Rapid isolation of monoclonal antibodies. Monitoring enzymes in the phytochelatin synthesis pathway.

Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecular levels. Furthermore, modern metabolic engineering requires accurate control of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these numerous proteins limits all proteomic and metabolic engineering projects. We describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptides were highly immunogenic in mice and crude protein extracts were effective sources of antigen, thus eliminating the time-consuming step of purifying the target proteins for antibody production. A case study was made of the three-enzyme pathway for the synthesis of phytochelatins. Enzyme-linked immunosorbent assays and western blots with the recombinant proteins in crude extracts demonstrated that the monoclonal antibodies produced to synthetic peptides were highly specific for the different target proteins, gamma-glutamyl cysteine synthetase, glutathione synthetase, and phytochelatin synthase. Moreover, immunofluorescence localization studies with antibacterial gamma-glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respective target proteins in chemically fixed cells from transgenic plants. This approach enables research to progress rapidly from the genomic sequence of poorly characterized target genes, to protein-specific antibodies, to functional studies.     

Production and characterization of peptide antibodies

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.     

Using structural analysis to generate parasite-selective monoclonal antibodies

Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B. malayi. B. malayi and human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 A crystallographic structure of B. malayi AsnRS was used to identify peptidyl regions that are surface accessible and available for antibody binding. Sequery and SSA (Superpositional Structural Analysis) software was used to analyze which of these peptides was most likely to maintain its native conformation as a synthetic peptide, and its predicted helical structure was confirmed by NMR. A 22-residue peptide was synthesized to produce murine mAbs. Four IgG(1) mAbs were identified that recognized the synthetic peptide and the full-length parasite AsnRS, but not human AsnRS. The specificity and affinity of mAbs was confirmed by Western blot, immunohistochemistry, surface plasmon resonance, and enzyme inhibition assays. These results support the success of structural modeling to choose peptides for raising selective antibodies that bind to the native protein.     

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the beta-chain in three host species by synthetic overlapping peptides representing the entire chain.

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.     

Conserved group-specific epitopes of the circumsporozoite proteins revealed by antibodies to synthetic peptides

The immunogenic properties of sporozoites are associated mainly with the circumsporozoite (CS) protein that covers the surface of mature sporozoites. This stage-specific protein has an immunodominant region with repetitive epitopes. Rabbits that are repeatedly immunized with sporozoites of Plasmodium knowlesi, a monkey malaria parasite, also recognize two synthetic peptides (N2 and C2) representing other polar domains of the CS protein. We show in this report that antibodies to the N2 and C2 synthetic peptides react not only with P. knowlesi but also with conserved regions of the surface membrane of other human, monkey, and rodent (but not avian) malaria sporozoites. Moreover, antibodies to N2 partially neutralize the infectivity of sporozoites of P. berghei, a rodent malaria parasite. In contrast, antibodies to synthetic peptides representing the repetitive epitope of P. knowlesi were strictly species specific.     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

Summary In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABP_c), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABP_c molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABP_c. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.     

Protein arrays for autoantibody profiling and fine-specificity mapping

Protein arrays provide a powerful approach to study autoimmune disease. Autoimmune responses activate B cells to produce autoantibodies that recognize self-molecules termed autoantigens, many of which are proteins or protein complexes. Protein arrays enable profiling of the specificity of autoantibody responses against panels of peptides and proteins representing known autoantigens as well as candidate autoantigens. In addition to identifying autoantigens and mapping immunodominant epitopes, proteomic analysis of autoantibody responses will further enable diagnosis, prognosis, and tailoring of antigen-specific tolerizing therapy.     

Profiling of myelin proteins by 2D-gel electrophoresis and multidimensional liquid chromatography coupled to MALDI TOF-TOF mass spectrometry

The myelin sheath is an electrically insulating layer that consists of lipids and proteins. It plays a key role in the functioning of the nervous system by allowing fast saltatory conduction of nerve pulses. Profiling of the proteins present in myelin is an indispensable prerequisite to better understand the molecular aspects of this dynamic, functionally active membrane. Two types of protein, the myelin basic protein and the proteolipid protein, account for nearly 85% of the protein content in myelin. Identification and characterization of the other "minor" proteins is, in this respect, a real challenge. In the present work, two proteomic strategies were applied in order to study the protein composition of myelin from the murine central nervous system. First, the protein mixture was separated by 2D-gel electrophoresis and, after spot excision and in-gel digestion, samples were analyzed by mass spectrometry. Via this approach, we identified 57 protein spots, corresponding to 38 unique proteins. Alternatively, the myelin sample was digested by trypsin and the resulting peptide mixture was further analyzed by off-line 2D-liquid chromatography. After the second-dimension separation (nanoLC), the peptides were spotted "on-line" onto a MALDI target and analyzed by MALDI TOF-TOF mass spectrometry. We identified 812 peptides by MALDI MS/MS, representing 93 proteins. Membrane proteins, low abundant proteins, and highly basic proteins were all represented in this shotgun proteomic approach. By combining the results of both approaches, we can present a comprehensive proteomic map of myelin, comprising a total of 103 protein identifications, which is of utmost importance for the molecular understanding of white matter and its disorders.     

Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.     

Monoclonal antibodies specific for African swine fever virus proteins.

We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles.     

Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies

We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.     

Preparation of antibodies capable of recognizing the Glu/Lys amino acid substitution at position 129 of the survivin sequence

Survivin is an oncofetal protein involved in the inhibition of apoptosis and the regulation of cell division. The functions of survivin are determined by its structural state (monomer or dimer). Owing to the natural polymorphism, either the Glu or the Lys residue can be at position 129 of the amino acid sequence of survivin. Lys has the capability for acetylation, and only the protein containing the acetylated residue Lys¹²⁹ tends to form a dimer. Thus, antibodies recognizing the amino acid substitution Glu129Lys can serve as a tool in the structural and functional investigations of survivin. For preparing the target antibodies, survivin fragments containing residue 129 were synthesized, rabbits were immunized with synthetic peptides, and the antibodies were purified by affinity chromatography on Sepharose conjugated with the corresponding peptides. It was shown by ELISA and immunoblotting that the affinity-purified antibodies are capable of recognizing the amino acid substitution Glu129Lys in the sequence of recombinant and endogenous survivin.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.