Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution

The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.     

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG₁/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.     

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.     

Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched ⁴⁶⁹KTPAEAR⁴⁷⁵ of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.     

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.     

Development and characterization of 1C6-203, a new monoclonal antibody specific to human thymidine phosphorylase.

Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma.     

Multiple epitopes in a dodecapeptide of myelin basic protein determined by monoclonal antibodies

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89.     

Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida.

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.     

Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids.

Monoclonal antibody (MAb) HNK-1 recognizes a carbohydrate epitope present in certain glycolipids, glycoproteins, and proteoglycans. Five different fixation methods, together with biochemical analyses of the antigens, were evaluated to study immunocytochemical localization of this epitope in layers of adult rat cerebellum; 4% paraformaldehyde/0.5% cetylpyridinium chloride was found to be optimal for overall immunoreactivity, and the antigens were apparent in all cerebellar layers. To differentially localize HNK-1-reactive carbohydrate epitope on proteins vs lipids in cerebellar layers, we tested the effect of 0.2%, 2%, or 4% glutaraldehyde combined with 2% paraformaldehyde (GT/PF) on HNK-1 and other MAb-reactive protein and lipid antigens; 2% or 4% GT/PF significantly reduced or abolished immunoreactivity of MAb HNK-1 and 5F9 (reacting with microtubule-associated protein 2) with cerebellar proteins analyzed on Western blots, but did not decrease HNK-1 reactivity to lipid antigens on HPTLC blots. In cerebellar tissue sections, HNK-1 and 5F9 immunoreactivity was reduced after 2% or 4% GT/PF fixation. However, significant amounts of HNK-1 immunoreactivity remained in molecular layer and deep cerebellar nuclei. GT/PF fixation did not cause significant changes in immunoreactivity patterns of other carbohydrate lipid antigens, such as those that react with MAb A2B5, 7A, and WCC4. Therefore, carbohydrate epitope on lipids, as opposed to that on proteins, may be preferentially detectable by immunocytochemistry after fixation with 2% or 4% GT/PF. The selective localization of HNK-1-reactive carbohydrate in the molecular layer and deep cerebellar nuclei with 2% or 4% GT/PF fixation correlates well with the observed presence of HNK-1-reactive lipids in these areas but not in the granular layer and white matter, as determined by microdissection of the individual layers and biochemical analysis. The application of 2% or 4% GT/PF fixation as a general method for differentiating the same carbohydrate epitope on proteins vs lipids in immunocytochemistry for other tissues and other antibodies remains to be further evaluated.     

Identification of a novel linear B-cell epitope in the M protein of avian infectious bronchitis coronaviruses

This report describes the identification of a novel linear B-cell epitope at the C-terminus of the membrane (M) protein of avian infectious bronchitis virus (IBV). A monoclonal antibody (MAb) (designated as 15E2) against the IBV M protein was prepared and a series of 14 partially-overlapping fragments of the IBV M gene were expressed with a GST tag. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using MAb 15E2 to identify the epitope. A linear motif, ¹⁹⁹FATFVYAK²⁰⁶, which was located at the C-terminus of the M protein, was identified by MAb 15E2. ELISA and western blotting also showed that this epitope could be recognized by IBV-positive serum from chicken. Given that 15E2 showed reactivity with the ¹⁹⁹FATFVYAK²⁰⁶ motif, expressed as a GST fusion protein, in both western blotting and in an ELISA, we proposed that this motif represented a linear B-cell epitope of the M protein. The ¹⁹⁹FATFVYAK²⁰⁶ motif was the minimal requirement for reactivity as demonstrated by analysis of the reactivity of 15E2 with several truncated peptides that were derived from the motif. Alignment and comparison of the 15E2-defined epitope sequence with the sequences of other corona-viruses indicated that the epitope is well conserved among chicken and turkey coronaviruses. The identified epitope should be useful in clinical applications and as a tool for the further study of the structure and function of the M protein of IBV.     

Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria.

The isolation and characterization of a monoclonal antibody (MAb 2G5) specific for the bacterial DnaK (HSP70) protein is described. The 2G5 MAb was initially selected because of its ability to bind to DnaK under denaturing conditions. Isotype analyses indicated that 2G5 was an immunoglobulin G2a. Dose-response Western blot (immunoblot) experiments with purified but unconcentrated 2G5 permitted detection of 10 ng of pure DnaK protein. The DnaK epitope was determined by Western blot analysis of a series of truncated DnaK fragments overproduced in Escherichia coli using 5' and 3' dnaK-deleted expression plasmids. The epitope mapped to a 22-amino-acid region spanning DnaK residues 288 and 310. Phylogenetic distribution of the epitope was examined by Western blot analysis of a wide variety of bacterial species and indicated that the epitope was uniquely present in gram-negative organisms. The proximity of the epitope to the presumed DnaK ATP-binding pocket suggested that MAb binding might inhibit DnaK ATPase activity. In vitro analysis supported this prediction and demonstrated that MAb-mediated inhibition of ATPase activity was antibody specific and occurred at stoichiometric molar ratios of MAb to DnaK. Possible mechanisms to explain the ability of the 2G5 MAb to inhibit DnaK activity are discussed.     

Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Analysis of a neutralizing antibody for human herpesvirus 6B reveals a role for glycoprotein Q1 in viral entry

Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.     

High-efficiency generation of monoclonal antibody for Vitreoscilla hemoglobin protein

Bacterial hemoglobin from Vitreoscilla (VHb) is recognized as a good fusion protein for the soluble expression of foreign protein. In this study, we generated a monoclonal antibody (MAb) against VHb for its detection. For the rapid screening of MAb, a protein chip technology based on the Alexa-488 (A488) dye labeling method was introduced. In order to fabricate the chip, the VHb protein was chemically coupled to the chip surface and then the culture supernatants of 84 hybridoma cell lines were spotted onto the VHb chip. The bound MAbs were measured by A488- modified anti-mouse IgG. A single spot (MAb A10) exhibited significantly high signal intensity. The immunoblot analysis evidenced that the MAb A10 can detect VHb-fused proteins with high specificity.     

Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver

We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein–protein or protein–lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340–384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.     

A chondroitin sulfate proteoglycan that is developmentally regulated in the cerebellar mossy fiber system.

It is known that the mammalian brain contains many kinds of proteoglycans, but almost all of them remain to be characterized. In this study, we prepared a monoclonal antibody against a phosphate-buffered saline-soluble brain proteoglycan (MAb 6B4). MAb 6B4 recognized a 600- to 1000-kDa chondroitin sulfate proteoglycan with a 250-kDa core protein (6B4 proteoglycan). The core protein of 6B4 proteoglycan carried the HNK-1 epitope. Immunohistochemical analysis of the adult rat brain indicated that this proteoglycan was expressed on the cell surfaces of a subset of neurons. In the hindbrain, 6B4 proteoglycan was highly expressed on the cerebellar Purkinje cells and Golgi cells, and at particular nuclei including the pontine nuclei and lateral reticular nucleus. Almost all of these nuclei were connected to the cerebellum through the mossy fiber system. A developmental study indicated that the expression of this proteoglycan changed dramatically during the formation of the cerebellar mossy fiber system. The mossy fibers from the pontine nuclei expressed 6B4 proteoglycan transiently from Embryonic Day 20 (E20) to Postnatal Day 30 (P30), during which time the axonal outgrowth and glomerular synapse formation occurred. The Purkinje cells, glomeruli, and Golgi cells began to be stained with MAb 6B4 from P10, P16, and P20, respectively. These expression stages correspond with the onset of their synapse formation. These results suggest that 6B4 proteoglycan is closely involved in the development of the cerebellar mossy fiber system.