共查询到20条相似文献,搜索用时 46 毫秒
1.
Lei Shi John C. Wheeler Raymond W. Sweet Jinquan Luo Brian Whitaker Raymond Brittingham Qiang Chen David M. Knight Ping Tsui 《Journal of molecular biology》2010,397(2):385-278
Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four VL and three VH domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on VH by keeping VL in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. VH diversity was sufficient for isolating Fabs to most antigens, whereas variability in VL was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling VL diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies. 相似文献
2.
Cristina de Carvalho Nicacio R. Anthony Williamson Paul W. H. I. Parren ke Lundkvist Dennis R. Burton Ewa Bjrling 《Journal of virology》2002,76(1):251-258
Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an approximately equal 80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an approximately equal 60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between approximately 2 and 7 microg x ml(-1). Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries. 相似文献
3.
Bernard A. Liu Andrea Uetrecht Jun Gu Nick Jarvik Vincent Nadeem Tony Pawson Jason Moffat Shane Miersch Sachdev S. Sidhu 《Protein science : a publication of the Protein Society》2015,24(11):1890-1900
Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high‐throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage‐displayed synthetic antigen‐binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high‐throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full‐length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high‐throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome‐wide scale. 相似文献
4.
Hutchins BM Kazane SA Staflin K Forsyth JS Felding-Habermann B Schultz PG Smider VV 《Journal of molecular biology》2011,406(4):595-303
Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered. 相似文献
5.
Hillig RC Urlinger S Fanghänel J Brocks B Haenel C Stark Y Sülzle D Svergun DI Baesler S Malawski G Moosmayer D Menrad A Schirner M Licha K 《Journal of molecular biology》2008,377(1):206-219
Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody. 相似文献
6.
Kempf E Weiss E Klein P Glacet A Spratt S Bourel D Orfanoudakis G 《Molecular biotechnology》2001,17(2):97-108
Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the
restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility,
for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation
of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their
display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection
by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by
phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation
by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones,
and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding
sequences expressed in Escherichia coli, amplified by a set of specific 5′ primers for different antibody germ lines, was similar to that observed with the original
untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein,
which proves the efficiency of this technique. 相似文献
7.
Kumar S Kalsi J Ravirajan CT Rahman A Athwal D Latchman DS Isenberg DA Pearl LH 《The Journal of biological chemistry》2000,275(45):35129-35136
The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody. 相似文献
8.
Jean Chatellier Nathalie Rauffer-Bruyre Marc H. V. Van Regenmortel Danile Altschuh Etienne Weiss 《Journal of molecular recognition : JMR》1996,9(1):39-51
Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134–146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli. They differ by 15 amino acid changes in the sequence of their variable region. The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcoreTM biosensor instrument. The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P. The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P. The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs. The location of amino acid changes was visualized on a model structure of the Fab. Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains. Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes. 相似文献
9.
Philippa M O'Brien Gavin Maxwell M Saveria Campo 《Protein expression and purification》2002,24(1):43-50
We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli. 相似文献
10.
Asanuma H Matsumoto-Takasaki A Suzuki Y Tamura S Sata T Kusada Y Matsushita M Fujita-Yamaguchi Y 《Biochemical and biophysical research communications》2008,366(2):445-449
Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have KD of 1.5 × 10−8 and 3.2 × 10−9 M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients. 相似文献
11.
Schneider EL Lee MS Baharuddin A Goetz DH Farady CJ Ward M Wang CI Craik CS 《Journal of molecular biology》2012,415(4):699-715
The type II transmembrane serine protease family consists of 18 closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular type II transmembrane serine protease, matriptase [MT-SP1 (membrane-type serine protease 1)], a phage display library was created with a natural repertoire of Fabs [fragment antigen binding (Fab)] from human naïve B cells. Fab A11 was identified with a 720 pM inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with a yield of ~ 200 mg/L. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic single-chain variable fragment library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å, respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role that the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting that this approach could be useful for clinical applications. 相似文献
12.
Eeva-Christine Brockmann Markus Vehniäinen Kim Pettersson 《Analytical biochemistry》2010,396(2):242-359
High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future. 相似文献
13.
Ruklanthi de Alwis Katherine L. Williams Michael A. Schmid Chih-Yun Lai Bhumi Patel Scott A. Smith James E. Crowe Wei-Kung Wang Eva Harris Aravinda M. de Silva 《PLoS pathogens》2014,10(10)
Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue. 相似文献
14.
Michael Hornsby Marcin Paduch Shane Miersch Annika S??f Tet Matsuguchi Brian Lee Karolina Wypisniak Allison Doak Daniel King Svitlana Usatyuk Kimberly Perry Vince Lu William Thomas Judy Luke Jay Goodman Robert J. Hoey Darson Lai Carly Griffin Zhijian Li Franco J. Vizeacoumar Debbie Dong Elliot Campbell Stephen Anderson Nan Zhong Susanne Gr?slund Shohei Koide Jason Moffat Sachdev Sidhu Anthony Kossiakoff James Wells 《Molecular & cellular proteomics : MCP》2015,14(10):2833-2847
15.
《Gene》1997,187(1):9-18
Phage display is now an established method to select antibody fragments specific for a wide range of diverse antigens. In particular, isolation of human monoclonal antibodies has become a reality and for most purposes bacterial expression of the selected recombinant antibody fragments is sufficient. However, there are some cases where the expression of complete human immunoglobulin in mammalian cells is, if not essential, at least desirable. For this reason we have designed and constructed a set of mammalian expression vectors which permit facile and rapid cloning of antibody genes for both transient and stable expression in mammalian cells. Immunoglobulin genes may be cloned into these expression vectors as V regions or as Fabs for expression as either complete antibodies or as Fab fragments, using restriction sites which are rare in human V genes. All the important elements in the vectors – promoter, leader sequence, constant domains and selectable markers – are flanked by unique restriction sites, allowing simple substitution of elements. The vectors have been evaluated using the variable regions from the neutralizing anti-nerve growth factor (NGF) antibody, αD11, and the V regions from 2E10, a scFv selected from a scFv phagemid library. 相似文献
16.
Escarlata Rodríguez-Carmona Olivia Cano-Garrido Martin Dragosits Michael Maurer Alexander Mader Renate Kunert Diethard Mattanovich Antonio Villaverde Felícitas Vázquez 《Process Biochemistry》2012,47(3):446-452
Production of recombinant antibody fragments (Fabs) in Escherichia coli has gained interest because of the recognised advantages of this expression system and because Fabs do not require glycosylation. However, more comprehensive studies on the factors that influence expression conditions and product yield are still required for full process development. In this work, the effect of growth temperature on the periplasmatic expression of the 3H6 Fab in E. coli was studied in carbon-limited continuous cultures operated at medium cell densities. Three different temperatures were assayed, namely 37, 33 and 30 °C. Results showed that biomass yield was not affected within this temperature range whilst product yield increased as temperature decreased. Periplasmic Fab secretion corresponded to 30% of the produced Fab protein and its efficiency was irrespective of the process temperature. Moreover, considerable product leakage to the culture supernatant was detected in all cases, ranging from about 40% at 37 °C to almost 70% at 30 °C. Besides, plasmid loss was observed along process time indicating a selective pressure against plasmid-bearing cells. This study supports the potential of continuous cultivations of E. coli at medium cell densities under well controlled conditions as a tool for characterising the impact of environmental parameters and cell physiology under protein production conditions. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2302-2311
The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease. 相似文献
18.
Francisco Ylera Stefan HarthDirk Waldherr Christian FrischAchim Knappik 《Analytical biochemistry》2013,441(2):208-213
The rapidly increasing number of therapeutic antibodies in clinical development and on the market requires corresponding detection reagents for monitoring the concentration of these drugs in patient samples and as positive controls for measurement of anti-drug antibodies. Phage display of large recombinant antibody libraries has been shown to enable the rapid development of fully human anti-idiotypic antibodies binding specifically to antibody drugs, since the in vitro panning approach allows for incorporation of suitable blockers to drive selection toward the paratope of the drug. A typical bottleneck in antibody generation projects is ranking of the many candidates obtained after panning on the basis of antibody binding strength. Ideally, such method will work without prior labeling of antigens and with crude bacterial lysates. We developed an off-rate screening method of crude Escherichia coli lysates containing monovalent Fab fragments obtained after phage display of the HuCAL PLATINUM® antibody library. We used the antibody drugs trastuzumab and cetuximab as antigen examples. Using the Octet® RED384 label-free sensor instrument we show that antibody off rates can be reliably determined in crude bacterial lysates with high throughput. We also demonstrate that the method can be applied to screening for high-affinity antibodies typically obtained after affinity maturation. 相似文献
19.
Weaver-Feldhaus JM Lou J Coleman JR Siegel RW Marks JD Feldhaus MJ 《FEBS letters》2004,564(1-2):24-34
Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library. 相似文献
20.
Atsushi Fukunaga Shingo Maeta Bajaj Reema Makoto Nakakido Kouhei Tsumoto 《Biochemistry and Biophysics Reports》2018
Antibodies are widely used not only as therapeutic agents but also as research tools and diagnostic agents, and extensive efforts have been made to generate antibodies that have higher affinity. It was recently reported that introduction of charged residues into the framework region of an antibody improved its affinity; however, the underlying molecular mechanism has not been elucidated. In this study, we used kinetic and thermodynamic analyses of the antibody–antigen interaction to investigate the molecular mechanism by which an antibody with introduced charged residues recognizes its antigen with higher affinity. The introduction of basic amino acid residues resulted in improvement of the affinity whereas the introduction of acidic residues weakened the interaction. For two mutant antigen-binding fragments (Fabs) with improved affinity (named K5- and R5-mutants), the balance between the association rate constant kon and the dissociation rate constant koff was distinct despite each mutant having the same number of charged residues. Moreover, thermodynamic analysis of the interactions in the transition state revealed a difference between the K5- and R5-mutants in terms of enthalpic energy change following formation of the encounter complex with the antigen. These results suggest that the affinity of the K5- and R5-mutants is improved by distinct mechanisms. Although the mutations destabilize the Fab and necessitate further studies, our strategy is expected to become a versatile and simple means to improve the affinity of antibodies to their antigens. 相似文献