Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies

Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.     

A Murine Monoclonal Anti-Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.     

Allergenic fragments of ryegrass (Lolium perenne) pollen allergen Lol p IV

To facilitate studies on establishing the nature of structure/function relationships of allergens, ryegrass pollen allergen, Lol p IV, was cleaved into smaller fragments by cyanogen bromide (CNBr) and the resulting peptides were further digested with trypsin. The resulting peptides were then fractionated by high performance liquid chromatography (HPLC) on a C-18 reverse phase column. The allergenic activity of the HPLC fractions was evaluated in terms of their ability to inhibit the binding of 125I-Lol p IV to serum IgE antibodies of a grass-allergic patient. Many of these fractions inhibited the binding between the native allergen and IgE antibodies in a dose-dependent manner. The inhibitions were specific, i.e., the fractions did not inhibit the binding between 125I-Lol p I (a group-I ryegrass pollen allergen) and the IgE antibodies present in the allergic human serum. The possibility that the allergenic peptide fractions were contaminated by the native undegraded allergen, which might have accounted for the observed inhibition, was ruled out by the fact that the native allergen could not be detected by SDS-PAGE and the elution profiles of allergenically active peptides did not coincide with that of native allergen. One of the allergenic sites recognized by monoclonal antibody (Mab) 90, i.e., site A, was located in HPLC fractions 90-100 while another allergenic site B (recognized by Mab 12) appeared to be lost following the sequential digestion of Lol p IV with CNBr and trypsin.     

Monoclonal antibodies that distinguish antigenic variants of canine parvovirus

Canine parvovirus (CPV) is classified as a member of the feline parvovirus (FPV) subgroup. CPV isolates are divided into three antigenic types: CPV type 2 (CPV-2), CPV-2a, and CPV-2b. Recently, new antigenic types of CPV were isolated from Vietnamese leopard cats and designated CPV-2c(a) or CPV-2c(b). CPV-2c viruses were distinguished from the other antigenic types of the FPV subgroup by the absence of reactivity with several monoclonal antibodies (MAbs). To characterize the antigenicity of CPV-2c, a panel of MAbs against CPV-2c was generated and epitopes recognized by these MAbs were examined by selection of escape mutants. Four MAbs were established and classified into three groups on the basis of their reactivities: MAbs which recognize CPV-2a, CPV-2b, and CPV-2c (MAbs 2G5 and 20G4); an MAb which reacts with only CPV-2b and CPV-2c(b) (MAb 21C3); and an MAb which recognizes all types of the FPV subgroup viruses (MAb 19D7). The reactivity of MAb 20G4 with CPV-2c was higher than its reactivities with CPV-2a and CPV-2b. These types of specificities of MAbs have not been reported previously. A mapping study by analysis of neutralization-resistant mutants showed that epitopes recognized by MAbs 21C3 and 19D7 belonged to antigenic site A. Substitution of the residues in site B and the other antigenic site influenced the epitope recognized by MAb 2G5. It was suggested that the epitope recognized by MAb 20G4 was related to antigenic site B. These MAbs are expected to be useful for the detection and classification of FPV subgroup isolates.     

Development and characterization of monoclonal antibodies to chicken riboflavin carrier protein

Several monoclonal antibodies (MAbs) specific to chicken riboflavin carrier protein (cRCP) were developed and characterized. Of the several MAbs analyzed, four were directed against nonoverlapping epitopes as demonstrated by MAb inhibition assay. Many of these epitopes appeared to be in close proximity and only three were situated at distinct part of the molecule as revealed by sandwich assay. A combination of chemical modification, peptide cleavage by chemical and enzymatic methods, was used to analyze the possible antigenic structure recognized by these MAbs. An assembled epitope spanning the region 22-87 forms the antigenic site recognized by 4999.1; while MAb 5555.3 interacted with the C-terminal peptide 203-219.     

Neutralization sites of type O1 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants

Monoclonal antibodies (MAbs) were derived from mice infected with foot-and-mouth disease virus type O1 Brugge (FMDV 01B) or immunized with inactivated virions (140 S) or viral subunits (12 S). A total of 19 neutralizing MAbs were characterized of which 17 recognized conformationally determined epitopes and two recognized amino acid sequences on isolated VP1. Neutralizing MAbs were used to select antigenic variants of FMDV O1B. Based on cross-neutralization and binding assays with MAbs the variants were divided into discrete groups demonstrating the presence of three unique neutralization sites on FMDV O1B. One site was present only on intact 140 S virions, a second was present on both 140 S virions and 12 S subunits, and the third was present on 140 S virions, 12 S subunits, and isolated VP1. Comparison of the deduced nucleic acid sequence of parental FMDV O1B with those of the O1B variants demonstrated that the epitope recognized by the VP1-reactive, neutralizing MAbs included amino acid residues 138, 144, and 148. Cross-neutralization assays demonstrated that these neutralization sites of FMDV O1B function on other type O1 strains of FMDV.     

Epitope mapping of horseradish peroxidase with use of monoclonal antibodies.

Nine mouse monoclonal antibodies (MAbs) to horseradish peroxidase (HRP) were used to develop an epitope map of the enzyme. The results of a competitive binding assay indicated three distinct patterns of reactivity. Two groups of MAbs (I and III) recognized epitopes located in separate antigenic regions on the molecule; another (II) bound to sites that overlapped with epitopes in either region I or III. Further definition of these regions was obtained by analyzing the MAbs for their binding to isolated heme, other peroxidases and heme-containing proteins, and to denatured and apo-HRP. None of the group I MAbs bound heme, suggesting that this region was removed from the active site of the enzyme. All of the group II and III MAbs bound heme as well as the other peroxidases and heme-containing proteins, indicating that they recognized heme-associated epitopes at or near the active site. Only one MAb (2A2) in groups II and III bound to apo-HRP but not to denatured HRP; it was also the only MAb in the entire panel that inhibited the catalytic activity of HRP. This suggests that the epitope recognized by 2A2 involves both the heme moiety and a conformationally dependent protein determinant near the active site.     

Antigenic characterization and analysis of the human immune response to outer membrane protein E of Branhamella catarrhalis.

Outer membrane protein E (OMP E) is a 50-kDa major OMP of Branhamella catarrhalis. Polyclonal antisera and four monoclonal antibodies (MAbs) to OMP E were generated to study its antigenic structure. All antibodies recognized epitopes in all 19 B. catarrhalis strains tested by immunoblot assays. By flow cytometry, it was determined that MAbs 1B3 and 9G10d recognized epitopes which are expressed on the surface of the intact bacterium, while MAbs IC11 and 7C10 recognized epitopes which were buried within the outer membrane. A competitive enzyme-linked immunosorbent assay showed that MAbs 1B3 and 9G10d recognize the same or closely related epitopes. Proteinase K treatment of whole bacterial cells revealed that MAbs 1B3 and 9G10d recognize a surface-exposed epitope located in the 17-kDa region towards the amino terminus of OMP E. The human serum and mucosal antibody responses to OMP E in adults with chronic bronchitis were studied. A majority of these patients had immunoglobulin A to OMP E in sputum supernatants. None of ten adults who experienced lower respiratory tract infections due to B. catarrhalis demonstrated a clear-cut rise in antibody titer to OMP E in serum or sputum supernatant. This study has demonstrated that OMP E has at least one surface-exposed epitope which is highly conserved among strains of B. catarrhalis and which is located in the amino-terminal 184 amino acids of the molecule.     

Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies

Summary Monoclonal antibodies (MAbs) were prepared to analyze antigens on the major inner capsid protein, VP 6 of avian group A rotavirus. Based on the results of a competitive binding assay using 15 MAbs directed against VP 6 of the PO-13 rotavirus strain, isolated from a pigeon in Japan, it was found that VP 6 of avian rotavirus possesses at least four spatially distinct antigenic sites. Two antigenic sites (I and II) were topologically distinct from the other two (III and IV), which were in close proximity. From the reaction of MAbs in indirect immunofluorescent antibody tests to a series of known rotaviruses, epitopes representing common antigens of all group A rotavirus including avian rotavirus were localized in sites II and III. One epitope in site IV appeared to have a subgroup antigenic specificity that reacted only with rotaviruses belonging to subgroup I. Interestingly, avian rotaviruses isolated from turkeys and chickens in Northern Ireland also reacted only with these subgroup I specific MAbs, but not with subgroup II specific MAb. This indicates that avian rotavirus has subgroup I specific antigen, which is antigenically similar to that of other mammalian rotavirus strains.     

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.     

Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies.

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.     

A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination

A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.     

An analysis of the properties of monoclonal antibodies directed to epitopes on influenza virus hemagglutinin

Summary Monoclonal antibodies (MAbs) specific for the hemagglutinin (HA) of the H3 subtype of influenza A virus were grouped according to their inability to bind to particular MAb-selected neutralization escape mutants of the virus having an amino acid substitution in one of the five postulated antigenic sites on the molecule. Additional residues critical to the binding of the MAbs were deduced from their patterns of reactivity with a panel of field strains and receptor mutants of the H3 subtype. The relationship of these residues to the actual epitopes recognized by the MAbs was inferred from their location on the three-dimensional structure of the HA molecule. In this way it was generally possible to identify a number of residues that are critical to the integrity of the epitope recognized by each of the MAbs examined. It was found that: (1) Several of these epitopes appear to be discontinuous and some may depend on residues contributed by more than one monomer. For example, residue 205, in the interface between monomers of the HA, was found to affect the integrity of the epitopes for several MAbs, possibly by stabilizing the conformation of residues around the receptor-binding pocket and/or in site B on the adjacent monomer. The activity of these particular MAbs was greatly decreased if the virus was exposed to pH 5. (2) All the MAbs tested neutralized viral infectivity and inhibited hemagglutination, although the single MAb directed to site C, which is the most distant from the receptor-binding site, was the least efficient. (3) Hemagglutination inhibition, and particularly neutralization tests, were more discriminating than ELISA in discerning subtle differences between the corresponding epitopes recognized by MAbs on different field strains. (4) Efficiency of neutralization of infectivity did not correlate consistently with hemagglutination inhibiting efficiency; MAbs postulated to bind to epitopes close to the receptor-binding pocket were very efficient at inhibiting hemagglutination, whereas neutralization efficiency tended to be more influenced by the affinity of binding of the MAb. (5) A MAb binding to any particular epitope could affect the binding of a second MAb directed to an epitope within the same or even a different antigenic site. The observed effect was most commonly inhibition of binding, which was not always reciprocal; enhancement of binding was also observed with certain combinations of MAbs. The relative affinity of the MAbs, in addition to steric constraints, were shown to be important factors in the ability to compete for interaction with HA.     

IgE and IgG cross-reactivity among Lol p I and Lol p II/III

In this study, the homologous C-termini of Lol p I Lol p II, and Lol p III were shown to contain cross-reactive B-cell epitopes. This was demonstrated by inhibition studies with purified Lol p I, II, and III and synthetic peptides of their C-termini. It was ruled out that the observed cross-reactivity was caused by cross-contamination of the purified allergens. Both human IgE and IgG bound to the C-terminus of Lol p I. These antibodies were cross-reactive with Lol p II and, more specifically, with its C-terminus. Within a small panel of allergic patients, no cross-reactivity with Lol p III was found. A hyperimmune polyclonal rabbit antiserum against Lol p I also recognized the Lol p I C-terminus. As for human antibodies, cross-reactivity with Lol p II and its C-terminus was demonstrated. Cross-reactivity with Lol p III was demonstrated with C-terminal peptides, but not with native Lol p III. A polyclonal rabbit antiserum against Lol p II bound to the C-terminal peptides of both Lol p II and III. This binding was inhibited with Lol p I, confirming that cross-reactive structures exist not only on the C-termini of Lol p II and Lol p I, but also of Lol p III and Lol p I. The existence of cross-reactivity between Lol p I and Lol p II and III possibly contributes to the frequently observed cosensitization for these allergens in grass-pollen-allergic patients.     

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA

BACKGROUND: Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. OBJECTIVES: To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. METHODS: Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. RESULTS: The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. CONCLUSION: This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.     

Immunological characterization of the lipooligosaccharide B band of Bordetella pertussis.

Two structurally and immunologically different components of Bordetella pertussis endotoxin can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining: a major A band and a faster-migrating minor B band. Certain mutant strains of B. pertussis express only the B band, while the wild-type strains produce both lipooligosaccharides (LOS). Two monoclonal antibodies (MAbs) directed against the minor LOS B band were generated, allowing the study of this surface molecule on different strains of Bordetella. These two MAbs, designated BL-8 and BL-9, reacted strongly with phenol-water-purified LOS obtained from a B. pertussis LOS B mutant strain. Sodium periodate treatment of the purified LOS prevented binding of the MAbs, indicating the carbohydrate nature of the epitope(s). Western immunoblotting experiments revealed that the epitope(s) recognized by these MAbs is conserved on all B. pertussis and Bordetella bronchiseptica Vir- (avirulent) variant strains tested but is not present on Bordetella parapertussis and B. bronchiseptica Vir+ (virulent) wild-type strains. Further studies showed that although present in the lipopolysaccharide B band expressed by Vir- strains, the epitope(s) recognized by the MAbs is not accessible on the surface of intact B. bronchiseptica cells. For B. pertussis, the density and accessibility of this epitope(s) are dependent on the virulence-associated or LOS phenotype expressed by the strain. Our data demonstrate that the expression and accessibility of the epitope(s) are significantly greater on the LOS B variant strains and LOS AB Vir- strains compared with fresh B. pertussis clinical isolates. For these latter strains, which are Vir+, this epitope(s) was barely detectable on the surface of intact bacteria, despite Western blot analyses that revealed specific reactions between the MAbs and the LOS B band. The two LOS B-specific MAbs had no bacteriolytic activity against a LOS AB wild-type strain, while the control MAb BL-2, which is specific for the B. pertussis LOS A band, significantly reduced the number of living bacteria in the same assay. Moderate lytic activity against a mutant strain expressing only the LOS B band was observed for MAb BL-8 but not for MAb BL-9 or BL-2. These data demonstrate that the type, amount, and surface exposure of the LOS are related to the phenotype expressed by a specific B. pertussis strain. In addition, the LOS B MAbs also reveal the antigenic conservation of carbohydrate epitopes among B. pertussis and B. bronchiseptica strains.     

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.     

Antigenic structure of transmissible gastroenteritis virus nucleoprotein.

A group of 11 monoclonal antibodies (MAbs) raised against transmissible gastroenteritis virus (TGEV) was used to study the antigenic structure of the virus nucleoprotein (N). To identify the regions recognized by MAbs, DNA fragments derived from the N-coding region of the TGEV strain FS772/70 were cloned into pUR expression plasmids and the antigenicity of the resulting fusion proteins was analyzed by immunoblotting. A major antigenic domain was identified, covering the first 241 amino acid residues of N, within which an epitope (residues 57-117) was also found. A second antigenic domain extended from residues 175 to 360 of the nucleoprotein, within which a subsite was characterized within the region covering residues 241-349. MAb DA3 recognized a linear epitope which mapped within residues 360 and 382 at the carboxy terminus of the nucleoprotein. The binding of the majority of the MAbs (8 out of 11) to large fusions, but not to smaller fragments included in them, suggests a conformational dependence of the MAb binding sites. Our data show that the use of fusions in Western blot experiments is a useful approach to map not only linear epitopes but more complex antigenic structures found in the nucleoprotein of the TGEV.     

Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes.

Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains.     

Characterization of monoclonal antibodies against the Escherichia coli hemolysin.

Twelve monoclonal antibodies (MAbs) produced against the Escherichia coli hemolysin (HlyA) encoded by the hemolysin recombinant plasmid pWAM04 were studied. HlyA derivatives from recombinant strains with different plasmids encoding HlyA amino-terminal and carboxy-terminal truncates, HlyA in-frame deletions, and HlyA frameshift mutations were used in immunoblots to localize the antigenic determinants for the anti-HlyA MAbs. The mapping of the MAb epitopes was also facilitated by immunoblotting analysis of HlyA polypeptide fragments derived by cyanogen bromide cleavage. The HlyA epitopes for 11 of the MAbs were mapped to relatively small linear regions of the cytolysin ranging from 28 to 160 amino acids. Five of the MAbs (C10, G8, E2, B7, and D12) neutralized HlyA hemolytic activity to varying degrees. The epitopes for these neutralizing MAbs were found to reside within the following HlyA regions: C10 and G8, amino acids 2 to 160; E2, amino acids 161 to 194; B7, amino acids 518 to 598; and D12, amino acids 626 to 726. Hemolytically active HlyA was dependent on the action of the hlyC gene product. The D12 MAb recognized only HlyA produced by strains with an intact hlyC function. MAb A10 recognized an epitope within the HlyA region from amino acids 728 to 829 where a glycine-rich repeat domain exists; however, this MAb did not neutralize HlyA hemolytic activity. A HlyA domain map showing the anti-HlyA epitope location was constructed.