Immunological study of a neurofilament protein variant (S150) with polyclonal and monoclonal antibodies

Ten polyclonal neurofilament antibodies were tested for domain specificity with immunoblots of chymotrypsin digests of a neurofilament protein of 150 kDa (NF 150K). In contrast to most monoclonal antibodies previously reported, the five polyclonal antibodies which showed domain specificity reacted with the 40 kDa α-helical rod domain of the molecule. (With one exception, monoclonal antibodies reacted with the 200 kDa carboxy-terminal peripheral domain.) Of these ten polyclonal antibodies only two reacted with an isoelectric variant of NK 150 K (S150) isolated by Liem and collaborators (Wong, J., Hutchison, S.B. and Liem, R.K.H. (1984) J. Biol. Chem. 259, 10867–10874) from bovine brain. 13 monoclonal antibodies were also tested for reactivity with S150 protein. With one exception, none of these antibodies reacted with this variant, not even a monoclonal antibody which we have previously shown to react with a non-phosphorylated epitope located in the rod domain of NF 150K. We suggest that either there are modifications other than dephosphorylation in the S150 isoelectric variant or, alternatively, that it is not derived from NF 150K.     

Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.     

Dipeptidyl peptidase IV is a target for covalent adduct formation with the acyl glucuronide metabolite of the anti-inflammatory drug zomepirac

The nonsteroidal anti-inflammatory drug zomepirac (ZP) is metabolised to a chemically reactive acyl glucuronide conjugate (ZAG) which can form covalent adducts with proteins. In vivo, such adducts could initiate immune or toxic responses. In rats given ZP, the major band detected in liver homogenates by immunoblotting with a polyclonal ZP antiserum was at 110 kDa. This adduct was identified as ZP-modified dipeptidyl peptidase IV (DPP IV) by immunoblotting using the polyclonal ZP antiserum and monoclonal DPP IV antibodies OX-61 and 236.3. In vitro, ZAG, but not ZP itself, covalently modified recombinant human and rat DPP IV. Both monoclonal antibodies recognized DPP IV in livers from ZP- and vehicle-dosed rats. Confirmation that the 110 kDa bands which were immunoreactive with the ZP and DPP IV antibodies represented the same molecule was obtained from a rat liver extract reciprocally immunodepleted of antigens reactive with these two antibodies. Furthermore, immunoprecipitations with OX-61 antibody followed by immunolotting with ZP antiserum, and the reciprocal experiment, showed that both these antibodies recognised the same 110 kDa molecule in extracts of ZP-dosed rat liver. The results verify that DPP IV is one of the protein targets for covalent modification during hepatic transport and biliary excretion of ZAG in rats.     

Lectin-like components in the macronuclear replication bands of Euplotes eurystomus

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar-binding components (i.e., lectin-like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar-binding components are co-localized in an eukaryotic cell.     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.     

Proliferating cell nuclear antigen/cyclin in the ciliate Euplotes eurystomus: localization in the replication band and in micronuclei

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.     

Large scale purification and immunological characterization of human placental nerve growth factor

Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which crossreared characterization. A protein species of approximately 26 kDa was obtained, which crossreacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF.     

Replication forms of the gene-sized DNA molecules of hypotrichous ciliates.

Using a method for obtaining DNA from 10 to 40 macronuclei for electron microscopy, we analyzed the structure of gene-sized, linear DNA molecules from S-phase macronuclei of two hypotrichous ciliates, Euplotes eurystomus and Styx sp. Three types of putative replicating intermediates were observed: (i) molecules with a bubble close to one end, (ii) molecules with single forks, and (iii) molecules with two forks. We conclude that: (i) each macronuclear DNA molecule replicates as an independent unit, (ii) the molecules contain an origin of replication close to one or both ends, and (iii) the mode of replication is bidirectional.     

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.     

Relationship between the two immunologically distinguishable forms of glucocerebrosidase in tissue extracts

Extracts of human spleen contain two immunologically distinguishable forms of glucocerebrosidase: form I is precipitable by polyclonal or monoclonal anti-(placental glucocerebrosidase) antibodies, whereas form II is not [Aerts, J. M. F. G., Donker-Koopman, W. E., Van der Vliet, M. F. K., Jonsson, L. M. V., Ginns, E. I., Murray, G. J., Barranger, J. A., Tager, J. M. & Schram, A. W. (1985) Eur. J. Biochem. 150, 565-574]. The proportion of form II glucocerebrosidase was high in extracts of spleen, liver and kidney and low in extracts of brain, placenta and fibroblasts. Furthermore, the proportion of form II enzyme was higher in a detergent-free aqueous extract of spleen than in a Triton X-100 extract of total spleen or splenic membranes. When form II glucocerebrosidase in a splenic extract was separated from form I enzyme by immunoaffinity chromatography and stored at 4 degrees C, a gradual conversion to form I enzyme occurred. The conversion was almost immediate if 30% (v/v) ethylene glycol was present. In the denatured state both forms of glucocerebrosidase reacted with anti-(placental glucocerebrosidase) antibodies. Form I glucocerebrosidase was stimulated by sodium taurocholate or sphingolipid-activator protein 2 (SAP-2), whereas form II enzyme exhibited maximal activity in the absence of the effectors. The pH activity profile of form II glucocerebrosidase was almost identical to that of form I enzyme in the presence of SAP-2. In the native state, form I glucocerebrosidase had a molecular mass of 60 kDa whereas that of form II glucocerebrosidase was about 200 kDa. After gel-permeation high-performance liquid chromatography of splenic extracts, the fractions with form II glucocerebrosidase contained material cross-reacting with both anti-(placental glucocerebrosidase) and anti-(SAP-2) antibodies. Preincubation of form I glucocerebrosidase with SAP-2 at pH 4.5 led to masking of the epitope on glucocerebrosidase reacting with monoclonal anti-(placental glucocerebrosidase) antibody 2C7. Furthermore, preincubation of form I glucocerebrosidase with monoclonal antibody 2C7 prevented activation of the enzyme by SAP-2. We propose that form I glucocerebrosidase is a monomeric form of the enzyme, whereas form II glucocerebrosidase is a high-Mr complex of the enzyme in association with sphingolipid-activator protein 2.     

The replication band of Euplotes is enriched with phosphoproteins

Summary— Employing several antibodies to phosphorylated protein epitopes, we demonstrate by immunostaining that the macronuclear replication band (RB) of the ciliated protozoan Euplotes eurystomus contains a high concentration of phosphoproteins. Enrichment is principally within the rear zone of the RB, the region of DNA synthesis and chromatin assembly. By immunoblot analysis, the various antibodies reacted with a diversity of macronuclear phosphoproteins, one of which was phosphorylated histone Hl. This diversity of phosphoproteins was also supported by examination of the macronuclear matrix generated by high NaCl extraction. Available evidence clearly indicates that the ultrastructural wave of chromatin modulation accompanying DNA replication is spatially correlated with a wave of localized nuclear protein phosphorylation.     

鼠冠状病毒核衣壳蛋白的抗体制备与抗原决定簇分析

核衣壳(nucleocapsid,N)蛋白有稳定病毒基因组、调控病毒复制及细胞状态的特殊作用。鼠肝炎病毒(murine hepatitis virus,MHV)为乙型冠状病毒属的原型病毒,是研究冠状病毒N蛋白功能的经典模型。本研究用去污剂处理鼠冠状病毒粒子暴露N蛋白,另用原核表达纯化的重组N蛋白分别免疫小鼠,制备多克隆及单克隆抗体。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹分析结果显示两类抗体均具有高灵敏度和特异度,与甲型冠状病毒猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)的N蛋白无交叉反应。原核表达缺失突变的N蛋白分析结果显示,多克隆抗体与单克隆抗体2E6识别的鼠冠状病毒N蛋白抗原决定簇完全一致,位于N端结构域(N-terminal domain,NTD)C端与SR之间的58个氨基酸残基内。此外,基于单克隆抗体2E6的ELISA及免疫荧光法能检测到感染细胞中和培养上清液中的N蛋白组分,且其含量与病毒复制的滴度一致。这些结果表明,鼠冠状病毒复制过程中粒子与细胞中的N蛋白可能维持相似的结构,使NTD与SR之间的部分氨基酸残基一直暴露在表面,从而形成了优势抗原决定簇。     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Purification of an antibacterial protein in the coelomic fluid of Nereis diversicolor (annelida,polychaeta). similitude with a cadmium-binding protein

1. Coelomic fluid of Nereis diversicolor shows antibacterial activity 24 hr after inoculation with Escherichia coll, while fluid from untreated control does not.2. This activity is detected by growth inhibition of E. coli or Micrococcus kristinae.3. The thermolabile protein has been purified by two distinct methods using gel filtration and exchange ion chromatography. By SDS-PAGE, the molecular weight of this protein has been estimated at about 10 kDa.4. The protein shows strong similitude with cadmium-binding protein (CBP) which is a dimer constituted by two 10 kDa subunits (Nejmeddine et al., 1988).5. In Western Blot, monoclonal antibody against CBP reacted with the antibacterial protein.6. Moreover, bacteriostatic activity of the coelomic fluid disappeared after incubation with polyclonal and monoclonal antibodies.     

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP auxin-binding protein - DEAE diethylaminoethyl - Ig immunoglobulin - kDa kilodalton - NAA naphthalene-1-acetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Genetic Variants of Plateins (Alveolar Plate Proteins) Among and Within Species of Euplotes1

ABSTRACT. In the ciliate Euplotes, each of the sub-plasmalemmal membranous sacs (the cortical alveoli) encloses a thin polygonal scale or alveolar plate (AP). Adjoining alveoli and their contained plates are tightly integrated into a confluent monolayer that appears to strengthen and help define the shape of the cell cortex. Recently the major proteins making up the AP have been identified. Monoclonal antibodies (MAb) prepared against the AP proteins (termed plateins) of E. aediculatus show reactivity by immunofluorescence with the plates of a wide variety of Euplotes species (including E. eurystomus, E. harpa, E. woodruffi, and E. patella). However, each species tested shows a different pattern of platein bands on immunoblots, in terms of the number and apparent molecular weights (M_r) of the reactive polypeptides. One species (E. gracilis) did not show reactivity with these MAb. Intraspecific platein variants were found within the E. woodruffi complex and among strains of different geographic origin in E. aediculatus. To study the heritability of these platein variants, we used two E. aediculatus clones of different mating type, collected at the same site, that show reproducible differences in the electrophoretic mobility of their lowest M_r platein. Both share common platein bands at 125 kDa and 99 kDa. One clone has its third platein band at 97 kDa, the other clone at 95 kDa. Fourteen F1 clones from matings of these two parental strains have been tested by immunoblotting (using anti-platein MAb). Each F1 clone has the lower Mr plateins of both parents, and hence displays (in addition to the 125-kDa band) a triplet of bands (99, 97 and 95 kDa) in this region of the gel/blot, rather than one of the alternative doublets exhibited by either parent clone. The simplest interpretation of these results is that the two lowest Mr plateins represent Mendelian allelic variants, co-dominant at this level of analysis. No phenotypic differences in cortical structure or properties have yet been noted that might correlate with the identified platein variants.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.