Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.     

Production and characterization of serovar-specific monoclonal antibodies to serovars 4, 8, and 9 of Mycobacterium intracellulare.

Serovar-specific monoclonal antibodies against Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex serovars 4, 8, and 9 were prepared. Nine, four, and one monoclonal antibodies, respectively, to the serovars were prepared by the usual cell fusion technique. All nine monoclonal antibodies to serovar 4 were monospecific for their homologous serovar and reacted with several native glycopeptidolipids (GPLs) and one major deacylated GPL from the homologous serovar. One of the four monoclonal antibodies to serovar 8 seemed to be monospecific for its homologous serovar, but the other cross-reacted with serovar 6 because serovar 6 organisms contain the same components as does the major deacylated GPL from serovar 8. One monoclonal antibody to serovar 9 was monospecific for its homologous serovar and reacted with one of the two major deacylated GPLs from this serovar. These antibody preparations proved useful for serovar identification.     

Development and characterization of monoclonal antibodies to Pneumocystis carinii.

Hybridoma-producing monoclonal antibodies against Pneumocystis carinii were produced by the fusion of nonsecreting mouse myeloma cells (P3X63-Ag8.653) with splenocytes from BALB/c mice that had been immunized with partially purified preparations of P. carinii. Of 227 hybridoma clones producing antibodies against P. carinii, as measured by an enzyme-linked immunosorbent assay, 12 monoclonal antibodies showing the highest reactivity in the enzyme-linked immunosorbent assay were further characterized. The majority (11 of 12) of the monoclonal antibodies did not cross-react with Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, or Mycobacterium avium as determined by absorption experiments. By using the indirect immunofluorescence assay, serological reactivity was shown for these antibodies with titers ranging from 1:40 to 1:10,240. By using a competitive binding assay, these 12 monoclonal antibodies could be divided into seven groups, each group reacting with a different antigenic determinant of P. carinii. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of P. carinii, followed by Western immunoblot analysis, allowed the identification of one major antigen with an apparent molecular weight of 110,000 by all 12 monoclonal antibodies. Other minor bands with molecular weights of approximately 116,000, 90,000, 55,000, and 35,000 were recognized by several of the monoclonal antibodies.     

Identification and characterization of serotype 4-specific antigens of Ureaplasma urealyticum by use of monoclonal antibodies.

Monoclonal antibodies against Ureaplasma urealyticum serotype 4 were produced by immunizing BALB/c mice with whole-cell antigens of the U. urealyticum serotype 4 reference strain. Ten monoclonal antibodies differentiated into two groups were found: one group included five monoclonal antibodies recognizing a band in immunoblotting that had a molecular mass of 81 kDa, and a second group included another five monoclonal antibodies recognizing three bands in immunoblotting that had molecular masses of 81, 75, and 71 kDa. Fifteen clinical U. urealyticum isolates were selected for serotyping with serotype 4-specific monoclonal antibodies and polyclonal antisera 1 to 14. The results obtained with polyclonal and monoclonal antibodies suggest the existence of heterogeneity of the serotype antigens among clinical isolates of U. urealyticum serotype 4.     

Generation of monoclonal murine anti-DNP-IgE, IgM and IgG1 antibodies: biochemical and biological characterization.

Monoclonal DNP-specific IgG (lambda 2 epsilon 2), IgM (kappa 2 mu2) and IgG [kappa 2 (gamma 1)2] were isolated fom the culture supernatant of hybridomas by affinity chromatography with 2,4-dinitrophenol bovine serum albumin (DNP-BSA) sepharose and characterized by biochemical and biological methods. The molecular weights were 84,200 for the epsilon chain, 55,400 for the gamma chain and 77,500 for the mu chain as determined by sodium dodecylsulphate polyacrylamide del electrophoresis (SDS-PAGE). The association constants for [3H]-DNP-lysine determined by equilibrium dialysis were 0 . 87 X 10(7) l/mol for IgE and 1 . 91 X 10(8) 1/mol for IgG1. The isoelectric focusing of the purified monoclonal antibodies revealed for IgG1 seven bands at a pH range of 6 . 3 - 7 . 2 and for IgE sixteen bands at a pH range of 4 . 5 to 6 . 8. the binding of 125I-anti-IgE to rat basophilic leukaemia (RBL) and rat mast cells which had been preincubated with various amounts of monoclonal IgE was studied. At saturation conditions of IgE, about 2 . 14 X 10(5) molecules of anti-IgE were bound per rat mast cell. Rat mast cells coated with monoclonal anti-DNP IgE were triggered for the release of histamine in the presence of either the antigen or guinea-pig anti-mouse IgE. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against ovalbumin or rat reaginic serum directed against Nippostrongylus brasiliensis with monoclonal mouse anti-DNP IgE was demonstrated.     

Precise characterization of norovirus (Norwalk-like virus)-specific monoclonal antibodies with broad reactivity

We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.     

Development and characterization of monoclonal antibodies reactive with paraquat

A set of three anti-paraquat monoclonal antibodies(MoAbs), named APM-1, APM-2 and APM-3, has been isolated. In order to evaluate the ability of these MoAbs to recognize various kinds of bipyridyl herbicides and similar congeners of paraquat, a competition enzyme-linked immunosorbent assay (ELISA) using avidin-biotin complex (ABC) was developed. All three antibodies strongly recognized paraquat and slightly did the other analogs. These three MoAbs are therefore advantageous to a toxicological study of paraquat and of its localization in tissues.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Development, characterization, and diagnostic applications of monoclonal antibodies against bovine rotavirus

Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.     

Minimal oligosaccharide structures required for induction of immune responses against meningococcal immunotype L1, L2, and L3,7,9 lipopolysaccharides determined by using synthetic oligosaccharide-protein conjugates.

The 12 types of meningococcal lipopolysaccharide (LPS) (immunotypes) contain immunotype-specific and cross-reactive epitopes situated on the oligosaccharide part of the LPS molecules. To identify useful cross-reactive epitopes and to determine minimal oligosaccharide structures required for the induction of an immune response against the most prevalent immunotypes, L1, L2, and L3,7,9, synthetic as well as native LPS-derived oligosaccharides were conjugated with tetanus toxoid. L3,7,9 phosphoethanolamine (PEA) group-containing oligosaccharide-tetanus toxoid conjugates evoked high immunoglobulin G (IgG) antibody levels in rabbits which were detected by an L2-, L3,7,9-, and, depending on the antiserum, L1-specific enzyme-linked immunosorbent assay (ELISA). Inhibition studies revealed that an identical antibody population was detected by L1 and L3,7,9 ELISA, indicating a similar tertiary structure of the inner core oligosaccharide of these two immunotypes. These antibodies recognize PEA group-containing epitopes present on the L1 and L3,7,9 LPS. An L2 PEA group-containing oligosaccharide-tetanus toxoid conjugate elicited L2- and L3,7,9-specific IgG antibodies, but in contrast with the L3,7,9 conjugates, no L1-specific IgG antibodies were evoked. These results indicate that L1 and L2 LPS do not contain cross-reactive epitopes, whereas both L2 and L3,7,9 LPS and L1 and L3,7,9 LPS possess common determinants. Three linear oligosaccharides and one branched oligosaccharide, representing partial structures of the inner core oligosacchardes of meningococcal LPS, were synthesized. Only the branched synthetic oligosaccharide-containing conjugate was able to induce and L1- and L3,7,9-specific immune response, whereas the linear oligosaccharide-protein conjugates evoked L2-specific immune responses. The branched oligosaccharide (beta-D-Glcp(1----4)-[L-alpha-D-Hepp(1----3)]-L-alpha-D-Hepp ) is therefore considered a minimal structure required for the induction of an immune response against L1 and L3,7,9 LPS and part of a cross-reactive epitope between these two immunotypes. For L2-specific immune responses, oligosaccharide structures terminating in beta-D-Glcp(1----4), alpha-D-GlcNAcp(1----2), or L-alpha-D-Hepp(1----5) are needed. The results suggest that it is possible to prepare an oligosaccharide structure with the ability to evoke an immune response against L1, L2, and L3,7,9 LPS. A feasible structure for such a "hybrid" oligosaccharide is discussed.     

Development and characterization of monoclonal antibodies to bovine brain calcineurin

Three monoclonal antibodies (MAb's) against bovine brain calcineurin, a Ca++-calmodulin dependent phosphatase, have been developed and characterized. Among these antibodies, two are alpha-subunit (60,000 Mr) specific and one is beta-subunit (19,000 Mr) specific. These antibodies also cross-react with similar proteins obtained from brains of human, rat and mouse. The methodology for raising these antibodies and their properties are described.     

Immunological properties and biological function of monoclonal antibodies to tobacco mosaic virus

Summary Monoclonal antibodies to TMVvulgare produced in hybridoma cultures as well as in ascitic fluid were characterized according to their reactivity with the virion and/or the coat protein monomer thus revealing specificity for epitopes, cryptotopes or neotopes. Different patterns of crossreactivity of these monoclonal antibodies with TMV strainsdahlemense and Holmes' Ribgrass occured. Some monoclonal antibodies showed stronger reactivity with these strains than with the immunizing strain. The monoclonal antibodies were TMV-specific as they did not react with ArMV and PLRV and proteins of healthy tobacco plants. The monoclonal antibodies were of the IgG_2a or IgM isotype. The specific activity (Ext_{405 nm}/hour/100 µg MCA) with the immunizing virus and its coat protein monomers was determined as characteristic property of each monoclonal antibody.A monoclonal antibody specific for the C-terminal epitope of TMV coat protein was selected by means of the corresponding chemically synthesized tetrapeptide. With this monoclonal antibody infectivity of TMV was neutralized.With 3 Figures     

Development, characterization and use of monoclonal antibodies against sTRAIL: measurement of sTRAIL by ELISA.

Two monoclonal antibodies against tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), designated VI10E and III6F, have been generated. These antibodies were useful in flow cytometry analysis, immunohistochemistry, immunoprecipitation and in the development of an immunoassay for the detection of soluble TRAIL (sTRAIL)in biological samples. The immunoassay was based on two monoclonal antibodies against TRAIL. VI10E was used as the capture antibody and bound TRAIL was detected with anti-TRAIL from R&D Systems which was digoxigenin (DIG)-labeled. This enzyme-linked immunosorbent assay (ELISA) was specific for TRAIL since a panel of other cytokines did not affect the signal. The immunoassay was suitable for the detection of sTRAIL in human serum and plasma samples, cell culture supernatants and cell lysates. In a preliminary screening, it was found that serum samples from human immunodeficiency virus (HIV)-infected patients contained sTRAIL, and all these positive samples were found in the AIDS group. Using the immunoassay, it was found that phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) to produce significant amounts of sTRAIL, the levels of which increased with exposure time. Thus, the immunoassay for TRAIL presented here represents a useful tool for measuring sTRAIL in various biological samples. It will also permit studies of release mechanisms as well as possible functions of the soluble form of this molecule.     

抗土拨鼠肝炎病毒核心蛋白单克隆抗体的制备、性质鉴定及初步应用

目的研制出能特异与土拨鼠肝炎病毒核心蛋白（WHc）结合的单克隆抗体,使之能特异性地对土拨鼠肝炎病毒（WHV）进行检测,并可能应用于相关肝炎病毒的筛查。方法以原核表达的土拨鼠肝炎病毒重组核心蛋白（WHc 1～149氨基酸）免疫BALB/c小鼠,常规杂交瘤技术进行细胞融合,有限稀释法克隆化,间接酶联免疫吸附试验（ELISA）和免疫组织化学（IHC）筛选、鉴定。结果筛选出5株（4B1E、6C5D、6C5C、6D1D、6D1G）能稳定分泌抗土拨鼠肝炎病毒核心蛋白抗体的杂交瘤细胞株。此5株单抗适用于ELISA、IHC、Western blot等方面的研究,与HBcAg有交叉反应,并且在部分中国旱獭的肝脏组织进行IHC检测呈现阳性反应。结论制备的5株单抗可用于土拨鼠肝炎病毒等嗜肝脱氧核糖核酸病毒的研究,可能在寻找新的相关肝炎病毒中起重要作用。     

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.     

Redesignation of a purported P1.15 subtype-specific meningococcal monoclonal antibody as a P1.19-specific reagent.

Two reference monoclonal antibodies against the meningococcal P1.15 subtype PorA, MN3C5C and 2-1-P1.15, showed only partial concordant recognition of meningococcal isolates. Cyanogen bromide cleavage of P1.19,15 PorA, peptide mapping, and sequencing of porA regions demonstrated that 2-1-P1.15 was specific for subtype P1.19, and henceforth it is to be redesignated as 2-1-P1.19.     

Development and characterization of human constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.     

Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.     

Selection and characterization of cyclic peptides that bind to a monoclonal antibody against meningococcal L3,7,9 lipopolysaccharides

There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.     

Production and characterization of monoclonal antibodies to Mobiluncus species.

Members of the genus Mobiluncus are anaerobic motile curved rods which are associated with bacterial vaginosis (BV). Murine monoclonal antibodies (MAbs) to the ATCC type strains of M. curtisii subsp. curtisii, M. curtisii subsp. holmesii, and M. mulieris were produced and characterized by enzyme-linked immunosorbent assay and indirect immunofluorescence assay. Four MAbs were subspecies specific and reacted with M. curtisii subsp. curtisii but not with M. curtisii subsp. holmesii; four were specific for M. mulieris. The remaining antibodies demonstrated some cross-reactivity: three were species specific and reacted with both subspecies of M. curtisii, and one defined an epitope shared by M. curtisii subsp. holmesii and M. mulieris but not by M. curtisii subsp. curtisii. None of the MAbs reacted with a panel of other bacteria commonly present in the vaginas of normal women or women with BV. Examination of the molecular specificities of the antibodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed four antibodies which were specific for an 82,000-dalton molecule of M. curtisii subsp. curtisii and five antibodies which bound a major band of M. mulieris at 93,000 daltons. Selected MAbs reacted in the indirect immunofluorescence assay with 24 of 25 Mobiluncus spp. clinical isolates from local women with BV and could be used for direct detection of Mobiluncus spp. in vaginal fluid from a patient with BV.