of IgE in the serum. 3.3. The anti-IgE mabs detect a serum immunoglobulin by immunoblotting Sera from ®ve horses affected with summer eczema, a disease with signs typical of an allergic type I hypersensitivity, and from three healthy control animals, were used to demonstrate the ability of the anti-IgE mAbs to detect native IgE. The sera were separated by SDS-PAGE under unreduced conditions and transferred to PVDF membranes by Western blotting. The immunoblot was incubated using aIgE-134 (Fig. 3a). A protein of approximately 210,000 Da could be detected in four out of ®ve sera from summer eczema affected horses and in one of the three control sera. The 210,000 Da serum protein in all likelihood represents the unreduced native IgE. A horse serum, containing a detectable amount of the suggested native IgE was separated by SDS-PAGE under reducing and non-reducing conditions. The ability of the different anti-IgE mAbs to detect the IgE was compared by immunoblotting. All monoclo-

51

nal antibodies, except the aIgE-132 mAb, recognized the non-reduced IgE of 210,000 Da, comparable to the aIgE-134 mAb (Table 2). In addition, the aIgE-176 mAb detected a protein of approximately 76,000 Da under reduced conditions (Fig. 3b), corresponding to the expected molecular weight of the equine IgE heavy chain (Wagner et al., 2002a). The result con®rmed that the anti-IgE mAbs, except aIgE-132, recognize an equine Ig of the expected molecular weight of IgE. It further con®rmed the differences we had observed for the epitopes recognized on rIgE by the aIgE-132, aIgE-134 and aIgE-176 mAbs by the competitive ELISA (see above). The aIgE-132 mAb detected only rIgE, but not native IgE. The epitope recognized by aIgE-176 is detectable on the whole putative IgE as well as on reduced IgE heavy chains, while all other 15 anti-IgE mAbs recognized epitopes by immunoblotting which are only detectable on unreduced IgE molecules. 3.4. Puri®cation of serum immunoglobulin by aIgE-134 Due to its ability to detect an immunoglobulin molecule of the expected molecular size of IgE in equine serum, the aIgE-134 mAb was coupled to CnBr-activated sepharose 4B. The serum was puri®ed in two steps: ®rst, using a protein G column for depletion of IgG isotypes and second, using the anti-IgE af®nity column to purify IgE from the IgG-depleted serum. Aliquots of the different serum fractions obtained were separated by SDS-PAGE (Fig. 4a) and their Ig content was determined by isotype speci®c ELISA. After passing the protein G column, a distinct depletion of serum IgG was observed (Fig. 4a, lane 2). The determination of isotypes by ELISA revealed, that IgG1 and IgG4, but not IgG3 were bound to the protein G column (data not shown). The fractions eluted from the protein G column contained IgG1 and IgG4 (pH 3.0; Fig. 4a, lane 3) or IgG4 alone (pH 2.5; Fig. 4a, lane 4). Accordingly, IgG1 and IgG4 were no longer detectable in the IgG-depleted serum by ELISA. The IgGdepleted serum fraction was applied to the anti-IgE af®nity column. After elution, no protein was found in the gel stained with Coomassie Blue (Fig. 4a, lane 6), although the IgE was detectable in the highly diluted eluate by ELISA. Thus, the eluate was concentrated

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B. Wagner et al. / Veterinary Immunology and Immunopathology 92 (2003) 45±60

Table 2 Characterization of monoclonal anti-equine IgE antibodies aIgE-

Detection of recombinant IgE by ELISAa

Characterization of IgE heavy chain epitopes by competitive ELISA 6ˆaIgE-134e

Cross-reactivity with other equine isotypesb

Detection of serum IgE by immunoblottingc

Detection of IgE‡ leukocytes by flow cytometryd

ˆaIgE-134f

Non-reducing

‡

‡ ‡

‡ ‡

‡ ‡ ‡

‡ ‡ ‡

‡ ‡ ‡

‡

‡

‡

‡

‡

‡

‡ ‡

‡ ‡ ‡

‡ ‡ ‡

Reducing

28 41

‡ ‡

43 112 119

‡ ‡ ‡

120 132 134

‡ ‡ ‡

150 151 176

‡ ‡ ‡

336 521 522

‡ ‡ ‡

‡ ‡ ‡

‡ ‡ ‡

‡ ‡ ‡

580 594 672

‡ ‡ ‡

‡ ‡ ‡

‡ ‡ ‡

‡ ‡ ‡

‡

‡

‡

‡

Positive: (‡); negative: ( ). a The anti-IgE mabs detected the recombinant NP-speci®c equi-murine IgE, but not murine NP-speci®c IgD. The murine IgD contained identical VH186.2 regions and l1 light chains like the recombinant IgE and differed only in the constant heavy chain region, con®rming that, the epitopes recognized by the anti-IgE mabs are located on the equine constant heavy chain region of the recombinant IgE. b Tested for equine IgM, IgG1, IgG3, IgG4, IgGc, IgA and equine light chains. c Detectable in sera of adult horses with high IgE concentrations. d Detectable on cells of adult horses. e The anti-IgE mab did not inhibit the binding of the aIgE-134 to the recombinant IgE heavy chain. f The anti-IgE mab inhibited the binding of the aIgE-134 to the recombinant IgE heavy chain.

(50- to 100-fold) and a distinct enrichment of the putative serum IgE was observed (Fig. 4a, lane 7). Aliquots of the concentrated fraction, which contained the putative IgE were compared by ELISA before and after heat treatment at 54 8C for 10 min. A signi®cant reduction …P < 0:01† of detectable IgE was observed for the heat treated sample, in which only 58% of the original IgE content could be measured (Fig. 4b). According to the equine isotype speci®c ELISA, low amounts of IgM and IgA were also detectable in this IgE enriched fraction (data not shown). The putative IgE content of the concentrated solution was determined using the equine rIgE for standardization. The IgE puri®cation was performed from sera of seven different horses with all seven yielding comparable results.

3.5. Quanti®cation of equine serum IgE with two anti-IgE mAbs recognizing different epitopes Serum IgE was quanti®ed by ELISA, using aIgE176 and aIgE-134 which recognize different epitopes of equine IgE for coating and detection. Puri®ed equine IgE was used to standardize the quanti®cation of IgE. To con®rm the speci®city for IgE we tested the different supernatants of heterohybridomas which contained monoclonal equine IgM and IgG isotypes (see above) in this assay. None of these equine isotypes could be detected by the ELISA based on aIgE176 and aIgE-134 (data not shown). Sera were obtained from 15 female Icelandic horses (aged between 5 and 10 years) with clinical symptoms of a summer eczema at the time of sampling, 11

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Fig. 1. Speci®city of various anti-IgE mabs for equine rIgE: (a) monoclonal antibodies, recognizing the equine rIgE were identi®ed by ELISA. The rIgE contains equine IgE heavy chain constant regions, fused to the murine VH186.2 variable regions and a murine l1 light chains. Murine IgD antibodies, expressed by the cell line B1-8d contain identical variable heavy chain regions and light chains. Both the equine rIgE and the murine IgD expressed by B1-8d are speci®c for the hapten 4-(hydroxy-3-nitro-phenyl)acetyl. The ability to detect equine rIgE (NPIgE, ®lled symbols) or murine IgD (NP-IgD, open symbols) was compared for aIgE-134 (circles), aIgE-132 (squares) and aIgE-176 (triangles). The IgD concentration was initially tested with an anti l light chain antibody; (b) some of the anti-IgE mAbs inhibited the additional binding of aIgE-134 to rIgE. A competitive ELISA was used to differentiate the epitopes on the equine rIgE heavy chain region, which were recognized by 17 anti-IgE mabs. For the detection of rIgE, supernatants which contained the various anti-IgE mAbs (aIgEsupernatants) were used. The aIgE-134 supernatant (aIgE-134 sup) and the puri®ed aIgE-134 mAb (pur. aIgE-134) [10 mg/ml] were used for positive controls. Negative controls consisted of phosphate buffer (buffer) and a control supernatant (sup. contr.) which lacked anti-IgE mAbs. The binding of the biotinylated aIgE-134 antibody is shown, which could bound in the negative controls or in well incubated with aIgE-41, aIgE-132 and aIgE-176 because the recognition epitope on the rIgE heavy chain was still accessible.

female control horses (aged between 2 and 9 years) without clinical signs and ®ve foals, aged between 5 and 6 months. The serum concentrations of IgE were compared in Fig. 5 for these three groups. In adult horses the serum concentration of IgE varied widely and was not signi®cant different between the groups of

allergic and healthy animals. For horses with summer eczema, serum IgE levels were 108:9  69:0 mg/ml …mean  S:D:†. Horses without clinical symptoms had serum IgE concentrations of 84:0  90:9 mg/ml. Interestingly, no IgE could be detected in the serum of any of the ®ve Icelandic horse foals.

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Fig. 2. The reactivity of anti-IgE mAbs with equine immunoglobulin isotypes other than IgE was tested by ELISA: (a) the pure equine isotypes IgM, IgG1, IgG3, IgG4 and equine light chains, obtained from supernatants of equi-murine heterohybridomas, as well as equine serum antibodies were not detectable with aIgE-134 and aIgE-176. The immunoglobulin content of all heterohybridoma supernatants and serum was con®rmed using a goat a horse IgG …H ‡ L† antibody [gah-IgG …H ‡ L†] instead of anti-IgE; (b) the equine serum used in (a) was tested for its content of various equine IgM, IgG and IgA antibodies.

3.6. Characterization of equine peripheral blood leukocytes recognized by anti-IgE mabs To obtain further evidence for the speci®city of the anti-IgE mAbs for native equine IgE, we used the mAbs to isolate and characterize equine leukocytes that bound IgE via Fce-receptors. Equine leukocytes were isolated from peripheral blood of Icelandic horses with and without summer eczema, stained for surface IgE and analyzed by ¯ow cytometry. An irrelevant murine IgG1 antibody was used as isotype control in the ¯ow cytometric analysis since all antiIgE mAbs are of the murine IgG1 subclass. Using aIgE-134, a small population of IgE‡ cells …1:3  0:5%† was found in adult horses (Fig. 6), which

differed distinctly from the isotype control …0:02  0:02%†. In addition, a highly signi®cant difference …P < 0:001† was observed between the IgE‡ leukocytes of adult horses and foals, in which only 0:1  0:06% of IgE‡ cells were detectable. Comparison of the frequencies of IgE‡ cells from adult horses with and without summer eczema showed no signi®cant difference between both groups. According to ¯ow cytometric phenotyping, the equine IgE‡ cells were granulocyte as well as large lymphocytes or monocytes (Fig. 7a). All anti-IgE mAbs were used for surface staining of equine leukocytes and detected IgE‡ populations similar to those detected by aIgE-134, except for the aIgE-132 mAb, which did not detect any equine leukocytes (Table 2).

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Fig. 3. Immunoblots to detect native IgE from horse serum using the anti-IgE mAbs: (a) equine sera were separated in by SDS-PAGE in a 7.5% gel under unreduced conditions and transferred to PVDF-membranes by Western blotting. Immunoblotting was performed using the aIgE-134 mAb. The sera were obtained from Icelandic horses with summer eczema (1±5) and from healthy control animals (6±8); (b) a serum, which contained a detectable amount of the suggested native IgE was separated in 10% gels under reduced (‡ME) and unreduced conditions ( ME). The proteins were detected by immunoblotting using aIgE-134 and aIgE-176. H2L2: unreduced IgE monomer, containing two heavy and light chains; H: IgE heavy chain.

To characterize the cells which were recognized by aIgE-134 surface staining in more detail, the IgE‡ leukocytes of an adult horse were enriched by two steps magnetic cell sorting. Around 1.5% IgE‡ cells could be detected before magnetic cell sorting (Fig. 7a). The ®rst sorting enriched the IgE‡ cells to about 30% in the positive fraction, but a distinct population of unstained cells was still detectable after elution from the column. After a second positive enrichment of this fraction, the IgE‡ cells were enriched to 85.2% (Fig. 7b). According to forward and side scatter (FSC/SSC) the enriched IgE‡ cells were predominately located in the large granulocyte and in the monocyte or large lymphocyte population. Aliquots of the two steps enriched IgE‡ cells were spun down on slides, stained by Wright and the cells were characterized by microscopy (Fig. 7c). The enriched IgE‡ equine leukocytes contained a total of 32%

basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils. The population of small lymphocytes could also be found in the ¯ow cytometric analysis of the enriched IgE‡ cells. They represent a cell population which is excluded from the IgE‡ gate in Fig. 7b, although they could be identi®ed as weakly IgE‡ cells compared to the negative granulocyte and lymphocyte populations (Fig. 7a). 4. Discussion The lack of monoclonal anti-equine IgE antibodies has severely hampered detailed investigations of the role of IgE in equine medicine and immunology. To overcome this situation, we have recently generated an equi-murine rIgE, and expressed it in the mammalian cell line J558L. The rIgE contains the entire equine

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Fig. 4. Puri®cation of putative IgE from equine serum: (a) the puri®cation was performed in two steps, ®rst using a protein G column to deplete serum IgG and second an IgE-speci®c af®nity column to purify IgE from the IgG-depleted serum fraction. The IgE-speci®c af®nity column was generated using aIgE-134. The different serum fractions were separated in a 7.5% non-reducing SDS-PAGE and stained with Coomassie Brilliant Blue: (1) equine serum before puri®cation; (2) IgG depleted serum; (3) protein G elution, pH 3.0; (4) protein G elution, pH 2.5; (5) ¯ow through antiIgE column; (6) elution anti-IgE column; (7) concentrated eluate, containing the putative equine IgE; (b) an aliquot of the concentrated IgE fraction obtained in (a) was heat treated at 54 8C for 10 min and the detectable IgE content was compared to that of the untreated sample by ELISA.

IgE heavy chain constant region, murine VH186.2 heavy chain variable regions and murine l1 light chains. It was shown to share a high structural and functional homology to native equine IgE (Wagner et al., 2002a). Using the rIgE as antigen, a total of 17 speci®c anti-IgE mAbs were generated, recognizing the rIgE. All mAbs except one (aIgE-132), detected native horse IgE as well as evidenced from data obtained by ELISA, immunoblotting, surface staining of equine blood leukocytes as well as by puri®cation of serum IgE using an aIgE-134 af®nity column. For the determination of the rare IgE antibodies in serum or other body ¯uids, like bronchial lavage, nasal secret etc., which always contain high amounts of IgG, IgM and/or IgA, the monoclonal antibodies against IgE must lack cross-reactivity to other equine Igs.

Fig. 5. Determination of total serum IgE by ELISA. Serum IgE concentrations of Icelandic horses with summer eczema (‡SE), animals without clinical signs ( SE) and healthy foals were compared. The mAbs aIgE-176 and aIgE-134, which recognized different epitopes on the IgE heavy chain, were used as af®nity matrix and for detection, respectively. The puri®ed IgE was used as reference to determine the concentration of IgE in equine serum.

Horses express Ig of nine different isotypes: IgM, IgG1 to IgG6, IgE and IgA, which correspond to the nine equine Ig constant heavy chain genes (IGHC genes; Wagner et al., 1998). The existence of an equine IgD has not yet been excluded. In contrast to the classical nomenclature for equine IgG isotypes

Fig. 6. Flow cytometric analysis of IgE‡ leukocytes from foals and adult Icelandic horses. Cells were obtained from peripheral blood and stained for surface IgE using the aIgE-134 mAb. For isotype control a murine IgG1 antibody was used, which did not recognized any surface antigen on equine blood leukocytes. A signi®cant difference …P < 0:001† in the number of IgE‡ cells was observed between adult horses and foals.

Fig. 7. Equine surface IgE‡ leukocytes were enriched by magnetic cell sorting (MACS). A total of 5  108 equine peripheral blood leukocytes were stained by aIgE-134 and sorted in two steps. The cell populations were controlled by ¯ow cytometric analysis (a) before cell sorting and (b) after two enrichment steps by MACS. The percentages of IgE‡ leukocytes before and after sorting are shown. The IgE‡ cells were mainly found in the granulocytes (1) and monocytes or large lymphocyte gate (2). After sorting, a distinctly enriched population of large granulocytes (3) was found. An additional fraction of low stained cells was detectable in the lymphocyte population (4); (c) Wright staining of the enriched IgE‡ cell fraction showed that most of these cells were basophils (B) and monocytes or large lymphocytes (M). In addition, a population of small lymphocytes (L) and a few eosinophils (E) were identi®ed.

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(IgGa, IgGb, IgGc, IgG(T), IgG(B), as reviewed by Montgomery, 1973), we have recently introduced the designation IgG1 to IgG6 which corresponds to the six IGHG genes (Wagner et al., 2002b). Although the six equine IGHG genes could hypothetically be expressed, not all IGHG genes have already been linked to serum IgG isotypes of the old IgG nomenclature (Table 1). It is however clear, that IgG1 corresponds to IgGa and IgG4 to IgGb (Wagner et al., 1998). The IgG(T) fractions described in earlier studies, most probably contained IgGs with two different heavy chain constant regions, namely IgG3 and IgG5 (Wagner et al., 2002b). The relation between IgGc and IgG(B) and IgG2 and IgG6 has not yet been determined. The anti-IgE mAbs were tested for reactivity with equine IgM and IgG isotypes expressed by heterohybridomas or found in equine serum. Out of eight equine isotypes other than IgE, isotype speci®c mAbs are available for sixÐIgM, IgG1, IgG3, IgG4, IgGc and IgA (Lunn et al., 1998; Sheoran et al., 1998; Wagner et al., 1998). None of these equine isotypes are recognized by any of the anti-IgE mAbs described here, as tested by ELISA. However, it is possible that the equine sera tested here also contained antibodies representing the remaining two IgG isotypes, and that their serum concentrations were too low for detection of a potential cross-reactivity with any of the anti-IgE mabs. It is also notable that in the assay using a polyclonal goat a horse IgG …H ‡ L† antibody as af®nity matrix bound on the plate, the anti-IgE mabs failed to detect any IgE in horse serum. This is probably due to the low IgE serum concentrations (as occurs in other species) and due to inhibition of its binding to the af®nity matrix by the excess IgG, IgM and IgA serum antibodies. However, in the same sera equine IgE could easily be detected using the aIgE-176 mAb as the af®nity matrix and the aIgE-134 mAb which recognizing a different IgE epitope as the detection reagent. This ELISA is the ®rst one available for quanti®cation of equine IgE in serum and other body ¯uids and can be used to investigate the role of IgE in medical disorders of horses. The sera which were used for detection of IgE by immunoblotting or ELISA, as well as the equine leukocytes, which were analyzed for surface IgE‡ cells, were obtained from Icelandic horses. All of these animals lived in the same herd and environment. Some of them had clinical symptoms of summer

eczema, others were apparently clinically unaffected. The foals investigated here were offspring of both clinically healthy and affected animals. Summer eczema is a disease with characteristic symptoms of an allergic, IgE-dependent type I hypersensitivity. Most probably the allergen which induces the disease is contained in the saliva of Culicoides spp. and is injected into tissues and blood during midge bites (Larsen et al., 1988; Wilson et al., 2001). Culicoides speci®c IgE antibodies can be detected on blood basophils of affected horses and the stimulation of such basophils with Culicoides extracts leads to a substantial release of histamine from these cells (W. Leibold, unpublished observations). We speculated that horses affected with summer eczema might have higher amounts of IgE in their serum or bound to their peripheral basophils, as compared to control animals. However, no signi®cant differences were found between levels of total serum IgE or numbers of IgE‡ cells in the blood between both groups. These indicates that both parameters and in particular the total serum IgE concentrations are not useful for the diagnosis of this disease. Both, serum IgE levels and cell-bound IgE, are apparently mostly speci®c for other ubiquitous antigens and not the allergens of Culicoides saliva. In foals, it was not possible to detect any IgE‡ leukocytes (foals 2.5-month old) or serum IgE (foals 5±6-month old), despite the fact that these animals spent the entire summer in an environment where their dams developed clinical symptoms of summer eczema. These ®ndings are in agreement with the observation that most horses, if not all, develop this disease at the age of 2±4 years if they are exposed to the allergen from birth on (Wilson et al., 2001). At least during their ®rst 6 months of life, horses do not develop an immune response to Culicoides saliva, which includes allergen-speci®c IgE antibodies. Thus, at this time, a preventive immunization with Th1 disposing adjuvant might be a useful strategy to prevent Culicoides-mediated allergy in horses with predisposition to develop the disease. The analysis of surface IgE‡ leukocytes identi®ed a mixed cell population. We suggest that these IgE‡ cells have bound IgE via FceRI on basophils, or via FceRII, on monocytes or B-cells. After two magnetic cell sorting steps, the basophils could be enriched from around 1% in the peripheral blood, to more than 30%

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in the positive enriched fraction. In all likelihood, these cells had bound the IgE via the high af®nity FceRI, although speci®c reagents to identify the equine FceRI are not yet available. We have recently shown that equine rIgE is able to bind to foal basophils and to mediate histamine release after cross-linking by antigen (Wagner et al., 2002a). After incubation with the rIgE for several hours, the number of IgE‡ foal leukocytes which could be stained using the anti-IgE mAbs was comparable to those of adult horses, as described here (data not shown). Thus, the foal basophils were able to bind IgE, suggesting that the absence of surface IgE‡ cells in foals is due to the lack of secreted IgE at this age. In addition to the basophils, we observed another population of about 50% of the IgE‡ equine peripheral leukocytes which were monocytes and/or large lymphocytes according to cytometric light scatter and Wright staining. We suggested that these cells had bound IgE via the equine low af®nity Fce-receptor (FceRII, human CD23) which is expressed by these cells in man and mice, and the nucleotide sequences of equine and bovine CD23 have been described, recently (Watson et al., 2000). Expression of the FceRII has been described for B-lymphocytes and monocytes (Melewicz et al., 1982), macrophages, folicular dendritic cells, platelets (Joseph et al., 1986) and eosinophils (Grangette et al., 1989). The IgE-FceRII interaction plays an important role in the regulation of IgE production as well as for antigen presentation (Gustavsson et al., 2000). Besides the aIgE-132 mAb which detects only heavy chains of rIgE, the monoclonal anti-IgE antibodies recognized at least three different epitopes on native equine IgE. As is evident from binding inhibition, the epitopes detected by aIgE-41, aIgE-134 and aIgE-176 are different, while the remaining anti-IgE mAbs recognized the same epitope as aIgE-134 or epitopes close by. A total of 15 anti-IgE mAbs detected epitopes only on the unreduced IgE molecule. Only aIgE-176 is speci®c for an epitope which is found on unreduced as well as on reduced equine IgE constant heavy chains. In conclusion, the different epitope speci®cities of the anti-IgE mAbs allow their use for IgE determination by various methods, including the highly speci®c ELISA based on two different anti-IgE mAbs, as described here. These anti-IgE mAbs are valuable tools for clinical and experimental investigations on

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