Pancreatic islet-cell epitope recognized by an anti-sulphatide monoclonal antibody.

Insulin-dependent (Type 1) diabetes mellitus is recognized as an autoimmune disease and islet-cell antibody (ICA) is present in the majority of patients at diagnosis. ICA labels both beta and alpha cells and is believed to be directed against a glycolipid. In this study we examine the presence of sulphatide (3'-sulphogalactosylceramide) or closely related structures (sulpholactosylceramide and seminolipid) in islet cells by means of a monoclonal antibody, Sulph I. Histological examination of pancreatic tissue from Lewis and BB rats, and BALB/c and NOD mice showed a pronounced labelling of the islets of Langerhans with Sulph I. No staining of the exocrine pancreatic tissue, the heart, the liver, the adrenals, the thymus, the spleen or lymph nodes was seen, but staining of some tubular cells and glomerular cells in the kidney as well as of myelin in nerve cells was found. Cytological examination of isolated Lewis islet cells and their cell subpopulations, separated using a fluorescence-activated cell sorter (FACS), showed positive surface labelling of 97.3 +/- 2.2% (SD) of the beta cells and 84.4 +/- 3.0% of the non-beta cells. Thus, the epitope on the glycolipid sulphatide or closely related structures is--with the exception of neural and certain kidney tissue--specifically present in islet cells. Furthermore, the staining pattern of the antibody used, Sulph I, was equivalent to that of ICA.     

免疫亲和质谱技术鉴定抗血管性血友病因子单克隆抗体SZ-125的抗原表位

目的 应用免疫亲和分离与质谱分析技术,结合合成肽法和氨基酸定点诱变技术,鉴定抗血管性血友病因子(vWF)单克隆抗体(mAb) SZ-125的抗原表位.方法 采用胰蛋白酶水解vWF A3区蛋白,产生一系列肽段,利用固定在琼脂糖珠上的SZ-125与其发生免疫亲和反应.用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术,对抗原决定簇肽段-抗体复合物进行分析.用人工合成分析得到的氨基酸序列并对其中的重要氨基酸位点进行了原位点突变.合成蛋白及突变蛋白均验证了其与抗体SZ-125间的结合力.结果 与SZ-125结合部位即连续表位的肽段为1001 EGGPSQIGDALGFAVR1016.免疫分析证实,合成肽段NH2-EGGPSQIGDALGFAVR-COOH可以与SZ-125结合.进一步通过定点诱变技术,分析得到决定抗原-抗体结合的关键氨基酸为E1001、F1013、V1015、R1016.结论 运用免疫亲和质谱、肽段合成、氨基酸定点突变等手段,精确定位了mAbSZ-125所结合的具体氨基酸位点.     

Identification of peptide motifs recognized by a human IgG autoanti-IgE antibody using a phage display library

BACKGROUND: The potential of murine monoclonal anti-IgE antibodies as long-term therapy for atopic diseases will have to rely, for the time being, on passive antibody administration. There is therefore considerable interest in developing a peptide-based vaccine for active immunization to elicit long-term protective anti-IgE antibodies in the patient. It has been shown that some human IgG autoanti-IgE antibodies have the ability to partially block the binding of IgE to Fc receptors such as Fc epsilonRI. Therefore, the epitopes recognized by such antibodies could have vaccine potential. OBJECTIVE: To determine the epitope specificity of one such human IgG anti-IgE antibody. METHODS: A 15-mer phage-peptide library was used to establish the epitope specificity of an IgG anti-IgE antibody isolated from the serum of an asthma patient. RESULTS: The SRPSP sequence, or part of it (i.e. RPS, RPSP, SPS or PSP), was present in all 18 phage-peptides that have been sequenced. This common motif was found to be within the human epsilon chain sequence Ser341-Thr355 near the N-terminus of the C epsilon3 domain. According to the human Fc epsilon model, the most accessible residues in this sequence are Arg342, Ile350, Arg351, Lys352 and Ser353. CONCLUSIONS: The present data should provide the molecular basis for the rational design of a suitable peptide immunogen (vaccine) for boosting the production of protective autoanti-IgE antibodies.     

从噬菌体短肽库中筛选单克隆抗体识别的抗原表位

将随机合成的寡聚核苷酸重组到噬菌体表面单价表达载体，构建了噬菌体随机八肽库。以抗原识别性明确的单克隆抗体９Ｅ１０固相化，对噬菌体短肽库进行了４～５轮“吸附－洗脱－扩增”的筛选，任选多个所获克隆进行ＥＬＩＳＡ检测。发现第４轮后６／２２个克隆可与９Ｅ１０结合，第５轮后１０／１０可与９Ｅ１０结合，此结合反应可被９Ｅ１０所针对的十肽抗原以及游离的９Ｅ１０特异性抑制。对所获阳性克隆进行ＤＮＡ序列测定发现，它们的序列可分为两种，其中一种序列与ｃ－ｍｙｃ十肽具有同源序列ＩＳＥｘｘＬ，而另一种的序列则完全不同。证实噬菌体表面单价表达体系用于构建噬菌体短肽库的有效性，为进一步筛选其它的具有高亲和力的特异性短肽打下了基础。     

Selection of an affinity-matured antibody against a defined epitope by phage display of an immune antibody library

In a previous study, we generated a murine hepatitis B virus (HBV)-neutralizing monoclonal antibody (mAb), KR127, that binds to an epitope (amino acids 37-45, NSNNPDWDF) of the preS1 antigen. Furthermore, an epitope tag, S1 (NANNPDWDF), was developed for protein tagging. The aim of the present study was to develop a high-affinity antibody to the same preS1 epitope. Mice were immunized with the N-terminal domain of human thrombopoietin fused to the S1 tag (nTPO-S1), and a phage-displayed chimeric Fab library was constructed and screened by panning against nTPO-S1. A high-affinity antibody (3-34) was selected that binds to the preS1 antigen. The IgG molecules of 3-34 showed approximately nine-fold higher affinity (K(D) 1.2 nM) for preS1 compared with KR127 (K(D) 10.4 nM), competed with KR127 for binding to the epitope, and bound to HBV particles. This study provides a simple and efficient way to develop a high-affinity antibody to a defined epitope by phage display of an immune antibody library.     

Identification of an epitope recognized by the monoclonal antibody PEP80 in the C-terminal cytoplasmic fragment of glycophorin A.

The monoclonal antibody PEP80 (IgG1) was raised by immunization of BALB/c mice with asialo-agalacto-glycophorin from human erythrocytes. The antibody is specific for glycophorin A (GPA) and reacts strongly with the GPA-derived tryptic peptide which is the C-terminal cytoplasmic portion of GPA, containing amino acid residues 102-131. Using the smaller chymotryptic fragments of this peptide and a set of solid phase-synthesized peptides allowed to establish that the MAb PEP80 is directed against an epitope comprising amino acid residues 112-121 of GPA. The peptides terminated with 120th or 119th amino acid residue were slightly less active, and the minimal structure which still gave a weak reaction with the antibody was the sequence of amino acid residues 112-118. The MAb PEP80 did not bind to live human erythroleukemic K562 cells, but showed a strong binding to the cells permeabilized with methanol.     

Identification of a novel linear epitope on EspA from enterohemorrhagic E. coli using a neutralizing and protective monoclonal antibody

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans and animals via the production of Shiga toxins, and injection of effectors into epithelia using type III secretion system (TTSS). E. coli secreted protein A (EspA) forms the filamentous conduits of TTSS, which extends into the translocation pore embedded in host cell membranes and aids in the transportation of bacterial effectors. In addition, EspA is closely associated with initial bacterial adhesion and the formation of biofilms. EspA in its various forms elicits protective immune responses, although the epitope responsible has not to be identified. Here we report the presence of a linear, immunogenic, conserved and partially protective epitope E07 (100Lys-120Val) on EspA, which is recognized by the novel monoclonal antibody 1H10. This antibody blocks EHEC-induced actin polymerization and confers protection in mice. These findings provide a better understanding of EspA-induced immune responses and could lead to epitope-based vaccines and antibody-based therapies.     

Identification of the conserved,conformation-dependent cytokeratin epitope recognized by monoclonal antibody (lu-5)

Summary The epitope recognized by the murine monoclonal antibody (mAB lu-5) recently described as a formaldehyde-resistant, pan-epithelial marker of great value in tumour diagnosis is located on the surface of cytokeratin filaments. It has been preserved during vertebrate evolution from amphibia to man. As this epitope is not reactive after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the epitope-bearing protein has been identified by a dot-blot antibody binding assay, using purified proteins in which the epitope is reconstituted. We show that the epitope is present in most cytokeratin polypeptides of both the acidic (type I) and basic (type II) subfamily but does not occur in other cytoskeletal proteins. The location of this widespread epitope is discussed with respect to homologies of amino acid sequences of cytokeratins and their conformations.     

Identification of Moraxella bovis by using a monoclonal antibody to a lipopolysaccharide epitope.

A monoclonal antibody to the lipopolysaccharide of Moraxella bovis is described. In an indirect fluorescent-antibody test, this monoclonal antibody reacted with 39 of 39 strains of M. bovis tested and did not react with 26 nonfermenting gram-negative coccobacilli other than M. bovis. When used in an indirect fluorescent-antibody test, it proved useful for rapid and easy identification of M. bovis.     

Identification of a target antigen recognized by a mouse monoclonal antibody to the bile canalicular surface of rat hepatocytes with a random phage display library

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.     

Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing.     

Selection for mutants improving expression of an anti-MAP kinase monoclonal antibody by filamentous phage display

It has recently been reported that single amino acid residues can strongly influence the expression of recombinant antibody fragments in Escherichia coli. Prediction of these critical positions can be difficult even with prior knowledge of the primary sequence and the three-dimensional folded structure of the antibody. To circumvent this, a Fab phage display library containing random point mutations was generated from a hybridoma specific for activated p44/p42 mitogen-activated protein (MAP) kinases. Clones that express Fab were selected by panning against the target antigen. It was found that a cysteine-to-serine substitution at position 91 in the CDR3 of the light chain was responsible for allowing expression of Fab. Site-directed mutagenesis was performed to effect this substitution and others at cysteine 91 on a nonexpressing clone. Mutants containing serine, glycine or alanine at position 91 expressed Fab and bound to target antigen. In contrast, tyrosine mutants had moderate Fab expression but no detectable binding to antigen. These results demonstrate that by using phage display, one can select for the expression of antibody fragments while retaining biological activity.     

Identification and characterization of tumor antigens by using antibody phage display and intrabody strategies

To generate a panel of antibodies binding human breast cancers, a human single chain Fv phage display library was selected for rapid internalization into the SK-BR-3 breast cancer cell line. Thirteen unique antibodies were identified within the 55 cell binding antibodies studied, all of them showing specific staining of tumor cells compare to normal epithelial cells. Two of the antibodies bound the ErbB2 oncogene while 6 bound the tumor marker transferrin receptor (TfR). By developing a scFv immunoprecipitation method, we were able to use LC-MS/MS to identify the antigen bound by one of the antibodies (3GA5) as FPRP (prostaglandin F2alpha receptor-regulatory protein)/EWI-F/CD9P-1 (CD9 partner 1) an Ig superfamily member that has been described to interact directly with CD9 and CD81 tetraspanins and to be overexpressed in adherent cancer cell lines. Although the 3GA5 scFv had no direct anti-proliferative effect, intracellular expression of the scFv was able to knockdown CD9P-1 expression and could be used to further define the role of the tetraspanin system in proliferation and metastasis. Moreover, the 3GA5 scFv was rapidly internalized into breast tumor cells and could have potential for the targeted delivery of cytotoxic agents to breast cancers. This study is the proof of principle that the direct selection of phage antibody libraries on tumor cells can effectively lead to the identification and functional characterization of relevant tumor markers.     

Production and characterization of monoclonal antibodies to human casein. A monoclonal antibody that cross-reacts with casein and alpha-lactalbumin

Four hybridomas have been produced that secrete monoclonal antibodies to human beta-casein, a protein synthesized by fully differentiated breast epithelial cells. The antibodies have been characterized on immunoblots and have been shown to react with methanol: acetone-fixed sections of human lactating mammary gland. Immunoblots show that three of these antibodies react with casein whereas one, F20.10, also recognizes an epitope present on human alpha-lactalbumin. Computer analysis of the amino acid sequences of these two milk proteins reveals very little sequence homology, leading to the conclusion that the three-dimensional shape, rather than the primary sequence, is important in this cross-reactivity.     

Methods for the generation of chicken monoclonal antibody fragments by phage display

Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be less immunogenic in mammalian animals commonly used for immunization. In order to optimize methods for constructing chicken immunoglobulin phage display libraries in the pComb3 system, we have immunized chickens with the hapten fluorescein, and generated combinatorial antibody libraries from spleen and bone marrow RNA. Herein we present methods for the isolation of scFv, diabody and Fab fragment libraries from chickens. Chicken Fab fragment libraries are constructed using human constant regions, facilitating detection with readily available reagents as well as humanization. Analysis of the selected V-genes revealed that gene conversion events were more extensive in light-chain variable region genes as compared to heavy-chain variable region genes. In addition, we present a new variant of the pComb3 phage display vector system.     

Definition of the epitope recognized by the Plasmodium falciparum-reactive human monoclonal antibody 33G2.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2-19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1-19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1-8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3-7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.     

Identification of Escherichia coli serotype O157 strains by using a monoclonal antibody.

The O157 antigenic determinant of Escherichia coli serotype O157:H7, an important bacterial pathogen, resides in the polysaccharide portion of its cellular lipopolysaccharide component which, from structural studies, was identified as a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, L-fucose, 2-acetamido-2-deoxy-D-galactose, and 4-acetamido-4,6-dideoxy-D-mannose residues (1:1:1:1). Hybrid cells producing monoclonal antibodies against the E. coli O157 antigen were obtained by fusion of myeloma cells with lymphocytes from BALB/c mice immunized with killed E. coli O157:117 cells. Clones were selected for binding specificity with purified O polysaccharide. One monoclonal antibody used in direct slide agglutinations or in coagglutination reactions with Staphylococcus aureus Cowan 1 cells sensitized with the affinity column-purified antibody accurately detected all strains of E. coli O157 tested. This selected monoclonal antibody did not agglutinate E. coli strains such as E. coli O7 and E. coli O116 or other bacteria which are known to give positive agglutinations with conventional polyclonal E. coli antisera. These results indicate that the monoclonal antibody is a superior specific-typing reagent.     

Characterization of a monoclonal anti-capsid antibody that cross-reacts with three major primate lentivirus lineages

Mouse monoclonal antibodies with varying specificities against the Gag capsid of simian and human immunodeficiency virus (SIV/HIV) were generated by immunizing mice with whole inactivated SIVagmTYO-1. Monoclonal antibody AG3.0 showed the broadest reactivity recognizing the Gag capsid protein (p24-27) and Gag precursors p38, p55, and p150 of HIV-1, HIV-2, SIVmac, and SIVagm. Using overlapping peptides, the AG3.0 epitope was mapped in capsid to a sequence (SPRTLNA) conserved among HIV-1, HIV-2, SIVrcm, SIVsm/mac, and SIVagm related viruses. Because of its broad cross-reactivity, AG3.0 was used to develop an antigen capture assay with a lower detection limit of 100 pg/ml HIV-1 Gag p24. Interestingly, AG3.0 was found to have a faster binding on/off rate for SIVagmVer and SIVmac Gag than for SIVagmSab Gag, possibly due to differences outside the SPRTLNA motif. In addition, the ribonucleic acid (RNA) coding for AG3.0 was sequenced to facilitate the development of humanized monoclonal antibodies.     

Recovery of an epitope recognized by a novel monoclonal antibody from airway lavage during experimental induction of chronic bronchitis.

Prolonged exposure of dogs to high concentrations of SO2 gas results in a syndrome with many of the characteristics of human chronic bronchitis, including cough and chronic mucous hypersecretion as well as airway obstruction. We developed and used a novel monoclonal antibody, GB-4B, raised against epithelial glycoprotein isolated from human hypersecretory mucus to probe airway lavage samples from dogs before and during prolonged exposure to SO2 gas. There were relatively low mean titers of the epitope recognized by GB-4B in airway lavage fluid as evidenced by enzyme-linked immunosorbent assay before exposure to SO2 gas. After 25 to 50 wk of SO2 exposure, the dogs showed a significant increase in pulmonary resistance and there was a significant increase in the titer of the epitope in the airway lavage fluid. Using the same antibody immunohistochemical analysis of airway tissues from SO2-exposed dogs revealed patchy staining of the mucous glands and airway secretory cells and dense staining along the airway surface; airway tissue from control dogs and one SO2-exposed dog whose lavage fluid did not contain the epitope showed little or no staining. These data demonstrate that similar mucin epitopes appear in airway lavage fluid under hypersecretory conditions in both animals and humans. The epitope may have utility as a marker of chronic mucous hypersecretion.     

Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody.

A monoclonal antibody (MAb) immunoglobulin G2a (2125) was produced against a 60-kDa Legionella heat shock protein (HSP), recognizing a unique epitope common to all species of the genus Legionella. The antibody reacted in the immunoblot with 59 Legionella species and serogroups that were tested and showed no cross-reactivity with other bacteria, including Acinetobacter spp., Bordetella spp., Pseudomonas spp., Mycobacterium spp., and Escherichia coli. Two other MAbs (2122 and 2130) reacted with the 60-kDa Legionella protein as well but showed different cross-reactivities with other gram-negative bacteria in the same molecular mass range. The genus-specific MAb 2125 as well as the cross-reacting MAbs 2122 and 2130 were shown to be reactive with the expressed protein of the cloned gene of the 60-kDa HSP of Legionella micdadei and Legionella pneumophila. These antibodies demonstrate that Legionella-specific and nonspecific epitopes are present on this protein. A sandwich enzyme-linked immunosorbent assay (ELISA) in which the genus-specific MAb is used both as a capture antibody and as a biotinylated second antibody has been established. With this test it is possible to detect Legionella whole cells, sonicated cells, and cell fractions containing the 60-kDa HSP. The main part of the 60-kDa HSP is found in the cytoplasmic fraction. The sandwich ELISA can be used to demonstrate the increased expression of the 60-kDa protein in Legionella cells following heat shock as well as marked differences in the detection of the 60-kDa HSP on whole cells of different Legionella strains. The high specificity and sensitivity of the sandwich ELISA for sonicated cells might be very useful to screen on a genus level for Legionella cells or the 60-kDa antigen in environmental isolates or body fluids of patients.