Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Monoclonal antibodies to the 27-34K insulin-like growth factor binding protein

Monoclonal antibodies were prepared against the 27-34K insulin-like growth factor (IGF)-binding protein purified from human placenta/decidua and designated placental protein 12 (PP12). Four different antibodies were characterized. Each recognized the major band at 32K on immunoblots of the purified PP12 preparation and amniotic fluid. In liquid phase RIA, IGF-I did not affect the binding of [125I] PP12 to one antibody (Mab 6303), it slightly increased the binding to two antibodies (Mab 6301 and 6304), and it slightly decreased the binding to one antibody (Mab 6302). All antibodies immunoprecipitated the cross-linked PP12-[125I] IGF-I complex, but Mab 6302 considerably less effectively than the others. Preincubation of PP12 with Mab 6302 completely inhibited the binding of [125I] IGF-I to PP12, whereas preincubation with Mab 6303 had no effect, and Mab 6301 as well as Mab 6304 increased it. These results suggest that Mab 6302 binds to an epitope at or near to the IGF-binding site, whereas the other antibodies react at other sites of the PP12 molecule. Conformational changes in PP12 probably account for the IGF-I-induced increase in the binding of Mabs 6301 and 6304 to [125I] PP12, and vice versa, for Mabs 6301- and 6304-induced increase in the binding of [125I] IGF-I to PP12.     

Two major human allergenic sites on ragweed pollen allergen antigen E identified by using monoclonal antibodies

Murine monoclonal antibodies specific for antigen E (AgE), the major allergen isolated from short ragweed pollen, have been produced and characterized. These monoclonal antibodies, when coupled to Sepharose and used as immunoadsorbents, specifically bound AgE when a crude pollen extract was passed through the column. Three antigenic sites (A, B, and C) on AgE were identified by using five of these monoclonal antibodies in both inhibition and double-bind solid-phase ELISA. These three antigenic sites appear to be nonoverlapping and nonrepeated, that is, present only once on each AgE molecule. Site C on AgE could readily be bound by the monoclonal antibody specific for that site, but only when AgE was in solution or "presented" by an anti-site A or anti-site B antibody. Site C appears to be only marginally available for binding when AgE is directly adsorbed to polyvinyl chloride microtiter wells. The majority of monoclonal antibodies isolated after immunization of BALB/c mice were specific for site A on AgE. In addition, the binding to AgE of pooled BALB/c polyclonal, hyperimmune antisera against AgE was blocked approximately 80% by a monoclonal antibody directed against site A, but was only blocked approximately 20% by an anti-site B monoclonal antibody. This suggests that site A on AgE is the predominant antigenic site in the BALB/c immune response and that site B represents a less dominant site. The binding of IgE in pooled human serum from ragweed-allergic individuals is blocked approximately 50% by a monoclonal antibody directed to site A on AgE and also approximately 50% by a monoclonal antibody directed against site B. A series of individual human short ragweed allergic antisera also showed significant, although varied, inhibition of IgE binding to AgE by both anti-site A and anti-site B monoclonal antibodies. Simultaneous addition of anti-site A and anti-site B was somewhat additive and inhibited up to 80% of the binding of human IgE specific for AgE. The conclusion from these data is that site A and site B defined by two murine monoclonal antibodies represent two very major allergenic sites in the human response to this molecule.     

Heterogeneous IgE glycoforms characterized by differential recognition of an endogenous lectin (IgE-binding protein)

IgE is highly glycosylated, but the function of the oligosaccharide side chains is largely unknown. The previous discovery of an animal lectin, IgE-binding protein (epsilon BP), affords an opportunity to study potential carbohydrate-dependent effector functions of IgE. epsilon BP is a beta-galactoside-specific lectin with binding affinity for IgE and is now known to be equivalent to carbohydrate-binding protein 35 and the Mac-2 Ag; thus, it may have multiple functions in addition to IgE binding. We have previously shown that rat r epsilon BP recognizes sialidase-treated human myeloma IgE to a much greater extent than the untreated IgE. In contrast, human epsilon BP binds essentially equivalently to a monoclonal murine IgE with or without sialidase pretreatment. To validate a possible role for epsilon BP in the IgE system, we investigated the pattern of recognition of epsilon BP for various polyclonal human IgE samples. We show that polyclonal IgE derived from four individuals with hyper-IgE syndrome or atopic dermatitis recognizes epsilon BP and that there is individual variation in the proportion of IgE recognized by epsilon BP, ranging from greater than 60% for one sample to almost undetectable levels in another. We conclude that epsilon BP does indeed recognize polyclonal IgE and that this recognition is modulated by sialylation of IgE oligosaccharides. Furthermore, there exist different IgE glycoforms, varying in the degree of sialylation, and these are distributed in a distinct manner in different individuals.     

Transfectomas expressing both secreted and membrane-bound forms of chimeric IgE with anti-viral specificity.

Because of the lack of a cell line expressing on surface and secreting human IgE of known Ag specificity, the construction of a transfectoma line possessing such properties would be useful for studying the roles of surface IgE and the effects of anti-IgE antibodies on IgE-producing B cells. Toward this goal, the human genomic DNA segment encompassing the two exons encoding the membrane anchor peptide of epsilon-chain and their flanking regions was sequenced. Hybrid epsilon and kappa genomic DNA comprising the C regions of human epsilon- and kappa-chains and the H and L chain V regions of the murine mAb BAT123, which reacts with the gp120 envelope protein of HIV-1, were constructed. Mammalian expression vectors containing these fusion genes were used to transfect murine myeloma Sp2/0 cells, and transfectants stably expressing on surface and secreting into culture medium chimeric IgE were obtained. The chimeric IgE showed identical Ag-binding properties as the murine mAb BAT123. Acting in concert with the specific peptide Ag polyvalently coupled to a protein carrier, the chimeric antibody could induce histamine release from human blood basophils. These results demonstrate the potential utility of the transfectoma cells and the chimeric IgE in studying the roles of membrane-bound IgE and effects of anti-IgE antibodies on IgE-producing B cells.     

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.     

The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I, of Dermatophagoides pteronyssinus. Relative binding site location and species specificity studied by solid-phase inhibition assays with radiolabeled antigen

The relative binding site location and species specificity of 19 mouse hybridoma antibodies, produced in four laboratories, to Dermatophagoides pteronyssinus major fecal allergen, Der p I, was studied by using immobilized mAb and inhibitions of radiolabeled Ag binding. Four mAb groups were defined, within which 4, 6, 8, and 5 mAb, respectively, cross-inhibited each other. Five mAb were members of both group 2 and 3, demonstrating a considerable overlap of epitopes between the corresponding antibody-binding regions. The degree of mAb species specificity, as assessed by inhibition with cold Der p I and Ag Der m I and Der f I from the related species, Dermatophagoides microceras and Dermatophagoides farinae, was highly variable even for mAb binding to the same region on the Ag. Five cases of cross-reactivity between Der p I and Der m I and one case of cross-reactivity between Der p I and Der f I were found. The N-terminal 30 amino acids of the three species showed 7 substitutions between Der p I and Der m I/Der f I and 2 between Der f I and Der p I/Der m I. Single mAb inhibited up to 65% of labeled Der p I binding to immobilized human IgE from allergic patients' sera and up to 24% of labeled Der p I binding to immobilized rabbit antibodies. The spectrum of species specificities in human IgE sera, as assessed by inhibitions with cold Ag, was similar to that of the mAb. No evidence for the presence of strictly sequential epitopes, reactive with either mAb or human IgE was found, as judged from the weak inhibitory activity of acid-denatured Der p I.     

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen,Cyn d 1

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.     

Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals

Using serum from human atopic individuals with a sufficiently high titre of IgE and IgG antibodies to birch- or hazel-pollen allergens and antigens, the localization of IgE binding sites in birch- and hazel-pollen grains was determined by pre- and post-embedding electron microscopic immunoautoradiography with 125I-anti-IgE, whereas the IgG binding sites were localized in ultrathin sections of birch-pollen grains by the protein-A/gold technique. Concerning the distribution patterns of both IgE/IgG binding sites within the pollen grains, no difference could be observed in the dormant pollen grain: Labelling was found in the exine part of the pollen wall and throughout the highly condensed cytoplasm except for starch grains and lipid droplets. The intine part and the germination pores were almost completely unlabelled. In pollen grains which had been soaked in a hypotonic buffer for 15 min, however, IgE binding sites were predominantly localized within the intine and the germination pores. The specificity of the labelling reactions and the observed differences in the localization patterns are discussed.     

Preparation and characterization of murine monoclonal antibodies that express both cold agglutinin and cryoglobulin activities

To obtain murine cold agglutinin (CA) and cryoglobulin antibodies, BALB/c mice were hyperimmunized with heat-killed type XIV Streptococcus pneumoniae. The spleen cells of these mice were fused with either the P3 NS1/Ag4. 1 or P3 X63/Ag.653 cell line. Several stable hybridomas were obtained that produced monoclonal antibodies (Mab) that reacted with rabbit and human erythrocytes only at temperatures below 37 degrees C. Three of these Mab were also cryoglobulins, as evidenced by their insolubility at reduced temperature. All of the antibodies studied were IgM(k) and reacted with purified type XIV S. pneumoniae polysaccharide at room temperature. With one exception, all antibodies were specific for N-acetyl-lactosamine, the immunodominant sugar residue expressed on type XIV polysaccharide. Inhibition experiments demonstrated that both CA activity and cryoprecipitation were inhibited by the same sugar compounds in the same order of efficiency. The data presented strongly suggest these CA antibodies are cross-reactive members of a S. pneumoniae-specific population. Cryoprecipitation persisted in antibodies purified under conditions that would exclude the presence of trapped serum antigens. It is therefore proposed that the cryoprecipitation observed is a result of the interactions of the antibody combining sites with carbohydrate residues of adjacent antibody molecules.     

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.     

The use of Fab-masking antigens to enhance the activity of immobilized antibodies

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water-soluble adducts of poly(2-methyloxazoline) polymers and a syntheticpeptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab-masking antigents (FMAs). The antibody used in this study is a Ca(2+)-dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre-equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA-Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC-Mab complexes formed by immunosorbents produced by either masked or unmaked antibody. These results provide evidence that orientgation may play an important role in the binding activity of immobilized antibodies.     

Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

Dissection of the combining site in a humanized anti-Tac antibody.

The genetically engineered "humanized" anti-Tac antibody (HAT) has been shown to bind the p55 chain of the human IL-2R with an affinity close to that of the murine anti-Tac. Although the HAT molecule contains all six mouse CDR, it was not known which, and to what extent, each of the CDR contributes to Ag binding. These questions were addressed by constructing a series of variant HAT antibodies, each substituting a single HAT CDR with a heterologous CDR. The association constants of the variant HAT antibodies to p55 were determined by competitive binding analysis. We find that CDR 1 and 3 of the H chain and CDR 3 of the L chain are essential for maintaining binding. The remaining three CDR appear to be involved to a lesser degree.     

Human pre-B and B cell membrane mu-chains are noncovalently associated with a disulfide-linked complex containing a product of the B29 gene.

B cell activation after Ag binding to membrane Ig (mIg) is mediated by a complex series of events that involves proximal activation of a tyrosine kinase and phospholipase C. Until recently it was unclear how mIgM and mIgD, with their limited cytoplasmic domains (three amino acids on each H chain), were able to couple to these secondary signal transducers. Studies of murine B cells conducted in several laboratories, including our own, suggest that products of the mb-1 (IgM-alpha or IgD-alpha) and B29 (Ig-beta, Ig-gamma) genes occur as disulfide-linked alpha/beta and alpha/gamma heterodimers that are noncovalently associated with mIgM and mIgD. Although studies utilizing Daudi and Raji cell lines indicate that human mIgM is also associated with a dimer containing the mb-1 gene product, the other molecules associated with the human receptor have not been identified. In this report we characterize the phosphoproteins that are noncovalently associated with mIgM on human tonsillar B cells and human pre-B cell lines. mIgM is noncovalently associated with a disulfide-linked heterodimer composed of variably glycosylated forms of two core proteins with apparent molecular mass of 26.5 and 27 kDa. Western blotting analysis reveals that the lower m.w. component of each of the mIgM-associated heterodimers and its 27-kDa deglycosylated core protein are reactive with antibodies against the murine B29 gene product. Thus, a product of the B29 gene is a component of the AgR complex in human and murine B cells, occurring as a disulfide linked dimer with product(s) of the mb-1 gene. Interestingly, mb-1 and B29 gene products expressed on human cells are much more heterogenously N-glycosylated than their murine B cell counterparts.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen.

Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.     

Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.