Human Immunoglobulin E Research Articles

Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings concomitantly with activation-induced cytidine deaminase expression and differentiation into plasma cells

Interleukin (IL)–21 downregulates immunoglobulin E (IgE) production in murine systems by inhibiting germline ε transcription in IL-4–stimulated B cells. We here sought to clarify the function of IL-21 in human B-cell IgE synthesis. IL-21 dramatically enhanced IgE production by human mononuclear cells, or purified total, naive, or memory B cells in the presence of IL-4 plus anti-CD40 mAb cross-linked with CD32-transfectants, and the production was strengthened with further addition of IL-10. It was concomitant to the enhancement of activation-induced cytidine deaminase (AID) mRNA expression, but no increase of germline ε transcription. We also observed that IL-21 promoted B-cell differentiation into plasma cells with increase of B-lymphocyte–induced maturation protein–1 (Blimp-1), but not X-box binding protein 1 (XBP-1), which was further accentuated by co-stimulation with IL-4 plus CD40 signaling. Thus, IL-21 is a strong inducer of IgE production in human beings concomitantly with AID expression and the differentiation into plasma cells. Our data suggest that IL-21 plays an important role in occurrence and the treatment of allergic disorders.

Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen

Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex-Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26-71, 119-280 and 119-291. However, residues at positions 26-199, 26-105, 26-86, 119-320 and 238-330 were found relevant in the IgE-binding. Our results indicate that (72)NISGHCSDCTFLGEE(86) and (292)VWNRYYDLKTNYSSSIILEYVNSGTKYLP(320) of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, (72)NISGHCSDCTE(86), (292)VWNRYYDLKT(301), (300)KTNYSSSIILEY(311) and (309)LEYVNSGTKYLP(320), were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to (292)VWNRYYDLKT(301) correlated significantly with the presence of allergic symptoms (P < 0.001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex-fruit syndrome.

Development of a FPW allergy biosensor for human IgE detection by MEMS and cystamine-based SAM technologies

Abstract Conventional allergy detecting kits usually require a large number of expensive testing samples, time-consuming procedures and complex instruments. To overcome these disadvantages, this study presents a high-sensitivity flexural plate-wave (FPW)-based allergy biosensor with integrated cystamine self-assembled monolayer (SAM) for detecting the concentration of immunoglobulin E (IgE) in human serum. The ultrasonic flexural plate-waves were respectively launched and received by two Cr/Au interdigital transducers (IDTs) located on the left and right side of the Si/SiO2/Si3N4/Cr/Au/ZnO floating thin plate. A 4.625-nm height cystamine SAM and a 1.078-nm height glutaraldehyde cross-linking layer were developed for the immobilization of IgE antibody and their surface chemical composition and structural perturbation changes were analyzed by Auger electron spectroscopy (AES) and Fourier transform-infrared (FT-IR) spectroscopy, respectively. Using atomic force microscopy (AFM), this work investigates the molecular topographies of the cystamine SAM, glutaraldehyde, IgE antibody and IgE antigen layers and the number of bonded IgE antibody/antigen pairs. A FPW-based allergy biosensor with very high mass-sensitivity (−8.52 × 107 cm2 g−1) and low operating frequency (8.75 MHz) of human IgE antigen was demonstrated.

Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen*

Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O-deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3-ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis. An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096-amino acid fragment of Ani s 7 (GenBank: EF158010) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained. The rAni s 7 fragment comprised 19 repeats of a novel CX(17-25)CX(9-22)CX(8)CX(6) tandem repeat motif not seen in any previously reported protein sequence. An internal (435)Met-(713)Arg fragment of the rAni s 7 (t-Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme-linked immunosorbent assay (ELISA) with t-Ani s 7 identified as positive the same 60 sera as UA3-ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O-deglycosylated nAni s 7. In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis-induced allergy.

Optimization of Critical Factors Affecting the Performance of an Allergen Chip for the Analysis of an Allergen-specific Human IgE in Serum

A sensitive and multiplexed assay of allergen-specific human immunoglobulin E (IgE) is of great significance in the precise diagnosis of allergies. We report on the optimization of critical factors for chip-based analysis of IgE in human serum with a high reliability. Extracts of two mite species were used as model allergens, and were spotted onto a glass slide for the construction of an allergen chip. Respective allergen-specific IgE in human serum was analyzed by using biotinylated anti-human IgE and a streptavidin-Cy3 conjugate. Factors affecting the performance of the allergen chip were investigated and optimized. Especially, the effect of additives, the concentrations of biotinylated anti-human IgE and the streptavidin-Cy3 conjugate, the serum dilution factor, and the concentration of allergen extract as a capturing agent were examined in detail. Under the optimized conditions, a chip-based analysis for sera from 43 patients revealed a reliable and reproducible diagnosis of respective allergies, showing a good correlation with a conventional CAP assay.

A Novel Piezoelectric Quartz Micro-Array Immunosensor for Detection of ImmunoglobulinE

A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.

Prostaglandin E2 potentiates the immunologically stimulated histamine release from human peripheral blood‐derived mast cells through EP1/EP3 receptors

Mast cells cultured from human peripheral blood have been widely used to study human mast cell function. Prostanoids are the important regulators of mast cell activity, however, there were no reports about the class of prostanoid receptors expressed on such cultured cells. The present study was to characterize pharmacologically the prostanoid receptors by investigating the effects of prostanoid receptor agonists on the immunoglobulin E (IgE)-mediated histamine release from the cultured mast cells. Mast cells cultured from human progenitor cells in peripheral blood were sensitized with human myeloma IgE, and then challenged with anti-human IgE following pretreatment with diverse prostanoid receptor agonists. The histamine content in supernatants and cell pellets were measured by histamine auto-analyzer. Of the prostanoid receptor agonists tested, the prostaglandin E2 (PGE2) receptor (EP receptor) agonist PGE2 (10(-7) to 10(-11) M) produced concentration-related potentiation of IgE-mediated histamine release from the cultured mast cells. Sulprostone, an EP1/EP3 agonist, SC-46275, a selective EP3 agonist, and 11-deoxy-PGE1, a selective EP2/EP3/EP4 agonist also caused a significant increase in histamine release induced by anti-IgE. BW245C, fluprostone, cicaprost and U46619 for the prostaglandin D2, F2alpha, I2, and thromboxane A2 receptors respectively, and the EP2/EP4 receptor agonist butaprost had little effect on anti-IgE stimulated histamine release from mast cells. The present results suggest that PGE2 potentiates the IgE-mediated histamine release from the cultured mast cell via EP3 and/or EP1 receptors.

Rejection of Primary Lymphoma Cells Derived from c-myc-Transgenic Mice after Haploidentical Transplantation: Treatment of the Tumor May Confer to Resistance Against GvL-Effect.

The long term prognosis of the High Grade B-cell-lymphoma in adults is still very poor, especially in the case of resistance to chemotherapy. To date allogeneic stem cell transplantation as a last treatment option results in poor outcomes due to high transplant related mortality or disease progression. This dilemma raises the question of whether transplant related mortality could be reduced by earlier transplantation, and whether such tumors can be recognized by an allogeneic graft providing a graft versus lymphoma effect. The immunology behind allogeneic rejection of high grade lymphomas is poorly understood due to a dearth of good animal models. Using a transgenic mouse model in which the human c-myc oncogene is driven by the human immunoglobuline lambda locus mimicking a t(8;22) translocation of Burkitt's lymphoma, we established primary lymphoma cell lines. C57BL/6c-myc tg mice were crossbred with DBA mice and lymph nodes from tumor bearing F1-mice were explanted and seeded on irradiated human MRC5 fibroblast feeder cells. Approximately 70% of all explanted lymphomas established long term cultures which could be maintained free of feeder cells after 4 to 6 weeks. FACS analysis of lymphoma cells displayed strong CD19 expression, reduced expression of MHC Class I and II and, in several cases, low expression of costimulatory molecules such as CD80, CD86 and CD54. The phenotype was maintained over time with a follow up of up to 4 months. As few as 100 lymphoma cells gave rise to orthotopic tumors or systemic disease within 40 days after s.c. or i.v. injection, respectively. On the contrary, when 50 Mio. F1-lymphoma cells were injected into haploidentical C57BL6 parental animals, no tumor growth could be observed after at least 100 days of follow up. Despite fast growth and low expression of MHC Class I and costimulatory molecules, lymphoma cells were easily rejected in a haploidentical setting. Ex vivo chemotherapy treatment of lymphoma cells resulted in resistance after 2 courses of 100nM Adriamycine. Increased doses up to 500nM Adriamycine further accelerated resistance and growth speed of the cells. Flow cytometric analysis indicated a reduced fraction of apoptotic cells in culture suggesting selection for cells bearing alteration in the apoptotic pathways. In further experiments altered cells will be tested in haploidentical settings to confirm clinical experience in our model.

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p 109–117, p 113–121, and p 103–141 (CHε2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2 d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2 d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE −/−) 129/SvEv mice (H-2 b). Comparable magnitude of CTL responses was observed in both strains immunized with p 109–117 or p 103–141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2 b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p 107–123, p 109–117, and p 113–121 expressed in CDR3 of VH62/human γ1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p 109–117 and p 103–141 while IgG responses were not suppressed. Since IgE peptide sequences of CHε2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.

The Importance of Lys-352 of Human Immunoglobulin E in FcϵRII/CD23 Recognition

The interaction of immunoglobulin E (IgE) with its low affinity receptor (FcepsilonRII/CD23) plays a central role in the initiation and regulation of type I hypersensitivity responses. We have previously identified the importance of amino acid residues in the A-B loop of the Cepsilon3 domain of human IgE and implicated a region close to the glycosylation site at asparagine 371 as contributing to IgE-CD23 interaction. These residues were now targeted by site-directed mutagenesis. The IgE-CD23 interaction was assessed by semiquantitative flow cytometry. Replacement of the entire Cepsilon3 A-B loop (residues 341-356) with the homologous rat IgE sequence resulted in complete loss of human CD23 recognition, as did replacement of residues 346-353, indicating that class-specific effector residue(s) are contained within these eight amino acids. Lysine 352 within the A-B loop was identified as contributing directly to human CD23 interaction. Mutation to the rodent homologue glycine or glutamate resulted in a significant reduction in binding compared with native IgE, whereas conservative substitution with arginine effected a small, but statistically significant, enhancement of CD23 binding. Mutation of the Cepsilon3 glycosylation site at asparagine 371 to threonine or glutamine did not significantly affect CD23 recognition. Our results yield new insights into the structural basis of the hIgE-CD23 interaction and hold promise for the rational design of drugs that can manipulate IgE-mediated regulation of the allergic response.

IgE antibody in the serum--detection and diagnostic significance.

IgE antibody in the serum--detection and diagnostic significance.

Human B immunoglobuline (HBIG) monotherapy or combined with lamivudine (LAM) in prophylaxis of HBV recurrence after liver transplantation (LT) in HBsAg-carriers

Human B immunoglobuline (HBIG) monotherapy or combined with lamivudine (LAM) in prophylaxis of HBV recurrence after liver transplantation (LT) in HBsAg-carriers

Monovalent fusion proteins of IgE mimotopes are safe for therapy of type I allergy.

By screening phage display random peptide libraries with purified immunoglobulin E (IgE) from birch pollen-allergic patients, we previously defined peptides mimicking natural IgE epitopes (mimotopes) of the major birch pollen allergen Bet v 1. The present study aimed to define a monovalent carrier for the IgE mimotopes to induce protective antibodies directed to the IgE epitopes, suitable for mimotope-specific therapy. We expressed the selected mimotopes as fusion proteins together with streptococcal albumin binding protein (ABP). The fusion proteins were recognized specifically by anti-Bet v 1 human IgE, which demonstrated that the mimotopes fused to ABP resemble the natural IgE epitope. Bet v 1-specific IgG was induced by immunization of BALB/c mice with fusion proteins. These IgG antibodies could inhibit IgE binding to Bet v 1. Skin testing of Bet v 1 allergic mice showed that the ABP mimotope constructs did not elicit type I skin reactions, although they possess IgE binding structures. Our data suggest that IgE mimotopes are safe for epitope-specific immunotherapy of sensitized individuals, when presented in a monovalent form. Therefore, ABP-fused mimotopes are promising candidates for a new type of immunotherapy based on the precise induction of blocking antibodies.

Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor

In vitro assays for allergen specific immunoglobulin E (IgE) are a convenient and reproducible alternative to intradermal skin testing in dogs. Such tests may be used to support a diagnosis of atopic dermatitis and to define appropriate allergens for immunotherapy. Current in vitro assays rely upon monoclonal or polyclonal antibodies as IgE detection reagents. However, in sera where allergen-specific IgG occurs in great excess, any IgE:IgG cross-reactivity of the detection reagent may result in lowered assay specificity. Therefore, we have developed an assay for canine IgE which uses a recombinant form of the extracellular part of the alpha chain of the human high affinity IgE receptor (FcεRIα). Biotinylated FcεRIα shows no significant binding to purified canine IgG, and recognizes a heat labile antibody in serum, with a detection limit of 73–146 pg/ml. Comparison of assay signals using the labeled FcεRIα and a highly specific anti-canine IgE monoclonal antibody (MAb) shows good agreement. The FcεRIα is therefore a sensitive and specific alternative to polyclonal or monoclonal antibodies for canine serum IgE measurement.

Immunoglobulin E antibodies of atopic individuals exhibit a broad usage of VH-gene families.

The term 'atopy' describes the genetically determined tendency to mount immunoglobulin E (IgE) antibody responses against per se harmless antigens (allergens). In this study we investigated the usage of VH families in the formation of IgE antibodies in 10 patients suffering from mucosal and/or skin manifestations of atopy. IgE antibody reactivities to exogenous allergen sources as well as to autoallergens were determined and, by immunoabsorption, it was demonstrated that allergen-specific IgE accounted for most of the total serum IgE levels in these patients. Using primers with specificity for the VH1-6 gene families and a primer specific for the first constant region of human IgE, cDNAs coding for IgE heavy chain fragments were amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from peripheral blood lymphocytes of the 10 atopic individuals. Hybridization of the heavy chain-encoding cDNAs with an IgE-specific internal oligonucleotide probe revealed a broad usage of all VH-gene families in the atopic individuals. The spectrum of VH families used in a given atopic individual was neither associated with the type or severity of clinical symptoms nor with the number of allergens recognized. The fact that allergen-specific IgE antibodies in atopic individuals originate from a broad variety of B cells thus reflects the activation of multiple B-cell clones during allergen sensitization. This finding should be borne in mind if therapeutic strategies for Type I allergy are considered that aim at a clonal elimination of allergen-specific B cells.

Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha.

The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, Fc epsilonRI. Crosslinking of Fc epsilonRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with Fc epsilonRI is a key recognition event of this process and involves the extracellular domains of the Fc epsilonRI alpha-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc-Fc epsilonRI alpha complex to 3.5-A resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one C epsilon3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to Fc epsilonRI for the treatment of allergy and asthma.

Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice.

CD23, the low affinity serum immunoglobulin E (IgE) receptor, is upregulated on B cells following interleukin (IL)-4 stimulation and is concomitantly cleaved to generate soluble CD23 (sCD23) fragments with cytokine-like activity. Compounds that selectively inhibit the proteolytic release of CD23 to generate sCD23 were assessed for their ability to inhibit IgE production in order to evaluate the contribution of sCD23 in the production of human IgE as well as the ability of such compounds to block IgE production. IgE production was measured in IL-4-stimulated human peripheral blood lymphocytes (PBL) and PBL-reconstituted SCID mice in the presence of a broad-spectrum matrix metalloprotease (MMP) inhibitor, a compound selective for inhibition of CD23 processing over MMPs and an anti-CD23 mAb, MHM6. The two compounds were equipotent in inhibiting IgE production without inhibition of IgG production by IL-4/anti-CD40-stimulated PBL. Soluble CD23 release was also shown to precede IgE accumulation in the cell-free medium. Addition of compound at later times other than day 0 in the 14 day assay resulted in progressively less inhibition of both IgE and sCD23, and exactly paralleled the effect of an anti-CD23 mAb, MHM6 on IgE levels. Both compounds also inhibited the release of CD23 from human RPMI 8866 cells adoptively transferred i. p. to mice. Doses required for inhibition of CD23 correlated well with the doses required for inhibition of IgE production in IL-4-challenged hu-PBL-SCID mice. IgE was selectively inhibited over total IgG in the SCID mice as well. Inhibition of CD23 processing alone is sufficient to inhibit IL-4-stimulated IgE production both in vitro and in vivo.

Evaluation of a Particle-enhanced Turbidimetric Immunoassay for the Measurement of Immunoglobulin E in an ILab 900 Analyzer

The measurement of immunoglobulin E (IgE) in serum is widely used in the diagnosis of allergic reactions and parasitic infections. We describe here a fully automated assay for human IgE suitable for routine application in a general chemistry analyzer. We used an ILab 900 analyzer. This instrument automates a particle-enhanced immunoturbidimetric assay with an analysis time of 9 min. The assay was linear in the range 4-1000 kIU/L (r = 0.9998). The intra- and interassay CVs at 57, 235, and 434 kIU/L were <3.5% and <7.4%, respectively. The detection limit was 4 kIU/L. Hemoglobin (</=16 g/L), bilirubin (</=250 micromol/L), and myeloma paraproteins did not interfere with the assay. The assay showed good correlation with a microparticle enzyme immunoassay (r = 0.998) with a mean difference between methods of -6 +/- 26 kIU/L. The new automated serum assay for IgE is an attractive alternative that avoids the need for dedicated instrumentation.

Technetium-99m labelling of human immunoglobulin and preliminary clinical evaluation in tumour patients.

In the present investigation we have human immunoglobulin labelled with 99Tc(m), applying two different systems. The radiochemical characteristics of the labelled antibody were studied by conventional, radioanalytical methods. Further on, pharmacokinetics of this 99Tc(m)-labelled biomolecule were investigated, by i.v. administration in women, checked for tumours of the ovaries, uterus and cervix. Scintigraphic findings were compared to the results of other imaging techniques (CT, US, X rays), as well as to surgical findings. Our studies indicated that 99Tc(m)-human immunoglobuline can be applied successfully for the scintigraphic detection of several malignant and benign tumours of the female genital system. Tumour accumulation is probably due to the activation of particular cells, as macrophages and lymphocytes, responsible for inflammatory and immunological responses.

Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.