Characterization of the Endothelial Surface Proteins Recognized by Anti-Endothelial Antibodies in Primary and Secondary Autoimmune Vasculitis

The antigenic structures recognized by anti-endothelial cell antibodies (AECA) in sera from 10 Wegener's granulomatosis (WG) and 12 systemic lupus erythematosus (SLE) patients with signs of vasculitis were characterized by immunoprecipitation of selectively radiolabeled surface membrane proteins from human umbilical vein endothelial cells. Electrophoretic analysis of the immunoprecipitated proteins revealed reactivities against endothelial antigens ranging in size from 200 to 25 kDa. AECA antigens were not cell specific, since the same sera also reacted, at least in part, with radiolabeled human fibroblast surface proteins. The majority of WG patients displayed a constant precipitation pattern of five proteins (180, 155, 125, 68, and 25 kDa). On the contrary, AECA from SLE sera reacted with a more heterogeneous series of endothelial proteins. A group of four proteins, however, was also found in the majority of SLE sera: 200, 180, 155, and 25 kDa. In addition, some endothelial antigens were immunoprecipitated only by WG (125 kDa) or by SLE sera (200 kDa), suggesting a different endothelial reactivity in different vasculitic processes. The reaction did not involve intracellular proteins as demonstrated by the lack of reactivity of SLE sera negative for AECA but positive for anti-cytoplasmic or anti-nuclear antibodies. These data confirming that AECA recognize surface endothelial determinants further support a potential pathogenetic role for these antibodies in autoimmune vasculitis.     

Antigen detection in enteropathogenic Escherichia coli using secretory immunoglobulin A antibodies isolated from human breast milk

Enteropathogenic Escherichia coli (EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children. The immune response to EPEC infection remains poorly characterized. The molecular targets that elicit protective immunity against EPEC disease are unknown. In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis. Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa. A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies. The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC). These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women. Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins. Such proteins reacted strongly with the purified sIgA pool. Moreover, nonvirulent E. coli strains were unable to generate a sIgA response. The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported. The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease.     

Multiplicity of mitochondrial inner membrane antigens from beef heart reacting with antimitochondrial antibodies in sera of patients with primary biliary cirrhosis

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular weights of 70 kDa, 54 kDa, 51 kDa and 45 kDa, as defined by their RF in SDS-PAGE gel. There was no correlation between the number of specificities and the titers of AMA as determined by immunofluorescence analysis. The 70-kDa protein was dissociated into a 36-kDa protein by trypsin digestion which still reacted with AMA. The reactivity to AMA of the 54-, 51- and 45-kDa proteins was abolished by trypsin digestion.     

Sulphonamide Antibodies: From Specific Polyclonals to Generic Monoclonals

Polyclonal antibodies (PAbs) against eight different sulphonamides were raised in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)) and enzyme (horseradish peroxidase (HRP)), using different coupling procedures. The competitive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjugates showed high sensitivity (0.2- 8.0 ng ml^-1 at 50% inhibition) and high specificity. The performances of these antibodies were compared with PAbs raised in mice against two sulphonamide derivatives (N¹ -[4-(carboxymethyl)-2-thiazolyl]sulphanilamide (TS) and N¹-[4-methyl-5-\[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA and KLH) in such a way that the common aromatic amino group was distal to the protein. In competitive indirect ELISAs (ciELISAs), these PAbs recognized several structurally different sulphonamides. The PAbs from mice immunized with TS-BSA reacted with sulphonamides containing thiazolyl, thiadiazolyl, pyridazinyl and isoxazolyl groups. The PAbs from mice immunized with PS-KLH reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxalinyl and pyridinyl groups. The spleen cells of the mice were fused with myeloma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So far, with only one of the mice (immunized with TS-BSA), this resulted in four different MAbs which recognized several sulphonamides. By use of the best MAbs (27G3A9B10 and 4E10B12B6E12) and an optimized ciELISA protocol, eight structurally different sulphonamides showed 50% inhibition at concentrations less than 100 ng ml^-1 or 5 ng/well. However, other relevant sulphonamides (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were detected at a high level only.     

A tubulin-related 55 kilodalton surface antigen recognized by different Trypanosoma cruzi stage-specific monoclonal antibodies from infected mice

Thirteen monoclonal antibodies (MAbs) specific for the membrane of live Trypanosoma cruzi have been obtained from BALB/c infected mice. Most of them had greater avidity for intact than for disrupted parasites. According to the staining by indirect immunofluorescence of the different live developmental stages of the parasite the MAbs could be divided into several groups. Three of them were trypomastigote specific, one amastigote-specific and two epimastigote-specific. The rest reacted with either all stage forms or with various combinations of the different stages. However, despite the fact that they seemed to correspond to stage-specific antibodies, ten of them reacted with the same 55/50 kDa antigen by immunoblotting. Similarly, a 55 kDa protein was immunoprecipitated from these MAbs. By contrast, a single band or a dimer of about 25 kDa was the predominant antigen(s) immunoprecipitated by the same MAbs in absence of protease inhibitors. This smaller protein may arise from proteolysis of the 55 kDa band. This protein is related to tubulin since tubulin (a 55 kDa protein) but not other cytoskeleton proteins blocked the binding of these MAbs to T. cruzi, and some MAbs react with pig alpha-tubulin by immunoblotting.     

Adhesion characteristics of lactobacillus is a criterion of the probiotic choice

A sampling of lactobacilli from the German National Collection of Microorganisms and L. fermentum 90 TS-4 (21) reference strain clone 3 (Russian Federation) were studied. The results indicate that the receptors on the surface of lactobacillus strains from the German collection had no structures complementary to type 1 fimbriae, though adhesins of some of them reacted with mannose and galactose receptors. Adhesion on a monolayer of continuous cell cultures showed that adhesion activity of lactobacilli was a function of many derivatives, and hence, the choice of a model for evaluation of the adhesion characteristics of the strain should be based on adhesins exhibiting universal properties in different test systems. One of them can be lectin-binding adhesin; its expression on the surface of cultures of lactobacilli from the German collection varies within the same range as was shown previously for lactobacilli, studied by the same criterion. The molecular weight of lectin-binding adhesin is 25–30 kDa, and the corresponding receptors are frequently present on various eukaryotic cells, and hence, cell models can be considered as the most adequate for studies of the competitive interactions between lactobacilli and adhesins of pathogenic microorganisms. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 2, pp. 192–195, February, 2008     

Determination of polyprotein processing sites by amino terminal sequencing of nonstructural proteins encoded by plum pox potyvirus

Nonstructural proteins of plum pox potyvirus were partially purified following a procedure described for the isolation of tobacco etch virus nuclear inclusion proteins. Plum pox virus proteins with electrophoretic mobilities corresponding to 49, 59 and 68 kDa reacted with antibodies against the 49 kDa and 54 kDa components of the nuclear inclusions and the 70 kDa component of the cylindrical inclusions of tobacco etch virus, respectively. Further purification by size exclusion high performance liquid chromatography or SDS-polyacrylamide gel electrophoresis, and amino terminal amino acid sequencing permitted the location in the plum pox virus polyprotein of the cleavage sites from which the 49 kDa (NIa-type, protease), 59 kDa (NIb-type, putative RNA replicase), and 68 kDa (CI-type) proteins originate. A 110 kDa protein which copurified with the plum pox virus inclusion proteins reacted with both anti-NIa and anti-NIb sera and had the same amino terminus as the plum pox virus 49 kDa protein, indicating that it is a non-processed 49-59 kDa polypeptide.     

The monoclonal antibody, Alz 50, recognizes tau proteins in Alzheimer's disease brain

The monoclonal antibody, Alz 50, is known to label many dystrophic neurites and neurofibrillary tangles in Alzheimer's disease (AD) brain. Using immunoprecipitation and immunoblotting, we have compared Alz 50 to monoclonal antibodies directed at two known components of neurofibrillary tangles, tau and ubiquitin, in order to characterize further the antigens recognized by Alz 50 in AD brain. Alz 50 labeled purified tau proteins in a highly similar fashion to two well-characterized tau monoclonal antibodies. Alz 50 precipitated proteins at the molecular weight of 50-70 kDa from AD but not normal brain; these proteins were reactive with the tau antibodies. In addition, Alz 50 precipitated proteins migrating principally around 160-180 kDa from AD but not normal brain; the relationship of the latter proteins to tau remains unclear. None of these proteins reacted with a ubiquitin antibody. We hypothesize that the proteins recognized by Alz 50 at much higher levers in AD than normal brain include modified and aggregated forms of tau.     

Immunoblot analysis of antibody responses to Sporothrix schenckii

The serologic response to Sporothrix schenckii was investigated in patients with sporotrichosis by solid-phase enzyme-linked immunosorbent assays (ELISAs) and Western immunoblot techniques. A soluble antigen preparation derived from an S. schenckii isolate contained 15 protein staining components ranging in molecular size from 22 to 70 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera from 40 patients with sporotrichosis demonstrated Sporothrix immunoglobulin G antibody by ELISA with titers between 128 and 65,200. No sera from 300 healthy individuals or 100 patients with various systemic mycoses other than sporotrichosis had ELISA titers greater than 64. By Western immunoblotting of the antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from 10 patients with cutaneous sporotrichosis reacted with 8 to 10 antigen components (range, 40 to 70 kDa), while sera from 15 patients with extracutaneous sporotrichosis reacted with a greater number of antigen components (15 to 20 bands) over a wider range of molecular sizes (22 to 70 kDa). Antibody to 40- and 70-kDa antigen components was detected by immunoblots in all sera tested from patients with sporotrichosis. Antibody to 22- to 36-kDa antigen components was present in sera from 13 of 15 patients with extracutaneous sporotrichosis, but these lower-molecular-weight components were not detected by sera from patients with cutaneous sporotrichosis. Antibody to these components was not detected by Western blotting in sera from 19 of 20 patients with other fungal diseases or from 30 healthy individuals. Purification of these specific antigen fractions could provide the basis of a sensitive and specific serodiagnostic test to indicate the presence and activity of extracutaneous sporotrichosis.     

Identification of the Clam Species Ruditapes decussatus (Grooved Carpet Shell), Venerupis rhomboides (Yellow Carpet Shell) and Venerupis pullastra (Pullet Carpet Shell) by ELISA

An ELISA has been developed for the identification of three clam species: Ruditapes decussatus (grooved carpet shell), Venerupis rhomboides ( yellow carpet shell) and Venerupis pullastra (pullet carpet shell). The assay was performed using polyclonal antibodies (PAbs) against clam soluble proteins in two different formats: microtiter plates and immunostick tubes. PAbs were rendered species-specific by blocking them with soluble proteins from heterologous clam species. For the indirect ELISA, clam soluble proteins were adsorbed onto polystyrene plates or immunostick paddles. Species-specific PAbs were then added to the wells or tubes. Goat anti-rabbit immunoglobulins conjugated to the enzyme horseradish peroxidase were used for the immunorecognition of the PAbs adsorbed to clam samples. Subsequent enzymatic conversion of the substrate allowed the identification of the three clam species analyzed.     

In situ immunocytochemical detection of cells containing antibodies specific for Eimeria tenella antigens.

A three-step immunocytochemical method for the in situ detection of antibodies specific for Eimeria tenella has been developed. The method is based on the binding of E. tenella antigens to antibodies in cryostat sections of chicken tissues and the recognition of these antigens by rabbit antiserum specific for E. tenella or mouse monoclonal antibodies specific for E. tenella. The rabbit antiserum and mouse monoclonal antibodies were revealed by the immunoperoxidase technique. Suspensions of sonicated sporulated oocysts, incubated with or without various concentrations of the non-ionic detergents Triton X-100 (TX-100) or Nonidet P-40 (NP-40), were used as antigen. Cells containing antibodies specific for E. tenella were detected only when detergent extracts of sonicated sporulated oocysts were used. After chickens were intravenously immunized with a suspension of sonicated sporulated oocyst antigen, cells containing antibodies specific for E. tenella antigens were detected in the red pulp of the spleen. After simultaneous immunoenzyme staining for isotype and antigen specificity, the E. tenella-specific antibody-containing cells were of the IgM isotype after the primary immunization and of the IgM and IgG isotype after the booster immunization. Immune complexes specific for E. tenella on the surfaces of follicular dendritic cells in the germinal centers were also stained. Chickens were also orally infected with sporulated oocysts. In these experiments, cells containing antibodies specific for E. tenella were detected in the lamina propria of the ceca and in the red pulp of the spleen. Specific immune complexes were also detected in the germinal centers of the cecal tonsils. When detergent extracts of sonicated sporulated oocysts were characterized by immunoblotting, rabbit antiserum specific for E. tenella reacted with proteins ranging in size from 16 kDa to 200 kDa, with major bands of 20 kDa, 24 kDa, 45 kDa, and 100 kDa. Monoclonal antibodies specific for E. tenella recognized only proteins of low molecular weight (20 kDa and 24 kDa) or high molecular weight (80-100 kDa). Immune chicken serum reacted with proteins of low and high molecular weight but especially with proteins of 100 kDa and 113 kDa. This method is the first by which immune complexes and cells containing antibodies specific for parasitic antigens can be detected in situ and may be of value for studies of the local humoral immune response to E. tenella in the mucosa of chickens.     

Antigenic characterization of the salmonid pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiological agent of salmonid rickettsial septicemia, was purified from infected immortal chinook salmon (Oncorhynchus tshawytscha) embryo cells by a combination of differential and Percoll density gradient centrifugation. Immune sera from rabbits immunized with purified whole cells of P. salmonis reacted with four protein antigens and two carbohydrate antigens with relative molecular sizes of 65, 60, 54, 51, 16, and approximately 11 kDa, respectively. The carbohydrate antigens appear to be mainly core region lipo-oligosaccharide with lesser amounts of lipopolysaccharide. Serum from convalescent rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) reacted with several minor immunoreactive protein antigens between 10 and 70 kDa in size and a carbohydrate antigen with a relative molecular size of approximately 11 kDa. The salmonid immune system did not appear to elicit a strong humoral response against this intracellular pathogen. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis cells suggest that the immunoreactive antigens identified with rabbit antisera are surface exposed and differ significantly from those identified with salmonid antisera.     

Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies.

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.     

Antigenic components of Gnathostoma spinigerum recognized by infected human sera by two-dimensional polyacrylamide gel electrophoresis and immunoblotting

Antigenic components of Gnathostoma spinigerum larval extract were revealed by two-dimensional gel electrophoresis (2-DE) and immunoblot analysis using sera from patients with 6 proven cases of gnathostomiasis, 5 presumptive cases of gnathostomiasis, 3 proven cases of angiostrongyliasis, 3 proven cases of cysticercosis, and pooled sera from healthy adults. By the 2-DE, the larval extract was highly complex and consisted of more than 75 polypeptides. Immunoblotting analysis of this larval extract after reaction with each of 6 proven gnathostomiasis sera revealed various numbers of antigenic spots ranging from 30 to 70 spots at the approximate molecular masses of less than 14.4 to more than 94 kDa with isoelectric points (pI) of less than 4.65 to 9.6. Antigenic spots at the approximate molecular mass of more than 30 kDa were recognized with the proven angiostrongyliasis, proven cysticercosis and healthy control sera but these sera did not react with the spots at approximate molecular masses of 23-25 kDa with pI of 8.3-8.5. The reacted spots, which consisted of at least 1 to 2 spots, were unique for the recognition of gnathostomiasis sera. Five out of 6 (83.3%) proven and 4 out of 5 (80%) presumptive gnathostomiasis sera reacted with these specific spots.     

Quality control Lactobacillus strains for use with the API 50CH and API ZYM systems at 37 degrees C.

The API 50CH and API ZYM systems fulfil an important role in the polyphasic taxonomic identification of lactobacilli. When the API 50CH fermentation profile of the quality control Lactobacillus casei var. alactosus (Lb. paracasei subsp. paracasei) strain NCFB 206 was determined at 37 degrees C, it was found to differ from that determined at 30 degrees C by BioMéreiux SA (Montalieu Vercieu, France) and the National Collection of Food Bacteria (Aberdeen, Scotland). In addition, the API 50CH fermentation and API ZYM profiles of Lb. casei strain ATCC 334T determined at 37 degrees C differed from those determined at 30 degrees C by Lee and Simard (1984). Strains NCFB 206 and ATCC 334T were thus assumed to exhibit temperature-dependent variation in fermentation profile, a phenomenon recently described by Nigatu et al. (2000). In contrast, Lb. rhamnosus strain ATCC 243T did not exhibit temperature-dependent variation in fermentation profile. Moreover, the fermentation profile obtained at 37 degrees C differed in only one respect (positive beta-gentiobiose utilisation) from that published by Collins et al. (1989). In addition, Lactobacillus strain GG produced a stable and reproducible API ZYM profile at 37 degrees C, although some variation in the level of enzyme activity was evident. Thus, strain NCFB 206 was replaced by strain ATCC 243T as the quality control strain of choice for use with the API 50CH fermentation system, and Lactobacillus strain GG adopted for use as a quality control strain with the API ZYM system for strain identification of lactobacilli at 37 degrees C. The API 50CH and API ZYM profiles of the commercially important Lactobacillus strains NCFB 1748, GG, KLD, F19, and ACA-DC 212.3 were determined at 37 degrees C after anaerobic growth in MRS broth. The fermentation and enzyme profiles of strain NCFB 1748 were almost identical to those of Lb. crispatus ATCC 33820T, those of strain GG were found to be more similar to those of Lb. rhamnosus strain 243T than Lb. zeae strain ATCC 15820T, those of strain KLD were most similar to those of Lb. fermentum DSM 20052T, while those of strains F19 and ACA-DC 212.3 were similar to those of Lb. casei strain ATCC 334T.     

Complete genomic sequence of the temperate bacteriophage PhiAT3 isolated from Lactobacillus casei ATCC 393

The complete genomic sequence of a temperate bacteriophage PhiAT3 isolated from Lactobacillus (Lb.) casei ATCC 393 is reported. The phage consists of a linear DNA genome of 39,166 bp, an isometric head of 53 nm in diameter, and a flexible, noncontractile tail of approximately 200 nm in length. The number of potential open reading frames on the phage genome is 53. There are 15 unpaired nucleotides at both 5' ends of the PhiAT3 genome, indicating that the phage uses a cos-site for DNA packaging. The PhiAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the PhiAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including PhiAT3 int and the attP (int-attP) region. It was found that PhiAT3 integrated at the tRNAArg gene locus of Lactobacillus rhamnosus HN 001, similar to that observed in its native host, Lb. casei ATCC 393.     

Latex allergy: allergen identification in Hevea braziliensis fractions by immunoblotting

Background Latex is the cause of several clinical symptoms of allergy, but the identification of allergens is not completely known. Objective The aim of this report was to study the immunoreactivity of puritied stable latex fractions from Hevea braziliensis. Methods We purified the cytoplasm of Hevea braziliensis and obtained three fractions: latex particles (LP), lutoids (L) and cytosolic serum (CS). Using Western blot, specific IgE directed to latex allergens was found in 80 patients with latex allergy. Results Five major groups of allergens migrating as 14, 25, 29, 37–45 and 50kDa were recognized. They were unequally distributed within the latex fractions: 37–45 kDa proteins were essentially recognized in CS and LP, whereas 14 and 29 kDa proteins were mainly labelled in the L fraction. As a control, aqueous glove extracts exhibited a more restricted pattern of reactivity, because only 14 and 29 kDa proteins were recognized by patient sera. The pattern of reactivity was not correlated with specific IgE levels, but sera from patients suffering from spina bifida reacted specifically with the minor protein of 25 kDa located in LP. Conclusions The present results show that latex allergic patients recognize several allergens which are differently distributed in subcellular fractions extracted from H. braziliensis and aqueous GE. The L fraction and GE were enriched in low molecular weight proteins and apparently contained the same allergens.     

Heterogeneity of the purified extracellular aspartyl proteinase from Candida albicans: characterization with monoclonal antibodies and N-terminal amino acid sequence analysis.

Three dominant proteins (41, 48, and 49 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in purified preparations of the extracellular aspartyl proteinase (AP) of Candida albicans. All three proteins bound to the specific carboxyl proteinase ligand, pepstatin A, and were associated with maximum AP activity. The N-terminal amino acid sequence for the 48- and 49-kDa proteins matched that reported by others for AP, whereas the sequence for the 41-kDa protein was unique and was not homologous to any known protein. Time course studies demonstrated the simultaneous presence of all three proteins, supporting evidence that the 41- and 48-kDa proteins were not breakdown products of AP. Previous studies did not detect carbohydrate in SDS-polyacrylamide gels of purified AP preparations stained with periodic acid and silver, making glycosylation an unlikely explanation for the observed differences in the molecular masses of the proteins. Some monoclonal antibodies directed against the 49-kDa protein reacted with the 41- and 48-kDa proteins, indicating cross-reactive epitopes. Other monoclonal antibodies, however, reacted only with the 49-kDa protein. We conclude that three pepstatin A-binding proteins occur in purified AP preparations: two have the same amino acid N terminus as that reported for AP, whereas the third has a unique sequence. All three proteins should be considered when undertaking studies to determine the role of AP in candidal pathogenesis or when preparing specific antibodies for antigen capture assays.