Thèses sur le sujet « CSF single chain library »

2007/2008
Multiple sclerosis (MS) is considered the prototype of inflammatory autoimmune diseases of the central nervous system (CNS). The typical feature of the disease is the plaques of demyelination. The evolution of the plaque lesion in MS implicates an inflammatory phase followed by a recovery of functional myelin; a second step is the chronic progressive disease with axonal loss. The earlier phase of MS may be mediated by an autoimmune reaction. Whereas the role of T cells in MS pathogenesis is well established, the role of B cells and autoantibodies in demyelination and plaque formation is still unresolved. However several evidences suggest a contribute of autoantibodies in MS pathogenesis. B cells and myelin specific autoantibodies are present in the sclerosis plaques, and there is an increased production of immunoglobulin (Ig) in the cerebrospinal fluid (CSF) of more than 90% of MS patients . Typically these Ig present an oligoclonal pattern and sequencing of oligoclonal IgG showed extensive somatic mutations suggesting B cell clonal expansion and a specific antigen-driven immune response. The most extensively studied putative autoantigens are components of CNS myelin (myelin basic protein MBP, proteolipid protein PLP, myelin oligodendrocyte glycoprotein MOG). The autoantibodies in MS recognize both linear and conformational epitopes, but at present the conformational epitopes of myelin proteins have not been identified. For example, in MS, the T-cell receptors of autoreactive T lymphocytes recognize various peptides of the MBP, and, in EAE, the anti-MOG antibodies recognize only conformational epitopes. Furthermore, the progression of MS is accompanied by the decline of primary T-cell autoreactivity and by the concurrent emergence of neo-autoreactivity (epitope spreading). However recent investigation have showed that no myelin antigens, like neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, may also have a role in MS pathogenesis. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Several strategies, involving the phage display technology, have been employed in the attempt to discover the antigen that drives the immune response in MS. A first strategy depends on the cloning of IgG repertoire of MS patients in a phage display library screened with brain sections or known antigens. Another strategy involves large phage display libraries of random peptides screened with IgG of CSF in order to identify peptides recognized by antibodies present in CSF of MS patients. Phage display is a technique which involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions and to select antibodies against a wide range of different antigens. In this project we have proposed: 1. to study the autoimmune response in MS by using the phage display for the expression of antibodies involved in the disease. We wanted to make a ScFv library from B cells of CSF of different multiple sclerosis patients, to employ as tool to select a phage display Human Brain cDNA library for the identification of new antigens recognized by the immune sistem in MS patients. 2. To produce single gene mini library of putative antigens (MBP,PLP, MOG) for the generation of epitope chips to use for serotyping the immune response in different patients 3. To investigate the feasibility to use a single gene phage display mini-library as tool for epitope mapping (both linear and conformational) of novels autoantigens 4. To investigate the role of NSE(neuron specific enolase), a new possible no myelin autoantigen in multiple sclerosis, in the pathogenesis of the disease and the usefulness as possible diagnostic marker. Results: Scope 1 B-cells from liquor of two MS patients were centrifuged and the total RNA was extracted from the pellets. Total RNA was retrotranscripted and variable region of heavy and light chain of the antibodies were amplified by PCR. Heavy chain and light chain were assorted and assembled before to be cloned in the phagemid vector pDAN 5. A 2x104 independent clones library was obtained and analyzed by PCR and fingerprinting. A diversity of 30,8% for heavy chain and 72,7% for light chain was established. ScFv library was used to select a phage display Human Brain cDNA library. 17 clones with an high reactivity were obtained and after sequencing 6 clones on 17 have shown to be the same antigen(antigen A ); the reactivity on other two antigens obtained with the selection (antigen B and C) of CSF from 18 MS patients and 16 patients with other neurological disease (OND) was tested by ELISA to evaluate diagnostic value of this protein. The results shown that SM response was statistically different from OND response; the ELISA test gave a specificity of 94,12% and a significance of 53,85 %. The reactivity for the antigen B was also evaluated on sera of MS patients and controls. The MS response was statistically different from OND response and shown a specificity of 97,44% and a significance of 58,62 %. Scope 2&3 We have generated three single gene mini libraries of the major antigens in MS (MBP, MOG and PLP); cDNA of each gene was obtained by RT-PCR and after fragmentation cloned in a phagemid vector (pEP1) to obtain a mini-library for each gene. We have obtained a 2x105 for MBP, 2.4x104 for MOG and 1.6x106 for PLP independent clones library. MBP and MOG libraries were characterized by PCR and fingerprinting. Sequencing analysis shown that the entire MBP transcript variant 7 mRNA (664-1177 nt) and MOG isoform alpha 1 mRNA (262-918 nt) were represented in the respective library. To testing the capacity of selecting a single epitope from our libraries, we have performed a selection test with a commercial monoclonal antibody that recognize MBP 82-98 epitope; after three selection panning all selected clones contain the nucleotidic sequence 906- 956 nt (MBP transcript variant 7 mRNA) which encodes the immunogenic epitope recognized by the monoclonal antibody. Scope 4 The reactivity of sera from 31 MS patients and 14 healthy controls was tested by ELISA on NSE ; statistical analysis of the results shown that the two populations were significantly different.
XXI Ciclo
1981

Studies suggest that natural autoantibodies may be part of an immunological network which maintains the normal homeostatic response seen in controls. Any defect in this network leading to autoimmunity may be represented in the anti-retinal antibody response observed in patients. Characterisation of the humoral autoimmune response occurring during active uveitis may provide valuable information on the immune mechanisms, both humoral and cellular, involved in uveitis. Serum titres and ELISA based tests can only partially describe an antibody response, a more complete description requires access to the B-cell repertoire constituting the response. In the past hybridoma technology has generated a wealth of vital information on antibody responses in animals, but with limited success when applied to humans, producing unstable cell lines with poor antigen affinity. Using scFv phage display antibody technology we attempted to isolate the immune response occurring during active uveitis using a phage display library derived from peripheral blood lymphocyte mRNA of a patient with active uveitis. In this study, we report the isolation and characterisation of human autoimmune recombinant scFv's from two libraries, a uveitis patient derived library and a healthy non uveitis donor derived library. Anti-IRBP and S-Antigen autoantibodies were successfully selected from both libraries. Sequence analysis of these selected autoantibodies revealed possible differential epitope targeting of disease associated anti-S-Ag autoantibodies, and exclusive use of the VH segment, DP49 was revealed among selected anti-S-Ag scFv's. In addition ELISA studies using the selected scFv's, and both patient and control serum, indicated that it may be possible to distinguish the 'natural' and disease associated anti-S-Ag responses at the idiotype/anti-idiotype network level.

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.

This thesis describes the construction and characterisation of an insulin-like growth factor (IGF-I)-binding single chain Fv (scFv) and the utilisation of this scFv as a model protein for the study of the application of DNA shuffling and ribosome display to antibody engineering. The variable domain genes were isolated from the hybridoma cell line producing the monoclonal antibody and successfully joined by PCR for the construction of the scFv, named anti-GPE. Sequencing of the gene revealed an unusually short heavy chain CDR2 region. The cloned scFv was expressed in E. coli and purified. Expression levels were low and the protein has poor solubility, most likely due to a reduction in folding efficiency caused by the abbreviated CDR2. The purified monomeric form of the protein was analysed for binding to IGF-I using surface plasmon resonance on the BIAcore 1000 with the specificity of the IgG version of the antibody for the three N-terminal residues of IGF-I - Gly-Pro-Glu - reproduced. The scFv's calculated dissociation constant of 3.68 µM is a low affinity for an antibody and is approximately 36-fold weaker than was calculated for the Fab version of the antibody, but it is concluded that the calculated affinity for the scFv was an apparent affinity that may be an underestimation of true affinity due to the presence of non-functional or misfolded scFv species within the gel-filtration purified monomer peaks. A mutant version of anti-GPE with residues inserted in the CDR2 to restore it to normal length produced a protein with improved expression and solubility characteristics while retaining IGF-I-binding. It was concluded that the short CDR2 was due to deletions generated during the somatic mutation process and a model for this is described. A ribosome display method using a rabbit reticulocyte lysate as a source of ribosomes was developed for specific selection of anti-GPE against IGF-I. Error prone PCR was used to produce a random point mutated library of anti-GPE (EPGPE). This was taken through several cycles of display and selection but selection for non-specifically binding scFvs was commonly observed. This was probably due to poor folding of ribosome-displayed proteins in the system used, possibly caused by the presence of DTT in the lysate and/or the low capacity of the anti-GPE framework to tolerate mutation while retaining stability. It is assumed misfolds, exposing hydrophobic regions, would have a tendency to non-specifically interact with the selection surface. Of the 64 EPGPE clones screened from four rounds of display and selection, many were shown to have poor or non-specific binding, but one scFv was characterised that was affinity matured 2.6-fold over anti-GPE wild type affinity for IGF-I. A DNA shuffling method was developed to produce libraries of chimaeric scFvs between anti-GPE and NC10 (anti-neuraminidase scFv) with the objective of isolating functional IGF-I-binding chimaeras. The NC10 scFv had its CDRs replaced with the anti-GPE CDRs prior to the shuffling to increase the likelihood of isolating IGF-I binders. Ribosome display was used for selection from the chimaera libraries. Selection strategies included elution of specific binders by GPE peptide and a GPE 10-mer peptide. Selection was also performed using IGF-I immobilised on a BIAcore sensorchip as a selection surface. Again, much non-specific selection was observed as seen for display of EPGPE, for what was expected to be the same reasons. Selected scFvs were genuinely chimaeric but with poor expression and solubility and mostly non-specific in their binding. One characterised selected chimaera, made up of three segments of each of the parental scFvs, was shown to bind specifically to IGF-I by BIAcore. Steps to improve the efficiency of the ribosome display system have been identified and are discussed.

Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.

Les anticorps catalytiques (ou abzyme) ont fait l’objet de nombreuses recherches et ont été produits pour réaliser de nombreuses réactions. Ces protéines ont été ensuite découvertes dans le sérum d’individus sains ou atteints de pathologies, dont les pathologies autoimmunes. Les études suggèrent que ces abzymes peuvent avoir des effets bénéfiques ou délétères sur la santé des individus. L’origine des anticorps catalytiques et leur rôle restent ambigus et doivent être approfondis. Nous avons développé une nouvelle stratégie visant à étudier les abzymes, basée sur la technologie du phage display. Nous avons construit 4 banques de fragments d’anticorps, chacune présentant un répertoire immun différent (fond génétique et état d’immunitaire) : saine et naïve, saine et immunisée, autoimmune et naïve, et autoimmune et immunisée. Les stratégies d’amplification et de clonage des régions variables des immunoglobulines ont été conçues afin d’optimiser la taille et la diversité des banques. Nous avons rassemblé les 4 banques en une banque unique élargie contenant 2.7×109 séquences. L’analyse des séquences a mis en évidence des différences dans les profils d’expression des sous-groupes de gènes selon la banque. Nous avons ensuite procédé à la sélection d’abzymes à activité β-lactamase en utilisant deux cibles : un peptide cyclique, et un dérivé de sulfone pénam, inhibiteurs de l’enzyme. Nous avons sélectionné 5 abzymes. Chacun de ces immunoglobulines ont des séquences protéiques propres, incluant un potentiel site actif. Ces résultats montrent que différents motifs peuvent assurer la fonction catalytique de la β-lactamase, confirmant la flexibilité moléculaire de cette enzyme
Catalytic antibodies (or abzymes) have been the focus numerous studies for some decades and have been produced with the ability to catalyze a wide range of reactions. They have also been discovered naturally in normal physiological and pathological conditions, notably on the background of autoimmune disease. Some have beneficial effects and others are detrimental to individual’s health. Hence, the origin of abzymes and their role in the immune response are ambiguous and must be enhanced. We have developed a novel strategy for the study of abzymes based on the phage display technology. We have constructed 4 libraries representing 4 murine immune repertoires with different genetic backgrounds and immunological states : healthy and naïve, healthy and immunized, autoimmune and naïve , and autoimmune and immunized. The strategies for the amplification and cloning of the immunoglobulin (lg) variable regions have been designed to optimize the size and diversity of the libraries. We have been able to pool the four libraries to create a large repertoire of size 2.7x109. After sequence analysis, we have found a number of statistically significant differences between the libraries. We have then used two strategically chosen targets to select for antibodies endowed with β lactamase activity : a cycle peptide and a penam sulfone, both inhibitors of the enzyme. We have selected for a total of 5 lgs endowed with β lactamase activity. The selected abzymes have different amino acid sequences. 3D modeling has provides insights on potential active sites demonstrating the ability of different structures to maintain the β lactamase activity and confirming the flexibility of the active site

Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry.
Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

碩士
國立交通大學
應用化學系碩博士班
102
The study is aimed to screen a single chain antibody fragment (scFv) from phage display library for cAMP recognition. The cAMP derivative (containing alkyl amine moiety) was conjugated on BSA and employed for scFv screening. After 6 rounds of panning, 3 clones (designated as 1D, 2E and 5H) with high titers were obtained. Since 2E exhibited higher titer than the other two clones, 2E clone, virtually composed of only the light chain variable region (VL), was mainly used in this study. The corresponding protein, named as cAMP-VL, was overexpressed and purified. The molecular weight was analyzed (14052.8 Da) by electrospray ionization mass spectrometry and confirmed to be consistent with the theoretical value (14054.3 Da). The binding features of cAMP-VL as free protein or on the surface of phage were further analyzed by ELISA or quartz crystal microbalance (QCM). The experiments were conducted using cAMP as probe for interacting with 2E phage and cAMP-VL. The detection limits of 2E phage and cAMP-VL are 200 phage particle/mL and 0.66 µg/mL, repectively. In the case of QCM analysis, cAMP derivative was immobilized on QCM chip, the dissociation constant (Kd) was estimated to be 74,900 phage particle/mL. In order to investigate the binding specificity of cAMP-VL, several nucleotides (adenosine,ATP,ADP and AMP) and nucleosides were used for competitive binding assessment. Results showed that cAMP-VL possesses high specificity to purine moiety. Further study on structure simulation attempted to sketch the binding domain of cAMP. P59, R61, and E81 were found to be the possible candidates for binding. P59R, R61K and E81Q mutants were constructed and purified for binding study. The outcome showed all three mutants moderately damaged the interaction with cAMP indicating these residues are somewhat important for cAMP binding. More studies will be proposed to improve the binding affinity of cAMP-VL toward cAMP based on the structure simulation or the complex of 3D protein structure.

碩士
國立嘉義大學
農業生物技術研究所碩士班
93
Odontoglossum ringspot virus (ORSV) is one of the commonest viral pathogens of the cultivated orchids. Infected plants appear unhealthy and may produce low quality flowers. In this experiment a set of ORSV-specific oligonucleotide primers were designed from the region of the coat protein (CP) gene of ORSV. The ORSV CP gene was cloned into the protein expression bacterial plasmid vector of the gutathione S-transferase（GST）fusion protein expression system. The recombinant ORSV CP was injected into mouse to induce immune response of the animal. The cDNAs of VH and VL of ORSV antibody genes were obtained by using reverse transcription polymerase chain reaction from the total the RNAs that were extracted from the spleen cells of immunized mouse. ScFv（single-chain variable fragment）library of ORSV were constructed with gene splicing by overlap extension. Thirty seven scFvs were selected from ORSV-scFv library following three rounds of affinity selection with ORSV CP as an antigen that was expressed in bacteria. Four scFv antibody have specific binding reaction against ORSV CP was selected. Comparing the sensitivity between scFv antibodies and ORSV polyclonal antibody tisted in enzyme linked immunosorbent assay (ELISA) to detect ORSV in leaf extracts of diseased Phalaenopsis plants. Unfortunately, the affinity between scFv antibody and ORSV was weaker than that between polyclonal antibody to the same antigen. The scFv antibodies and polyclonal antibody appeared to have similar sensitivity limit as low as 10 ng/μl. The results highlight the potential of applying the scFv antibodies in the diagnosis of virus diseases.

碩士
國立嘉義大學
農業生物技術研究所碩士班
93
The cultivated orchids commonly infected Cymbidium mosaic virus (CyMV) that causes the symptoms of leaf necrosis and flower malformation, the prevalent disease may results in enormous loss of orchid growers. For preventing the disease from spreading, the growers should immediately remove the infested plants from greenhouse. For finding the infested plants ELISA is commonly applied to detect the virus due to the immunodetection is simple and economical. In the experiment, the scFv (single chain fragment variable) antibodies against CyMV were developed with recombinant phage antibody system. The primer pair for cloning CyMV coat protein gene was designed based on the gene sequence, the cloned coat protein (cp) gene 672 bp of CyMV was ligated into the expression vector pGEX of the (glutathione S-transferase) expression system, then CyMV cp was purified from the fusion protein of GST-CyMV cp and it was used as antigen to immunize mouse. The total RNAs was extracted from spleen of the immuned mouse and was reversed transcribed into cDNAs, and the VH and VL were cloned from the cDNAs and were assembled with linker into scFv fragments by fill-in reaction. These scFv fragments were ligated into the phagemid vector pCANTAB 5E that transfected competent E. coli cells, so that the phage-displayed antibodies were bio-panned on immobilized antigen from the phage display scFv antibody library. These recombinant phagemids 5-E12, 1-A4, 1-H1, 1-E11, 2-D5, 4-A3 were screened out based on these vectors expressing the scFv antibodies that showed strong affinity with antigen. Both 5-E12 and 2-D5 clones expressed their scFv antibodies that appeared high affinity with CyMV. The sensitivity of immunodetection of scFv antibody 2-D5 was better 5-E12 due to the immunoassay of 100 ng 2-D5 and 5-E12 scFv antibodies bound to 108 particles of CyMV per well were OD405 0.27 and 0.19, respectively. Comparison of the amino sequences both scFv antibody 2-D5 and 5-E12 that appeared very similar, and there are only 3 amino acids different of 250 amino acids between 2-D5 and 5-E12.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.
Dissertation

碩士
慈濟大學
微免暨分子醫學研究所
94
A large naïve human single-chain Fv (scFv) phage library was used to search for human monoclonal antibodies against tumor-associated antigens by panning with the purified recombinant proteins expressed in E. coli. The cell surface adhesion molecules MUC18, which is overexpressed in several types of human tumors and an Epstein-Barr virus (EBV) nuclear antigen ENBA-1, which is expressed in all EBV-associated tumors, were selected for this study. After five to six rounds of selection-amplification-rescue, 74 of 82 monoclones of MUC18 (90 %) and 14 of 30 (47 %) of EBNA-1 analyzed bound selectively to MUC18 and EBNA-1 in a phage ELISA. The criteria used to assess the specificity of each monoclone was robust binding to the antigen with ELISA reading at A405 > 0.5. In Western blot analyses, the MUC18 scFv fragment antibodies recognized the MUC18 molecules expressed in a variety of tumor cells including HeLa, NPC, melanoma, and breast cancer cell lines. Similarly, EBNA-1 scFv fragment antibodies detected the EBNA-1 molecules only in EBV-positive cells, but the intensity of bands was very weak. The reason for weak detection could be attributed to the low titers of the scFv antibodies, which were obtained after five rounds of selection, in contrast to MUC18 that had gone through six rounds of selection. Diversity analyses of these antigen-selective individual clones by BstNI fingerprinting and nucleotide sequencing revealed a single distinct scFv fragment of MUC18 clones and 7 distinct scFv fragments of EBNA-1 clones. Protein sequences were deduced from the DNA sequences and fragment regions (FR) and complementarity determining regions (CDR) were also identified. The deduced amino acid sequences from the nucleotide sequences of the MUC18 clones analyzed also revealed a single pattern of both light and heavy chains of V-gene. It appears that most of the MUC18 scFv clones isolated use identical or very closely related V-gene sequences. This may reflect that the isolated clones were highly specific and all recognized a single epitope. Despite the variations in BstNI fingerprintings, nucleotide sequences, and deduced amino acid sequences of 11 EBNA-1 binding clones, only the light chain of the V-gene was detected. These results indicated that EBNA-1 binding clones use only VL sequences. The isolation of soluble scFv fragment phages specific for MUC18 was attempted. Even though we could detect by ELISA the soluble forms of scFv antibodies for MUC18, we were unable to detect the MUC18 molecules by Western blot using the soluble scFv antibodies. These results provide a lead for further development of diagnostic assays and selective tumor targeting immunotherapy. Thus, the scFv fragment phage antibodies selected from the phage display library can be used to deliver highly cytotoxic moieties such as radionuclides, toxin, or chemotherapeutic agents to the tumors expressing MUC18 and EBNA-1.