Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells.

IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cvarepsilon3-domain are crucial for FcvarepsilonR-binding. In order to further study structures of importance for the function of IgE, such as the carbohydrates, fragments with unmodified amino acid sequence were blunt-end cloned and expressed in baculovirus-infected Sf9 cells. Two fragments of human IgE, one encompassing the entire Fc-region (rCvarepsilon2-4) and a smaller one comprising the second and third domain (rCvarepsilon2-3), were produced and characterised with respect to epitope expression, glycosylation and FcvarepsilonR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cvarepsilon2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCvarepsilon2-4 mainly formed a dimer, whereas rCvarepsilon2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCvarepsilon2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cvarepsilon2-, Cvarepsilon3- and Cvarepsilon4-domains, whereas rCvarepsilon2-3 only reacted with anti-Cvarepsilon2 mAbs. Moreover, in FACS analysis rCvarepsilon2-4 interacted with two cell-lines constitutively expressing FcvarepsilonRI or FcvarepsilonRII, whereas rCvarepsilon2-3 lacked reactivity. A substantial reduction in the ability of rCvarepsilon2-4, following endoglycosidase treatment, to react with recombinant alpha-chain of the high affinity receptor for IgE in sandwich ELISA, indicated a role of N-linked oligosaccharides in stabilising receptor binding structures. Taken together, our results show that rCvarepsilon2-4, but not rCvarepsilon2-3, will be useful in studies of structure-function relationships of IgE, including the role of N-glycosylation, since it demonstrated appropriate epitope expression, conformation and ability to bind Fcvarepsilon-receptors.

N-glycosylation influences epitope expression and receptor binding structures in human IgE.

Although human IgE is relatively rich in carbohydrates, there are few studies concerning their structural and functional importance. The low serum concentration of IgE has limited carbohydrate characterisation to a few IgE myeloma proteins. Four to six of the seven potential N-glycosylation sites in the constant region of the epsilon chain seem occupied together with some residual microheterogeneity. We have used a panel of 28 anti-Cepsilon2, 7 anti-Cepsilon3 and 18 anti-Cepsilon4 domain-specific anti-IgE mAbs, and rFcepsilonRIalpha to examine the effect of N-glycosylation on epitope expression of human IgE. Myeloma proteins IgE(DES)-kappa, IgE(ND)-lambda and IgE(UD)-kappa as well as polyclonal IgE were deglycosylated with PNGF and/or sialidase and tested in different ELISA. In all ELISA approaches, the reactivity of most domain-specific anti-IgE mAbs was independent of the glycosylation state of IgE(DES), except for one-third of the anti-Cepsilon2 mAbs. These mAbs reacted better with deglycosylated IgE(DES) in the order of treatment PNGF/sialidase > PNGF > or = sialidase > buffer control. In sharp contrast, the reactivity of IgE(DES) with rFcepsilonRIalpha was not influenced by sialidase but markedly reduced following PNGF or PNGF/sialidase treatment. These findings were neither myeloma restricted nor caused by aggregation, since monomeric IgE demonstrated the same reactivity pattern. Thus. N-glycosylation seems to influence both structure and function of human IgE. The oligosaccharides modulate epitope expression, mainly in the Cepsilon2-domain, as revealed by a subset of mAbs. They also promote subtle changes in the Cepsilon3-domain, leading to a reduced FcepsilonRIalpha binding. These findings suggest physiological implications of carbohydrates in human IgE.

Human IgG is substrate for the thioredoxin system: differential cleavage pattern of interchain disulfide bridges in IgG subclasses.

Thioredoxin, a 12,000 mol. wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity. Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH)2. It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherichia coli thioredoxin systems. The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH)2, which directly reduced IgG disulfides in a time and dose-dependent manner. Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy light chain and inter-heavy chain disulfides, and SDS PAGE corroborated that the buried intrachain disulfides were left intact. The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4. In sharp contrast, IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times. A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide. Consistent results were obtained for several IgG myeloma proteins within each subclass. The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.

Antigen-binding sites dominate the surface properties of IgG antibodies.

A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen-binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties.

Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Inhibition of idiotype-anti-idiotype reaction in particle counting immunoassay as a tentative assay of IgE.

Rabbit antibodies were raised against a purified mouse monoclonal IgG1 antibody having specificity for the D epsilon 2 determinant of human IgE. After appropriate absorption this antiserum was anti-idiotype specific and was used to set up a particle counting immunoassay for human IgE. Latex particles coated with F(ab')2 fragments of the anti-idiotypic antibodies were agglutinated by the monoclonal anti-IgE antibody (20 ng/ml). This reaction was inhibited up to 100% by human IgE and the assay took 30 min to perform with a sensitivity of 40 IU/ml. The coefficient of correlation with a routine IgE assay was r = 0.96, and the mean analytical recovery tested on 10 sera was 95.8%.

Typing of subclasses and light chains of human monoclonal immunoglobulins by particle counting immunoassay (PACIA).

The subclasses of monoclonal IgGs and IgAs were identified by particle-counting immunoassay. The principle of the test is the inhibition of the agglutinating activity of either specific antisera or monoclonal antibodies (for IgA only) on latex particles coated with a monoclonal IgG or IgA of known subclass. The feasibility of assay of polyclonal Ig subclasses was demonstrated. However, the anti-IgG2 antiserum cross-reacted with an allotype (nG4m(b)) of IgG4. The possibility of typing monoclonal Igs for light chains by the same technique was also demonstrated. Results are obtained in 30 min, and the method requires only small amounts of purified immunoglobulins (Igs) and antisera or monoclonal antibodies.

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Immunoglobulin isotypes reveal a predominant role of type 1 immunity in multiple sclerosis.

IgG, its subclasses and IgE concentrations were measured in cerebrospinal fluid (CSF) and serum of multiple sclerosis (MS) patients and matched controls as surrogate markers for type 1 and type 2 immunity. IgE indices were significantly reduced in MS patients compared to controls. In contrast, IgG1 was elevated in CSF of MS patients and elevated indices indicated intrathecal synthesis. Because isotype switching to IgE and IgG4 is driven by type 2 immunity and occurrence of IgG1 has previously been found in type 1 immunity-dominated diseases, the results underscore a role of type 1 immunity in MS.

IgE in the cerebrospinal fluid.

The level of IgE in the cerebrospinal fluid (CSF) was determined by particle counting immunoassay. With a limit of sensitivity of 0.2 IU/ml, this immunoglobulin was detected neither in CSF of non-neurological patients (n = 27) nor of patients with sciatica (n = 13). IgE was present in samples from some patients with either multiple sclerosis (MS) or various infections of the central nervous system. In these cases, an IgE index [CSF IgE/serum IgE: CSF albumin/serum albumin] was calculated as 0.29 (SD 0.12). This value is not abnormal as the mean IgG and monomeric IgA indices are 0.45 and 0.34, respectively. Therefore, the IgE detected in most of the CSF samples was not locally produced. However, most patients with tuberculous meningitis had clearly an increased IgE index suggesting a local biosynthesis, but we failed to detect any IgE antibody activity against purified protein derivatives.

Are increased levels of von Willebrand factor in chronic coronary heart disease caused by decrease in von Willebrand factor cleaving protease activity? A study by an immunoassay with antibody against intact bond 842Tyr-843Met of the von Willebrand factor protein.

Low levels of von Willebrand factor (VWF) in von Willebrand's disease type 2A (VWD 2A) result from increased cleavage of the bond 842Tyr-843Met in the VWF protein by VWF cleaving protease. On the other hand, decreased levels of this protease result in unusually large VWF in thrombotic thrombcytopenic purpura with thrombotic complications. In the present study, we designed an enzyme-liked immunosorbent assay of VWF cleaving protease activity to be used to assess whether the high levels of VWF in coronary heart disease (CHD) relate to a deficiency of this protease. Plasma samples with added Pefabloc and CaCl(2) were incubated with purified VWF coated on a microtiter plate. The remaining undigested multimers were quantified by an antibody directed against the intact 842Tyr-843Met bond of the VWF protein. Phosphate-buffered saline (PBS), instead of plasma, was used to obtain the initial level of coated undigested VWF. The reduction in absorbance at 492 nm between PBS and the unknown sample was taken as a measure of the protease activity. The assay was applied to plasma samples from 21 senior women with chronic CHD (cases) and 34 age-matched controls, as well as to samples from three patients with VWD 2A. The protease activity was similar in the two women groups (P>.05), although the VWF antigen levels were higher in the cases (P<.01). The VWD 2A patients had similar plasma levels of the protease to that in normal pooled plasma (NPP). In the senior controls, the protease activity correlated with the subject age (r's=-.61, P<.01, n=34). In conclusion, the developed method is specific for evaluating the protease function on VWF cleavage. The moderate increase of VWF antigen in chronic CHD may not depend on the protease activity. The age influence on the protease levels supports earlier findings of higher VWF levels in healthy older subjects. A high sensitivity of the mutated protein of VWF for the protease effect rather than increases in activity or quantity of the enzyme is probably involved in the pathogenesis of VWD 2A.

Naturally occurring human IgA autoantibodies against IgE-DES myeloma protein. Prevalence and specificity.

The prevalence and specificity of naturally occurring human IgA anti-IgE autoantibodies (a-E Ab) were studied by ELISA with anti-IgA monoclonal antibodies (mAb) and a purified myeloma IgE as solid-phase protein, i.e., IgE-DES(kappa). Such detected IgA a-E Ab were common among adults, and significantly increased geometric means (GM) were found in patients with atopy (P = 0.006; n = 41; GM = 79.3 arbitrary units (AU)/ml) and filariasis (P = 0.02; n = 41; GM = 75.9 AU/ml), as compared with nonatopic controls (n = 42; GM = 48.8 AU/ml). No such difference was observed between age-matched nonatopic (n = 22; GM = 36.7 AU/ml) and atopic (n = 22; GM = 38.6 AU/ml) children. Children had significantly (P < 0.001) lower IgA a-E Ab concentrations than adults, probably as a result of age, because IgA a-E Ab concentrations and age of children were significantly correlated (n = 44; P < 0.05; r = 0.30). IgA a-E Ab concentrations were very low in cord serum (n = 32; median < 0.1 AU/ml). Sex did not influence IgA a-E Ab concentrations in any study group. The specificity of IgA a-E Ab in nine sera was studied by ELISA inhibition assay using IgE-DES myeloma as solid-phase protein and inhibitory proteins of the IgG, IgM, IgD, and IgE classes, including five different IgE myeloma proteins, as well as three enzymatic fragments of IgE-DES. The inhibitions indicated that all IgA a-E Ab tested reacted in a low-affinity reaction with determinants restricted to IgE-DES, i.e., the solid-phase protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cord serum IgE in relation to family history and as predictor of atopic disease in early infancy.

Cord serum IgE was assayed by particle counting immunoassay (PACIA) in an unselected series of European newborns (n = 190; geom mean = 0.37 IU/ml) and a cut-off limit established (greater than or equal to 1.20 IU/ml) for prediction of atopy. At control follow-up by questionnaire 18 months after birth, 38 infants (20.0%) had developed definite (9.5%) or probable (10.5%) atopy with a significant predominance of boys (P less than 0.03). Infants with a positive immediate family history (IFH) had a higher risk of developing atopy (P less than 0.0025) and also had a higher incidence of elevated cord IgE (P less than 0.02) than infants with a negative IFH. Maternal atopy influenced cord IgE levels significantly (P less than 0.00005), whereas paternal atopy did not (P = 0.23). No fetal IgE antibodies against five common allergens could be demonstrated in 36 cord sera tested. Breast-feeding for 3 months was not sufficient to prevent atopic symptoms. The predictive value of cord IgE was high since 26 of 36 newborns (positive predictive value = 72.2%) with elevated cord IgE had developed atopic symptoms before follow-up. Of the 38 infants who developed atopic symptoms, 26 had elevated cord IgE (sensitivity = 68.4%) compared to only 10 (6.6%) of the 152 atopy-free infants (P less than 0.00005). The data indicate that elevated cord IgE as determined by PACIA is a good predictor of early-onset atopy, better than family history (P less than 0.008), and that primarily maternal atopy seems to affect fetal IgE synthesis.

Maternal smoking influences cord serum IgE and IgD levels and increases the risk for subsequent infant allergy.

The effects of parental smoking on IgE and IgD levels in cord serum and subsequent infant allergy were investigated in an unselected series of 186 European newborn infants. Maternal smoking caused a significant rise in both IgE (p less than 0.05) and IgD (p less than 0.05), a finding that was most apparent in newborn infants with a negative biparental history of allergy (p less than 0.025 and p = 0.005, respectively). Furthermore, newborn infants of nonallergic parents had a more than threefold (p less than 0.01) higher incidence of elevated cord IgE (greater than or equal to 1.20 IU/ml) and a fourfold (p = 0.005) higher risk of developing definite or probable atopic disease before 18 months of age if the mother smoked than if she did not. Paternal smoking did not influence, in whatever the subgroup, cord IgE or subsequent infant allergy but increased cord IgD (p less than 0.001) among newborn infants with a negative family history even after controlling for maternal smoking (p less than 0.04). These results suggest that parental smoking in some way affects the fetal immune system, probably via substances in tobacco smoke. Maternal smoking in particular appears to exert a pronounced effect on the IgE system already in fetal life, predisposing even "low-risk" infants to subsequent sensitization, probably in synergy with a later acquired mucosal damage that would facilitate penetration of foreign matter. Pregnant women and mothers should be encouraged to try and give up smoking that might help to prevent allergic disease in their infants.

Major differences in specificity among naturally occurring human IgG-subclass anti-IgE autoantibodies.

BACKGROUND: The specificity of naturally occurring IgG-subclass anti-IgE autoantibodies (a-IgE Ab) was studied by ELISA inhibition assay with a highly purified IgE-DES myeloma protein as a solid-phase antigen. Because the IgG isotypes differ in effector functions, detailed specificity studies of IgG subclass anti-IgE antibodies might clarify their putative role. METHODS: Selected sera containing a high concentration of a single IgG a-IgE Ab subclass were allowed to react with purified immunoglobulins of all five classes including five different IgE myeloma proteins and papain-derived Fab epsilon and Fc epsilon fragments of IgE-DES. RESULTS: The inhibition results indicated that IgG1, IgG2, and IgG3 a-IgE Ab reacted in a low-affinity reaction with IgE myeloma protein-restricted determinants because only IgE-DES was inhibitory. Moreover, the reacting epitopes were heat resistant (2 hours, 56 degrees C) and localized in the Fab epsilon-DES fragment. In sharp contrast, IgG4 a-IgE Ab reacted with high affinity to all five IgE myeloma proteins involving heat-susceptible epitopes confined to the Fc epsilon fragment. CONCLUSIONS: It seems that a majority of IgG a-IgE Ab have a specificity for nonisotypic myeloma-restricted determinants whereas IgG4 a-IgE Ab are mainly isotype specific. These findings ought to be taken into account in the consideration of physiologic implications of IgG a-IgE Ab and in the interpretation of previous investigations in which intact IgE myeloma proteins have been used to detect IgG a-IgE Ab.

Autoantibodies of the IgM class against a human myeloma protein IgE(DES). I. Occurrence.

We report on the natural occurrence of human serum antibodies with specificity for a human monoclonal myeloma IgE(DES). These antibodies were of the IgM class, based on their susceptibility to reduction, sedimentation in sucrose gradients, gel filtration and inhibition of agglutination by anti-IgM antiserum. Autoantibody levels were studied in several groups of patients by particle-counting immunoassay using latex particles to which purified monoclonal IgE(DES) was coupled. Only sera of patients suffering from parasitosis had significantly higher levels (p less than 0.0005) than those of healthy blood donors. Cord sera had very low levels, followed by an age-dependent increase during early infancy. There was no relation (p greater than 0.10) between serum IgE and IgM antibody level. On the other hand, significant relations between IgM anti-IgE(DES) levels and serum IgM (p less than 0.0005), serum IgA (p less than 0.001) and serum IgG (p less than 0.05) were observed suggesting that high levels were caused by or related to polyclonal activation of the immune system.

Maternal smoking leads to increased cord serum IgG3.

IgG subclass levels in cord serum of unselected European newborns were studied in relation to parental smoking. Cord IgG3 was significantly (P less than 0.01) higher in newborns of smoking mothers (n = 18; geom.mean = 87.0%) compared to those of non-smoking mothers (n = 66; geom.mean = 61.0%). Maternal smoking did not influence (P greater than 0.10) cord IgG2 or cord IgG4. Paternal smoking did not affect any of the three immunoglobulins in cord serum (P greater than 0.10). These data suggest that tobacco smoking affects IgG3 synthesis in some direct or indirect way.

Particle counting immunoassay of immunoglobulin E antibodies after their elution from allergosorbents by pepsin: an alternative to the radioallergosorbent test.

The absorption of specific antibodies on allergen-coated cellulose discs, followed by pepsin digestion to release the IgE Fc" fragment, which is then assayed by particle counting immunoassay (PACIA), is a new practical and reliable method to quantitate IgE antibodies and to standardize allergens. The coefficient of variation of repeated assays does not exceed 12.8% and the correlation coefficient with RAST was r = 0.96. Some discrepancies could be explained by differences in the antiserum specificities and the presence of anti-IgE autoantibodies. The determination of IgE antibodies by PACIA offers the following advantages: (1) no radioisotopes; (2) stability of reagents, since antibody-coated latex particles and the standards keep their activity for more than 1 yr; (3) low cost of reagents due to ease of preparation --PACIA does not require purified antibodies to measure IgE but only F(ab')2 fragments of the IgG fraction of the anti-IgE antiserum to coat latex; (4) avoidance of nonspecific absorption of the labeled anti-IgE on the solid phase; (5) prevention by elution of the inaccuracies observed in RAST at very high titers of IgE antibodies; (6) expression of the results in international units per milliliter to calculate the ratio of specific IgE Ab vs total IgE, with a high ratio suggesting that the patient is sensitive to a few allergens; (7) standardization of allergenic extracts; and (8) the particular specificity of the anti-IgE antiserum directed against a heat- and protease-resistant fragment of IgE.