A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin

Two hybridoma cell lines were selected after the fusion of the myeloma cell line X-63 Ag8-653 with spleen cells from mice immunized with bovine brain microtubules. These lines, clones 3F3 and 16D3, secrete IgM antibodies both staining a fibrillar network in fibroblasts. Autoradiography of immunoblots of SDS gels showed that the antigenic determinants defined by these antibodies are present on tubulin and also on several other polypeptides in mammalian cells. In contrast, they were found to react only with tubulin in Trypanosoma brucei, parasitic protozoan which are the causative agent of sleeping sickness. By immunofluorescence microscopy, 3F3 bound only to a subpopulation of microtubules associated with the flagellum of these cells when, under the same conditions, 16D3 stained other microtubule populations including sub-pellicular microtubules. These results show that flagellar tubulin differs from tubulin of other locations in the same cell by at least one antigenic determinant which could be involved in microtubule specialization.     

Heterogeneity of rat macrophages recognized by monoclonal antibodies: an immunohistochemical and immunoelectron microscopic study

Three monoclonal antibodies, designated RM-1, TRPM-1, and TRPM-2, were raised against rat peritoneal macrophages. By the immunoperoxidase method, antigens recognized by these antibodies were distributed throughout most tissue and free macrophages examined, including those of splenic red pulp, lymphatic sinus, connective tissue, and peritoneal cavity, as well as Kupffer cells of liver and alveolar macrophages. The numbers of positive cells were different for each antibody. RM-1 and TRPM-1 were also reactive with interdigitating cells (IDCs) in the thymus-dependent area and with Langerhans cells in the skin, whereas TRPM-2 failed to demonstrate IDCs in thymic medulla and Langerhans cells. The reactions of each antibody were observed by immunoelectron microscopy in the different ultrastructural compartments of the cells. RM-1 recognized a cell surface antigen; reaction products for TRPM-1 were found on a part of the cell membrane and in the cytoplasmic vacuoles; and those of TRPM-2 were present along the nuclear envelope and intracytoplasmic vacuoles. These antibodies seem to be useful not only for the detection of macrophages in tissue sections but also for investigation of macrophage heterogeneity in different tissues.     

鉴定单克隆抗体识别蛋白质抗原表位方法的建立

为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法,选择程序死亡受体-1(PD-1)作为目的蛋白。基于丙氨酸扫描策略,建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法,确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体,鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位,与已报道的基于晶体结构的抗原表位高度一致,表明本方法操作简单、准确性高,可用于治疗性单克隆抗体的抗原表位作图。     

Dinitroanilines bind alpha-tubulin to disrupt microtubules

Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in alpha-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of alpha-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma alpha-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus alpha-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate alpha-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on alpha-tubulin function.     

Study of antigenic epitopes recognized by monoclonal antibodies to recombinant interferon-gamma

Seven hybridomas (BG 1-7) which secreted monoclonal antibodies against recombinant interferon-gamma were produced. The ascites fluids containing four of the seven monoclonal antibodies (BG 1-4) neutralized the antiviral activity of both natural and recombinant interferon-gamma. Competition between labeled and unlabeled monoclonal antibodies for interferon-gamma in a solid phase immunoassay showed that BG 1 was competed by both BG 3 and BG 4 but not by BG 2; BG 2 was competed by BG 3 but not by BG 1 nor by BG 4. These results suggest that human interferon-gamma has at least two antigenic epitopes; one of the epitopes reacted with BG 1 & BG 4 while the other reacted with BG 2; BG 3 either binds to a region overlapping with the other two epitopes or reacts with both epitopes. The antigenic epitopes recognized by these four neutralizing monoclonal antibodies are likely at or closely related to the active sites of interferon-gamma.     

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.     

Structural analysis of the epitopes recognized by monoclonal antibodies to angiotensin II

Six clones were obtained that secrete anti-angiotensin II antibodies after somatic cell fusions between splenocytes of immunized BALB/c or outbred OF1 mice and NS-1 myeloma cells. The dissociation constants for angiotensin II ranged from 0.3 to 2.9 nM. A panel of 20 structural analogs of the hormone were used as probes to analyze the specificity of binding. From the binding studies and the putative three-dimensional structures of the tested peptides, three families of antibodies could be distinguished that recognized overlapping epitopes; the conservation of the native conformation of the angiotensin II molecule in the analogs appeared essential for the preservation of a high affinity to the antibodies. With one antibody, the affinities of the angiotensin II analogs have been correlated with their intrinsic biologic activities (as measured by in vivo pressor tests), and not with their binding affinity to the membrane receptor. These results are interpreted as mimicry, by the antibody binding site, of the active conformation of the receptor site.     

Allotypic specificities of murine IgD and IgM recognized by monoclonal antibodies

Hybridomas generated from mice immunized with allotype and H-2-incompatible spleen cells were screened by flow cytometry. Monoclonal antibodies (MAb) to four of the five known specificities of IgD were identified. The location of these specificities on the IgD molecule was determined by the ability of a given MAb to bind to cells stripped of the Fab fragment by trypsin proteolysis. Igh-5.1 (present on IgDa and IgDe) and Igh-5.5 (unique to IgDe) were both located on the Fab fragment. Results from cross-blocking experiments suggest that, except for Igh-5.3, MAb which recognize the same specificity bind to the same antigenic determinant. Both the trypsin digest and blocking studies indicate that Igh-5.3 is composed of at least two antigenic determinants. AF6-78.25, a MAb specific for IgM of the b, d, and n haplotypes, was also identified; this MAb defines a new specificity, Igh-6.6. On the basis of the reactivities of these MAb, it appears that although the Igh-Ce and Igh-Cd haplotypes are virtually identical at the Igh-3(gamma 2b), Igh-1(gamma 2a), and Igh-2(alpha) loci, they are highly divergent at the Igh-5(delta) and Igh-6(mu) loci. This indicates that the Igh-Cd haplotype may have resulted from a recombination between Igh-Ce and another haplotype.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Characterization of epitopes recognized by anti-Streptococcus mutans P1 monoclonal antibodies

Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.     

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.     

A monoclonal antibody to alpha-tubulin: purification of functionally active alpha-tubulin isoforms

Both alpha- and beta-tubulin exist as numerous isotypic forms that originate from different primary sequences as well as a variety of posttranslational modifications. Recent studies show that tubulin dimers differing in the beta-subunit differ significantly in their subcellular distribution as well as in their functional properties such as assembly, dynamics, conformation, and interaction with antimitotic drugs; however, very little is known about the functional significance of the different alpha-tubulin isoforms and their posttranslational modifications. In an effort to get a better understanding about the alpha-tubulin isoforms, a monoclonal antibody, AYN.6D10, was prepared against the mammalian alpha-tubulin C-terminal sequence Glu-Glu-Gly-Glu-Glu-Tyr. Using an immunoaffinity column, bovine brain tubulin was fractionated into three functionally active alphabeta heterodimers which were identified by immunoblotting with alpha-tubulin-specific antibodies and sequence analysis. Assembly studies in the presence of glycerol and Mg2+ show that one of the fractions, that contains mainly the tyrosinated form of alpha1/2, assembled poorly, while the nontyrosinated form assembled normally. The results indicate that tubulin dimers differing in their alpha-tubulin may differ in their functional properties. Future studies with the isoforms may yield valuable information regarding the role of alpha-tubulin and its posttranslational modifications in regulating microtubule assembly and function in vivo.     

Heterogeneity of mouse Thy 1.2 antigen expression revealed by monoclonal antibodies

A different sensitivity of T cells from C57B1/6 and DBA/2 mice to treatment with the monoclonal anti-Thy 1.2 F7D5 serum as compared with a conventional alloantiserum is reported. Depletion of T helper cells, Con A-, PHA-, MLC-, and GVH-reactive cells from a DBA/2 or C57B1/6 spleen cell population was readily achieved with the conventional alloserum. In contrast, the F7D5 antiserum abolished all T functions studied in C57B1/6 spleen cells whereas it was totally or partially ineffective on DBA/2 spleen cells when T helper, MLC, or GVH reactivity were assayed. It did however eliminate the capacity of DBA/2 spleen cells to respond to stimulation with Con A or PHA. Analysis in an Ortho-Cytofluorograf of thymocytes and sIg^? lymphocytes labeled with either GAMB-F or F7D5 + RAM Ig-F showed no difference at the level of the thymocytes: Thy 1.2 antigen as revealed by either GAMB or F7D5 is similarly expressed in the two mouse strains. The fluorescence profiles of splenic T lymphocytes indicated a reduced representation per unit cell basis of the Thy 1.2 antigenic determinant recognized by F7D5 in DBA/2 mice. Moreover, this same determinant is expressed in only 70% of all Thy 1.2-positive cells detected in DBA/2 sIg^? population. This implies that, in DBA/2 mice, maturation of T cells is accompanied by a complete or partial loss of the F7D5 Thy 1.2 determinant and that T helper functions and MLC and GVH reactivity are mediated by T cells which express little or none of this F7D5 Thy 1.2 determinant.