Production and characterization of monoclonal immunoglobulin A antibodies directed against Salmonella H:g,m flagellar antigen

Hybridomas were generated after intragastral immunization of BALB/c mice with live Salmonella suberu and subsequent fusion between isolated spleen lymphoblasts and myeloma cells. Three monoclonal antibodies (MAbs) of immunoglobulin A (IgA) isotype were selected and characterized. All of them were found to recognize the H:g epitope in enzyme-linked immunosorbent assay and immunoblotting but did not react with all H:g-expressing strains in slide agglutination test. All MAbs strongly agglutinated Salmonella enteritidis type strain and a large number of S. enteritidis clinical isolates. They were not bactericidal in the presence of complement. All hybridoma clones produced secretory IgA forms, which were found in the gastrointestinal tract of mice bearing hybridoma as a subcutaneous 'backpack' tumor or after intravenous application of purified MAbs. The IgA MAbs stability demonstrated in different tests together with their antigen specificity and strong agglutination ability make them a useful diagnostic tool for serotyping of Salmonella strains.     

Protective efficacy of IgA monoclonal antibodies to O and H antigens in a mouse model of intranasal challenge with Salmonella enterica serotype Enteritidis

Protective properties of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis (S. enteritidis) were evaluated in a model of generalized infection after intranasal (i.n.) inoculation of BALB/c mice. Passive i.n. instillation of antibodies 1 h before i.n. challenge did not prevent infection, and mice developed rapid inflammatory response in the lower respiratory tract. The passive systemic immunization was partially protective and a single intravenous (i.v.) injection of both O and H antigen specific IgA antibodies prolonged survival period of the infected animals. Permanent secretion of O:9 specific IgA MAb 177E6 into the respiratory tract in a "backpack" tumor model protected 50% of animals infected i.n. with a high dose of virulent S. enteritidis strain. Thus, secretory IgA (S-IgA) directed against O:9 antigen alone can prevent bacterial invasion in the respiratory epithelium.     

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa.

Serotypes O2, O5, and O16 of Pseudomonas aeruginosa are chemically related, and the O antigens of their lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the O2, O5, and O16 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum O-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks O antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the O repeat that is common to all three serotypes of the O2-O5-O16 group, while the O-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more O-antigen repeat units are present on the LPS molecule. Thus, the O-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the O side chains.     

Monoclonal antibodies for detection of the H7 antigen of Escherichia coli.

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.     

Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies

Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.     

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Lipopolysaccharide expression and virulence in mice of Australian isolates of Salmonella enteritidis

The lipopolysaccharide (LPS) of 54 Australian isolates, nine isolates acquired or isolated overseas, and two reference strains of Salmonella enteritidis was studied to assess its relation to pathogenicity. LPS was extracted by proteinase K digestion of whole cells, and analysed by polyacrylamide gel electrophoresis. All isolates possessed an LPS structure identical to that of a reference strain of Salm. enteritidis phage type 4. Representative strains of the clinically prevalent phage types 4, 14 and 26, which express long chain LPS, were assessed for their pathogenicity in mice. Salmonella enteritidis phage type 4 produced a lethal infection in BALB/c mice, but not in C3H/HeJ or Quackenbush (outbred) strains. Phage types 14 and 26 did not produce an obvious infection in any mice, suggesting Australian strains of phage type 4 are more virulent than phage types 14 and 26.     

Interrelationships between strains of Salmonella enteritidis belonging to phage types 4, 7, 7a, 8, 13, 13a, 23, 24 and 30

Salmonella enteritidis strain P278849 expressed long-chain lipopolysaccharide (LPS, termed 'smooth'), carried plasmids of 38, 34 (pDEP 44, incompatibility group N, R-type AS), 2.0 and 1 MDa, and belonged to phage type (PT) 23. Introduction of pDEP 44 into a smooth strain of Salm. enteritidis PT 4 produced a smooth strain of Salm. enteritidis of PT 24. Transfer of this plasmid into a strain of PT 8 also resulted in formation of a smooth strain of Salm. enteritidis of PT 24. Moving pDEP 44 into strains of Salm. enteritidis of PTs 7 or 7a which did not express LPS (termed 'rough') resulted in rough strains of PT 23. In contrast, transfer of this plasmid into a smooth strain of Salm. enteritidis PT 7a resulted in a smooth strain of PT 23. Introduction of pDEP 44 into strains of Salm. enteritidis of PT 13 or PT 13a did not change the phage type, whereas transferring the plasmid into strains of PT 30 caused strains to become resistant to lysis by the typing phages and therefore untypable. The possibility of strains of Salm. enteritidis of PT 8 being the progenitors of strains of Salm. enteritidis of PT 24 must now be considered when investigating the epidemiology of Salm. enteritidis of PT 24 infections in areas where Salm. enteritidis PT 8 is common.     

Murine monoclonal antibodies specific for lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

Role of Myxococcus xanthus cell surface antigen 1604 in development.

The inhibition of development of Myxococcus xanthus by monoclonal antibody (MAb) 1604 has been further investigated with two MAbs produced against the affinity-purified cell surface antigen (CSA) 1604. Both of these second-generation MAbs, 4070 and 4054, reacted with the same band at 150 kilodaltons (kDa) on Western immunoblots of lysed and reduced cells. This band was also identified by MAb 1604. However, the affinity-purified CSA was a complex of the two proteins (51 and 23 kDa) and lipopolysaccharide (LPS) that the 150-kDa material comprised. One of the three MAbs, 4070, reacted with LPS on Western immunoblots. Another MAb, 4054, reacted with the 23-kDa protein, and MAb 1604 reacted with the 51-kDa protein found in the CSA complex. Competitive binding studies verified that MAbs 4054 and 1604 identified different epitopes, and MAb 4070 probably reacted with a third epitope of the CSA 1604 complex. MAb 4054 blocked development, although not as thoroughly as MAb 1604 did, when added at 60 micrograms/ml to cells undergoing submerged development. In contrast, MAb 4070 prevented sporulation in submerged development and induced the cells to reaggregate in rings around the initial aggregation centers. A mutant strain of M. xanthus that is deficient in the epitope for MAb 1604 retained the epitope for MAb 4054. The affinity-purified antigen 1604, when added to cells at greater than or equal to 550 ng/ml, altered the appearance of the fruiting bodies and at higher concentrations prevented fruiting body formation. The CSA 1604 moiety responsible for this inhibitory effect is apparently a peptide constituent and not the LPS.     

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately 80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunoglobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studied in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD50 of five strains of P. aeruginosa was increased greater than or equal to 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MAbs in mice challenged with approximately 10 LD50 of the homologous LPS serotype ranged from less than 0.01 mg/kg to 1.00 mg/kg.     

Variation in Salmonella core lipopolysaccharide as detected by the monoclonal antibody M105

L.P. MANSFIELD, E. BILLETT, E. OLSEN AND S.J. FORSYTHE. 1996. The lipopolysaccharide antigenicity of 22 Salmonella strains (representing nine serogroups) and four non-salmonellae Enterobacteriaceae to the Salmonella genus specific monoclonal antibody M105 was analysed. The monoclonal antibody M105 reacted with all 22 Salmonella strains. Probing SDS-PAGE separated LPS molecules with MAb M105 revealed that the antibody reacted with the core region of all Salmonella serovars. However, no reaction was obtained to the long-chain LPS of serovars O ( Salm. adelaide and Salm. ealing ), C₁ ( Salm. infantis, Salm. livingstone and Salm. virchow ) or Salm. arizonae . It is plausible that the presence of a second core antigenic type results in the lack of reaction between long-chain LPS and the Salmonella genus specific monoclonal antibody M105.     

Conversion of Salmonella enteritidis phage type 4 to phage type 7 involves loss of lipopolysaccharide with concomitant loss of virulence

Three strains of Salmonella enteritidis phage type 4 (PT4) and 33 strains of S. enteritidis phage type 7 (PT7) were examined for the ability to produce lipopolysaccharide (LPS) and for plasmid carriage. The LPS of all strains of PT4 gave a typical 'ladder' pattern by SDS-PAGE and silver staining, and on serotyping these strains were shown to express the O-antigens 9, 12. In contrast, strains of PT7 did not express long-chain LPS and were autoagglutinable. All strains of PT4 and the majority of strains of PT7 carried a single plasmid of 38 MDa, indistinguishable when characterised by restriction endonuclease fragmentation analysis. Epidemiological and experimental observations have demonstrated a relationship between strains of S. enteritidis PT4 and PT7, and our results, using mice, show that the loss of ability of strains of PT4 to snythesise LPS is responsible for the conversion of highly virulent strains of PT4 to avirulent strains of PT7. From epidemiological data of human infections in England and Wales, we suggest that strains of S. enteritidis PT7 may be less virulent for humans.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

A conserved coronavirus epitope, critical in virus neutralization, mimicked by internal-image monoclonal anti-idiotypic antibodies.

Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Ab1) have been selected. All six MAbs inhibited the binding of Ab1 to TGEV and specifically cross-linked MAb1-6A.C3. Four of these hybridomas secreted gamma-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.E11) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as beta-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two beta-type MAb2s and in 25 to 50% of mice immunized with gamma-type MAb2. Both beta- and gamma-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.     

Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains

AIMS: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format. METHODS AND RESULTS: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E. coli O157. A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E. coli (EHEC) O157 strains and also with two enterotoxigenic E. coli O157 strains. Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected. CONCLUSION: A MAb-based sELISA to detect E. coli O157 was produced. Its application to field samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-specific interference of the cross-reacting strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay produced is not wholly specific to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E. coli O157.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.