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。     

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.     

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.     

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.     

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.     

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.     

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50–200 by in length and cloning these into the 5 terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.     

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.     

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.     

Comparative study of epitopes recognized by two monoclonal antibodies that protects mice against Trichomonas vaginalis challenge

Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.     

Identification of epitopes on cytochrome P450 3A4/5 recognized by monoclonal antibodies

In this study we describe the mapping of epitopes on CYP3A4/5 recognized by a panel of monoclonal antibodies (MAbs). CYP3A4 and CYP3A5 cDNAs were cloned in GST expression vectors and the fusion proteins were subjected to Western blot. Eight MAbs reacted with the full-length GST-3A4 fusion protein as well as baculovirus cDNA-expressed CYP3A4, while six of these reacted with baculovirus cDNA-expressed CYP3A5. Five (MAb 347, 351, 352, 354, and 357) out of 8 MAbs were inhibitory in a metabolic assay using quinine as substrate. MAbs 352, 354, and 357 brought about a moderate inhibition of quinine metabolism (60-70%) while MAb 347 inhibited quinine 3- hydroxylation in human liver microsomes (n=6) by more than 70%. MAb 347 was a potent inhibitor of baculovirus-expressed CYP3A5-catalyzed metabolism of quinine (95%) at 相似文献   

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.     

Transfer of polymorphic monoclonal antibody epitopes to the first and second domains of HLA-DR beta-chains by site-directed mutagenesis.

We used site-directed mutagenesis of HLA-DR beta-chains to localize the binding sites for two polymorphic DR-binding mAb to residues in the first and second external domains, respectively. Transfer of three first domain alpha-helical residues, G73, R74 and N77, normally present in DR3a and DRw52a, to a DR4 beta-chain was sufficient for recognition of this mutant DR molecule by a DR3-specific mAb, NDS 9. A polymorphism controlling the binding of a DR4-specific mAb, GS 359-13F10, was mapped to a tyrosine at position 96 of the DR4 beta-chain second domain by the construction fo a chimeric DR molecule containing a DR2-first domain and DR4-second domain. The mapping of these two polymorphic epitopes to specific positions on the DR beta-chain will allow further structural and functional analysis of the DR molecule.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Relationships between epitopes on IgE recognized by defined monoclonal antibodies and by the FC epsilon receptor on basophils

The present study investigated whether the sites on the FC region of the IgE molecule, recognized by different anti-IgE monoclonal antibodies (mAb), are identical to those recognized by the Fc receptor (Fc epsilon R). The anti-IgE mAb recognize different clusters of epitopes on the Fc region of IgE and could interfere to different degrees with the binding of IgE to mast cells and basophils, but still recognized cell-bound IgE. Analysis of the stoichiometry and affinity binding of 125I anti-IgE mAb Fab' to free IgE have revealed that anti-IgE mAb of one group (51.3) recognized three repetitive determinants on the IgE Fc portion, and another group (95.3) recognized only one determinant. When these stoichiometric studies were performed with cell-bound IgE, it was found that only one of the sites recognized by 51.3 mAb was involved in the Fc epsilon R binding site. On the other hand, the site recognized by 95.3 mAb was not the Fc epsilon R binding site. Such findings establish mAb 51.3 as a useful tool for isolating the IgE peptides involved in the binding site to the receptor.     

Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.     

Chain specificity assignment of monoclonal antibodies to human laminins by using recombinant laminin beta1 and gamma1 chains.

In the present study, the chain specificity of 16 commonly used monoclonal antibodies to human laminin(s) was analysed by using recombinant laminin beta1 and gamma1 chains. By ELISA, all antibodies reacted with purified placenta laminin, and most antibodies recognised either recombinant beta1 or gamma1 chains. Reactivity and chain specificity was confirmed against the recombinant chains in Western blotting under non-reducing conditions, and only a few antibodies were reactive under reducing conditions. Most antibodies were able to immunoprecipitate associated laminin beta1/gamma1 chains from platelet lysates. Based on these results and data from the literature, a tentative epitope map is presented.     

Epitope regions in the heavy chain of Clostridium botulinum type E neurotoxin recognized by monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) were previously established against the heavy chain (Hc) of botulinum type E neurotoxin in BALB/c mice immunized with the type E toxoid. Five MAbs (LE15-5, LE34-6, EK19-7, EK21-4, and AE27-9) showed toxin-neutralizing activity in mice. Two of the five MAbs, EK19-7 and EK21-4, recognized the regions located at amino acid positions 731 to 787 and 811 to 897, respectively. One of the remaining three antibodies (LE34-6) reacted with the amino acid sequence VIKAIN, at amino acid positions 663 to 668, closed by the ion channel-forming domain. It is suggested that the ion channel-forming domain may also be associated with the blocking of acetylcholine release. Furthermore, the amino acid sequence YLTHMRD within 30 residues of the C-terminal region of the Hc component seemed to be recognized by LE15-5. It has been reported that the binding domain of the type E toxin is located on the C-terminal half of the Hc component. Therefore, the neutralizing activity of LE15-5 antibody may be attributed to its ability to block the binding of neurotoxin to the receptor of target cells.