Mouse IgE Research Articles

Proteolytic digestion of mouse IgE

Conditions are described for the preparation of F(ab′) 2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab′) 2 fragments with M r ≊ 130,000 which yielded Fab fragments on further disgestion. The release of Fab fragments from F(ab′) 2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab′) 2 produced by papain. The best yields of difficulty of separating a 93 kDa by-product from the F(ab′) 2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin ≊ trypsin ⪢ papain .

Rapid Detection of Antigen-Specific Mouse IgE by Enzyme-Linked Immunosorbent Assay (Elisa)

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.

Comparison of allergenic potency of five batches ofAlternaria alternata for preparation of reference standard

Five batches of 34-016 isolate were grown separately on synthetic revised tobacco medium for 28 days. Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. The extracts had similar isoelectric focussing (IEF) patterns. In crossed-radioimmuno-electrophoresis (CRIE) using human atopic sera, each showed two dominant and two to three minor allergens. In direct radioallergosorbent (RAST) and RAST inhibition tests with human atopic sera and passive cutaneous anaphylaxis (PCA) tests with mouse IgE, all samples were of similar potency. In IgE immunoblots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, each extract showed strongly reactive bands at 16, 18, 30, 33 and 59–100 kDa MW. These results indicate that a reference preparation ofA. alternata suitable for standardization purposes can be obtained from any batch of 34-016 isolate.

Lymphoid cell subsets in colonie mucosa and HLA-DR antigens on colonic epithelia in colitis excluding ulcerative colitis and crohn’s disease

Lymphoid cell subsets, including T cells as well as Ig-containing cells in the colonic mucosa and HLA-DR antigens on colonic epithelia, were examined in non-IBD colitis (colitis excluding ulcerative colitis (UC) and Crohn's disease) by the indirect immunoperoxidase staining method. Mouse anti-CD5, CD8, CD4, IgG, IgA1, IgA2, IgM, IgD, IgE, HLA-DR, and NuIa monoclonal antibodies were used as the first antibody. The results were compared to those of the normal controls and UC. T cell subsets in non-IBD colitis were almost similar to those of the controls and UC. The number of Ig-containing cells of all classes, except for IgA, tended to be increased in non-IBD colitis. In particular, both IgG- and IgE-containing cells were significantly increased compared to those in the controls. Compared to UC, IgG-containing cells were decreased in non-IBD colitis. Namely, in non-IBD colitis, as well as in UC, the change of Ig-containing cells (B cell lineage) was more pronounced than that of T cells. The frequency of the expression of HLA-DR antigens on colonic epithelia in non-IBD colitis was 70%, which was significantly higher than that in controls (0%), but significantly lower than that in UC (100%). Whether the differences in the number of IgG-containing cells, and the frequency of epithelial HLA-DR expression between non-IBD colitis and UC was due to the differences of the degree of local inflammation or due to the differences of the nature of the two diseases was not elucidated in this study.

Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig.

Abstract We have investigated the interaction of mouse (m) IgE with its Fc epsilon RI on rat basophilic leukemia cells using a set of chimeric Ig that were constructed by exchanging homologous H chain C domains between human (hu) IgG1 and mIgE. Binding affinities were examined with equilibrium and kinetic measurements, and we found that epsilon/C gamma 3 (mIgE with C epsilon 4 replaced by C gamma 3) was indistinguishable from mIgE. The huIgG1 and the other chimeric Ig, which did not contain both C epsilon 2 and C epsilon 3, did not bind detectably to rat basophilic leukemia cells (Ka less than 10(6) M-1). The ability of these chimeric Ig to stimulate a cellular response (degranulation) in the presence of multivalent Ag was also tested. The epsilon/C gamma 3 was indistinguishable from mIgE in eliciting a high level of degranulation, whereas the other chimeric Ig stimulated no response even when they were preaggregated to enhance their binding avidity. These results demonstrate that C epsilon 4 may be replaced by C gamma 3 without affecting the binding and cell activating properties of mIgE. The lack of binding by the other chimeric Ig indicates that both C epsilon 2 and C epsilon 3 are necessary for the binding interaction.

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli.

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.

Antibody organization on lipid films. Influence of pH and interchain disulphide reduction

Two distinct lattice structures are observed for two-dimensional (2-D) antibody organization on phospholipid films. A low-order, small-unit-cell, square lattice is obtained at pH 7 and below for mouse IgE, mouse IgG2a and IgG2b and rabbit IgG. At pH 7.5 and above, the observed lattice structure switches to a large-unit-cell, hexagonal type for rabbit IgG and mouse IgE. Interchain disulphide reduction by exposure to 2 mM-dithiothreitol results in the formation of the compact 2-D lattice for all cases and for all pH conditions.

Suppression of mouse IgE response by immune complexes

The effect of antibodies alone or complexed to antigen on an IgE response was compared in mice immunized with a low dose of antigen. The results demonstrated that immune complexes (ICs) at equivalence were more efficient than antibodies in promoting a suppression of this response when they were injected simultaneously or before, but not after, the antigen. This effect was dose dependent, antigen specific, and long lasting. Furthermore, only isologous ICs were suppressive. ICs prepared with IgG1 antibodies were more effective than ICs prepared with IgG2. Digestion of the Fc fragment of the antibody eliminated completely the suppressive action of antibodies alone, but not of ICs. These results indicate that, whereas an Fc-dependent mechanism is responsible for the effect of antibodies, an additional mechanism is also operating in suppression induced by ICs.

Purification of human basophils using mouse monoclonal IgE

A method was developed to purify human blood basophils using monoclonal mouse IgE. Enriched suspensions of basophils were sensitized with IgE anti-trinitrophenol (TNP) hapten which was prepared from hybridoma culture supernatants. Rosettes were induced with TNP-coupled red cells and separated from contaminating cells on Percoll gradients. Basophils were dissociated from red cells using cold hypotonic lysis. Using this technique the percentage of basophils was increased from less than 1% to an average of 83.5 ± 7.8% with a recovery of 58.0 ± 12.7%. Final purities were highest when enriched sensitized suspensions contained ≥ 10% basophils. Using a radioenzymatic assay, purified basophils were shown to contain an average of 1.3 ± 0.3 pg histamine. Anti-human, but not anti-mouse IgE, released histamine at an optimal dilution. Using this technique, in 3–4 h, we are able to prepare highly purified suspensions of basophils for further study.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters: An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10 5−10 6 in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Enhancing effect of malondialdehyde modification on the mouse IgE response to protein antigens.

To investigate the immunochemical properties of proteins reacted with malondialdehyde (MDA), which is a major degradation product of lipid oxidation, the antibody responses of mice immunized with MDA-modified bovine serum albumin (BSA) were examined. The specific IgE response to MDA-BSA was 4 to 8 times higher than that to native BSA, suggesting that the specific IgE antibody response to MDA-modified protein was enhanced by MDA bound to the surface of the protein. The antibodies to MDA-BSA did not distinguish the modified protein from the native one, and the antibodies raised against the modified proteins were not specific for the MDA portion but were for the carrier proteins.

Molecular structure and expression of the murine lymphocyte low-affinity receptor for IgE (Fc epsilon RII).

The cDNA encoding the murine low-affinity receptor for IgE (Fc epsilon RII) has been isolated from a cDNA library prepared from B cells activated with lipopolysaccharide and interleukin 4. It encodes a 37-kDa protein of 331 amino acids with two potential N-linked glycosylation sites. Analogous to its human counterpart, there is no signal sequence and the putative transmembrane region is close to the amino terminus, indicating an inverse membrane orientation with the carboxyl terminus at the cell exterior. The predicted murine Fc epsilon RII amino acid sequence demonstrates a 57% identity with its human counterpart. The murine sequence has an additional internal repeat motif of 21 amino acids giving four repeats as compared to three in the human sequence. Furthermore, the murine Fc epsilon RII is truncated at the carboxyl terminus and the Arg-Gly-Asp sequence, a common recognition site of integrin receptors, which is found in the reverse configuration in human Fc epsilon RII, is missing. B cells activated with interleukin 4 and lipopolysaccharide have an increased amount of Fc epsilon RII mRNA as compared with resting or lipopolysaccharide-stimulated B cells. Con A-activated normal T cells, the TH-2 cell line D10, as well as the macrophage cell line J774 have no detectable Fc epsilon RII mRNA. Expression analysis using transiently transfected COS cells revealed that recombinant murine Fc epsilon RII binds anti-Fc epsilon RII as well as mouse and rat IgE but does not bind human IgE or mouse IgG. Fc epsilon RII expressed in COS cells has a molecular mass of 45 kDa whereas the Fc epsilon RII from B-cell lines is a 49-kDa protein.

Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells.

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.

Trypanosoma musculi co-express several receptors binding rodent IgM, IgE, and IgG subclasses.

The present work demonstrates the expression of receptors for the Fc portion of rodent Ig by the murine parasite Trypanosoma musculi. By using a rosette assay adapted to the parasite morphology and by flow cytometry analysis, three distinct receptors were identified. A receptor binding rabbit or rat polyclonal IgG and mouse monoclonal IgG1, IgG2a, and IgG2b was found on parasites purified from the blood and the peritoneal cavity of infected mice and on parasites maintained in culture conditions. This IgG receptor was degraded by pepsin. A separate receptor, binding only mouse monoclonal IgG3 was observed on cultured parasites. A receptor binding rabbit, rat, and mouse IgM was found on cultured and peritoneal parasites, but not on blood parasites. This receptor did not bind IgG or IgA but it bound mouse and rat IgE as well as IgM. It was degraded by trypsin. IgG and IgM/IgE receptors were co-expressed on single parasites. They were not of host origin but synthesized by trypanosomes as shown by reexpression in vitro after proteolytic degradation. Their expression was variable with the development of trypanosomes both in vitro and in vivo.

Comparative studies of allergens from mycelia and culture media of four new strains ofAlternaria tenuis

Abstract Four new isolates, identified as Alternaria tenuis, 34–016, 34–039, 35–056 and 35–193 were grown on revised tobacco medium. Mycelial and broth extracts were prepared and their biochemical and immunological properties were examined. Compared to the mycelial extracts, the four broth extracts had lower protein contents and showed fewer protein bands in isoelectric focusing, crossed-immunoelectrophoresis and sodium dodecylsulphate polyacrylamide gel electrophoresis. However, direct radioallergosorbent and passive cutaneous anaphylaxis tests with mouse IgE revealed that mycelial and broth extracts were of similar potency and had extensive cross-reactivity. In crossed-radioimmunoelectrophoresis using human atopic sera, the mycelial extracts showed 1–2 dominant and 3–4 minor allergens while the broth extracts had one dominant and one minor allergen. In RAST inhibition tests, two isolates, 34–039 and 35–056, had 100-fold more allergen content in the broth extracts compared to the mycelial extracts, whereas the levels were similar for broth and mycelial extracts of the other two isolates. The results indicated that culture broths of 34–039 and 35–056 were excellent starting materials for purification of some of the major allergenic components of A. tenuis as they combined good yield with high allergenic activity, and lower level of other protein components. In addition, extracts containing wide spectrum of allergens and suitable for diagnostic purposes, can be prepared from mycelia and culture fluid of these isolates.

Implanted IgE-Fc.epsilon.R complexes elicit IgE-mediated activation of RBL-2H3 cells

The high-affinity receptor for IgE (Fc epsilon R) is the cellular trigger of the antigen-induced activation of mast cells and basophils. To examine the functional integrity of Fc epsilon R, we have adopted a protein implantation procedure whereby the purified receptor complex was coreconstituted with Sendai virus envelopes. The latter promoted fusion of the hybrid vesicles with recipient cells such as rat basophilic leukemia, RBL-2H3, thus serving as a vehicle for the receptor. The implanted Fc epsilon R was complexed with 125I-labeled mouse IgE (anti-DNP) to permit receptor quantification as well as specific triggering by DNP20BSA. Implantation in the presence of unlabeled rat IgE, which blocked the native receptors on the recipient RBL-2H3 cells, resulted in incorporation of up to 15 ng of receptor-bound IgE/10(6) cells. This was roughly equivalent in amount to 10-20% of the native receptors on such cells. The exocytosis which was triggered in the recipient cells by reagents that specifically recognized the implanted IgE reached between 15 and 50% of the maximal response. Various treatments that interfered with the activities of the viral envelopes reduced both receptor incorporation (3-5-fold) and cell degranulation (3-10-fold). These included separation of the receptor from the reconstituted envelopes, addition of serum to the incubation mixture (to inhibit vesicle-cell binding), and trypsinization of the virus (to inhibit vesicle-cell fusion). Poly(ethylene glycol) 8000 (4%) enhanced both the incorporation of the receptor and its functional responses. These treatments distinguished between real incorporation of IgE-Fc epsilon R complexes and other mechanisms of 125I-IgE association with the recipient cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA

A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then β- d-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-β- d-galactosidase-conjugated streptavidin were added and, of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.

Antigen-induced bronchopulmonary alterations in the guinea pig: a new model of passive sensitization mediated by mouse IgE antibodies.

A selective model to study the IgE-mediated anaphylactic bronchoconstriction (BC) in the guinea pig is needed, since human asthma involves mainly this class of antibody. However, most procedures presently available for passive homologous or active sensitization lead to responses which are mediated both by IgE and IgG antibodies. In this study, we developed an anaphylactic model in which guinea pigs are passively sensitized with mouse ascitic fluid containing dinitrophenol (DNP)-specific IgE antibodies. Challenge of sensitized animals with DNP coupled to bovine serum albumin evokes a bronchoconstrictor response that is maximal 5 h after sensitization. The resulting anaphylactic BC is not blocked by the H1 histamine antagonist mepyramine, by the peptido-leukotriene antagonist FLP 55712 nor by the platelet-activating factor antagonist BN 52021 alone. However, when the sensitized animals are pretreated with the three drugs in combination, significantly reduced BC was observed upon challenge with the antigen. This latter result indicates that IgE-dependent BC involves the participation of different mediators, a characteristic shared in common with allergic asthma in human.

Terasaki-ELISA for murine IgE antibodies: II. Quantitation of absolute concentration of antigen-specific and total IgE

A Terasaki tray-based ELISA system was developed for the quantitative measurment of antigen-specific and total IgE antibodies in 5 μl samples of mouse serum dilutions. The assay was based upon non-competitive binding of mouse IgE antibodies between the immobilized appropriate antigen or capture antibodies and the detecting rabbit antibodies. A conjugate of protein A-labelled β-galactosidase and the fluorigenic substrate methylumbelliferyl-β- d-galactoside were used as a detecting system. The resulted fluorescence could be measured rapidly and automatically using an inverted micro-fluorimeter. These measurements were automatically transformed into absolute concentrations by a microprocessor-based program using a four-parameter logistic function and an absolute IgE standard. The assay was shown to have a detection limit of 0.04 ng/ml and a range of linearity of 0.04–20 ng/ml, which is sufficient to measure IgE concentrations in mouse serum.