Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 Mr surface exposed region of the molecule.     

幽门杆菌Catalase/GST融合蛋白的表达、标签切除及鉴定

旨在利用GST融合基因表达系统表达幽门螺杆菌Catalase融合蛋白,并利用凝血酶切除GST标签.将重组质粒Catalase/pGEX-4T-1转化大肠杆菌BL21( DE3)感受态中,用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,Catalase/GST融合蛋白以部分可溶性的形式表达在上清中.采用谷胱甘肽琼脂糖树脂Glutathione Sepharose 4B对其进行纯化,得到Catalase/GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western blotting鉴定.高效表达出Catalase/GST融合蛋白的相对分子质量约85 kD,凝血酶成功地切除了GST标签,Western blotting证实Catalase蛋白能被鼠抗Catalase单克隆抗体识别.     

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.     

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.     

Linear B-cell epitopes of the major core protein of human immunodeficiency virus types 1 and 2.

Nine murine monoclonal antibodies directed to the major core protein p24 of human immunodeficiency virus type 1 (HIV-1) were obtained and then tested by using an epitope mapping system (Pepscan) covering the whole p24HIV1 protein to characterize antigenic domains. Four different linear epitopes were identified. Monoclonal antibodies recognizing three of these epitopes also reacted to p26HIV2 in Western blotting (immunoblotting). A monoclonal antibody specific for the fourth epitope, located at position 179 to 188 of the gag polyprotein p55HIV1 (human T-cell lymphotropic virus type 3B strain), did not react with HIV type 2 (HIV-2) core proteins. The corresponding sequence is constant in all known HIV-2 and simian immunodeficiency virus (SIV) isolates, including a very divergent SIV strain from African green monkeys (SIVagm/tyo). This observation may be relevant to the phylogeny of primate lentiviruses. Two of the conserved epitopes might be immunogenic during natural infection and could therefore be used for diagnosis and prognosis purposes. These two epitopes are AAEWDRVHP and EIYKRWII, starting at positions 209 and 260 of the polyprotein p55HIV1, respectively.     

Identification of two antigenic epitopes on SARS-CoV spike protein

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1. The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. A number of monoclonal antibodies were developed against the fusion protein. In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively. Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively. Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.     

SOX4单克隆抗体的制备及其在肿瘤细胞表达分析中的应用

应用分子生物学技术, 构建了含SOX4编码序列的原核表达载体, 在大肠杆菌 DH5a中获得了GST-SOX4融合蛋白的可溶性表达。应用谷胱甘肽-Sepharose 4B对重组蛋白进行了纯化, 利用纯化的融合蛋白免疫小鼠, 制备了可特异性识别SOX4的单克隆抗体。通过间接 ELISA 法鉴定了抗体的效价为1 × 10-5, Western blotting 分析证实了抗体的特异性。结果显示, 该抗体可识别细胞内外源性过表达及内源性的SOX4蛋白。在培养细胞系、小鼠不同组织中, SOX4蛋白的表达存在显著的差异。本研究制备的SOX4单克隆抗体具有良好的特异性, 为进一步研究SOX4在肿瘤发生中的作用提供了重要的工具。     

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.     

A monoclonal antibody directed against the head region of vimentin

The production and identification of a monoclonal antibody directed against an epitope in the aminoterminal head region of vimentin is described. Enzyme-linked immunosorbent assay, protein blotting and indirect immunofluorescence were used. The wide range of cross-reactivity within cytoskeletal proteins observed for this antibody gives evidence for a determinant in an evolutionarily conserved region. Computer comparison of aminoacid sequences of the immunoreactive proteins and biochemical cleavage of vimentin provide possible clues to some antigenic determinants.     

Linear and Conformation-Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.     

The cGMP-gated cation channel of bovine rod photoreceptor cells is associated with a 240-kDa protein exhibiting immunochemical cross-reactivity with spectrin.

A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.     

β-Galactofuranoside glycoconjugates on conidia and conidiophores of Aspergillus niger

Galactose in the furanoic conformation appears to be limited to bacteria and lower eukaryotes. Galactofuranoic (Galf)-containing glycoconjugates that occur in organisms pathogenic or allergenic to man are frequently antigenic and immunodominant. We have used an immunochemical approach, employing a monoclonal antibody that recognises Galf epitopes, to investigate the presence of Galf-containing glycoconjugates within conidia and conidiophores of Aspergillus niger. ELISA and immunofluorescence microscopy indicated that specific and saturable binding sites were found on both. Inhibition studies confirmed that this binding was to Galf-containing glycoconjugates. Interestingly, the conidiophore heads were particularly rich in these glycoconjugates. Western blotting identified a Galf glycoprotein of 150-200 kDa from disrupted conidia.     

Development and partial characterization of monoclonal antibodies to the hemolymph juvenile hormone binding protein of Manduca sexta

Murine monoclonal antibodies were made against the hemolymph juvenile hormone binding protein (JHBP) of Manduca sexta. Binding studies in conjunction with Western blot analysis of native and sodium dodecyl sulfate gels confirmed that antibodies from 10 hybridoma lines interacted with the juvenile hormone binding protein. The pattern of cross-reactivity among the hybridoma lines suggests that different epitopes are recognized. The cross-reactivity pattern for monoclonal antibody 9 suggested a common epitope in three different hemolymph proteins: JHBP, insecticyanin and a 40–45 kDa protein. Western blot analysis of a two-dimensional gel using monoclonal antibody 6 revealed interaction with JHBP and with several proteins that may be precursors or degradation products of the binding protein. An enzyme-immunoassay was developed that detects JHBP in the hemolymph at nanogram levels.     

抗HCMV Pp65蛋白单克隆抗体的制备及性质研究

人类巨细胞病毒（Human cytomegalovirus,HCMV）是一种机会性感染疱疹病毒,在人群中感染比较常见,但在免疫缺陷个体与新生儿中可引起严重的疾病。HCMV Pp65是HCMV活动感染的主要标志,也是临床检验检测HCMV感染的重要靶标。为研发抗HCMV Pp65蛋白单克隆抗体作为临床免疫检测HCMV感染的关键原料,本研究采用重组表达的HCMV Pp65蛋白免疫BALB/c小鼠,将免疫小鼠淋巴结细胞与sp2/0细胞融合,采用间接ELISA法筛选阳性克隆与效价测定,再用Western blotting进行抗体特异性鉴定,最后用免疫捕获PCR和免疫荧光法评价其应用前景。最终获得了1株能稳定分泌高效价的抗HCMV Pp65单克隆抗体的杂交瘤细胞株,命名为8D6。Western blotting及间接ELISA检测其效价分别达1∶4 000和1∶105;细胞免疫荧光与免疫捕获PCR实验结果说明,该杂交瘤细胞株分泌的单克隆抗体具有良好的亲和力和特异性,具有作为外周血细胞免疫荧光细胞化学和免疫捕获PCR检测HCMV临床感染的关键原料,也可作为双抗体夹心法检测HCMV临床感染的关键原料,为建立HCMV感染快速灵敏的临床诊断试剂打下了重要的基础。     

Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus.

Human cytomegalovirus contains an envelope glycoprotein of 58 kilodaltons (gp58). The protein, which is derived from a glycosylated precursor molecule of 160 kilodaltons via proteolytic cleavage, is capable of inducing neutralizing antibodies. We have mapped the epitopes recognized by the neutralizing monoclonal antibody 7-17 and a second antibody (27-287) which is not neutralizing. Overlapping fragments of the carboxy-terminal part of the open reading frame coding for gp58 were expressed in Escherichia coli as beta-galactosidase fusion proteins. The reactivities of antibodies 7-17 and 27-287 were determined by Western blot (immunoblot) analysis. Both antibodies recognized sequences between amino acids 608 and 625 of the primary gp58 translation product. The antibodies almost completely inhibited one another in a competitive binding assay with intact virus as antigen. Moreover, antibody 27-287 was able to inhibit the complement-independent neutralizing activity of antibody 7-17.     

Epitope mapping of Escherichia coli cell division protein FtsZ with monoclonal antibodies.

A fusion between lacZ and ftsZ of Escherichia coli was constructed to obtain a beta-galactosidase-FtsZ fusion protein. This fusion protein was used to raise antibodies against cell division protein FtsZ. Six monoclonal antibodies were obtained, and they reacted with FtsZ from cytoplasm and membrane fractions. The epitopes in FtsZ were localized by studying the reactions of the monoclonal antibodies with fusion proteins truncated at the carboxy terminus and with fragments that were obtained by CNBr cleavage of purified FtsZ. Five different epitopes were defined. Epitopes I and III reacted with the same monoclonal antibody, without showing apparent amino acid homology. Epitope II was defined by monoclonal antibodies that cross-reacted with an unknown cytoplasmic 50-kDa protein not related to FtsZ. Epitopes IV and V were recognized by different monoclonal antibodies. All monoclonal antibodies reacted strongly under native conditions, so it is likely that the five epitopes are situated on the surface of native FtsZ. By using these data and computer analysis, a provisional model of FtsZ is proposed. The FtsZ protein is considered to be globular, with a hydrophobic pocket containing GTP-binding elements. Epitopes I and II are situated on each side of the hydrophobic pocket. Because the carboxy terminus contains epitope V, the carboxy terminus of FtsZ is likely oriented toward the protein's surface.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.