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1.
Affinity maturation is a process that leads to the emergence of more efficient antibodies following initial antigen encounter and represents a key strategy of the adaptive immunity of vertebrate organisms. Earlier and detailed sequence studies of the antibody response to a model antigen, the hapten 2-phenyl-5-oxazolone (phOx), define three different classes of antibodies. Class I antibodies use the V(H)Ox1/V(kappa)Ox1 gene pair and dominate the early stages of the anti-phOx response, class II antibodies use the V(kappa)Ox1 gene but a different V(H) segment and are common in the intermediate stages, and class III antibodies use the TEPC15/V(kappa)45.1 genes and play the greatest role in the late stages. Only the crystal structure of one anti-phOx antibody, the class II NQ10/12.5 Fab fragment, has been described. Here we report the crystal structures of the scFv form of the low and high affinity anti-phOx class III antibodies NQ10/1.12 and NQ16/113.8 complexed with the hapten. The two antibodies differ by nine amino acid substitutions, all located in the V(H) domain. Analysis of the two structures shows that affinity maturation results from an increase in surface complementarity, as a consequence of a finely tuned and highly concerted process chaperoned by the somatic mutations, and implies a more efficient hapten-induced fit in the mature antibody. The data also demonstrate that class III antibodies respond in a completely different way to the architectural problem of binding phOx compared to the class II antibody NQ10/12.5. 相似文献
2.
Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response. 总被引:10,自引:2,他引:8
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P M Alzari S Spinelli R A Mariuzza G Boulot R J Poljak J M Jarvis C Milstein 《The EMBO journal》1990,9(12):3807-3814
The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family. 相似文献
3.
Suk-yoon Song Byung-ung Hur Kyung-woo Lee Hyo-jung Choi Sung-soo Kim Goo Kang Sang-hoon Cha 《Molecules and cells》2009,27(3):313-319
The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously,
but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created
two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted.
Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 × 107 combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific
for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II,
an antibody library of a larger size, named DVFAB-131L, which has a 1.5 × 109 combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 × 107 heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our
results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort,
and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule 相似文献
4.
We report on the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragments from a murine monoclonal anti-cardiotoxin antibody M gamma 2-3 directed against a cobra cardiotoxin. The Fab fragment has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution, X-ray crystallographic studies. The crystals are monoclinic, space group C2, with a = 161.2 A, b = 40.4 A, c = 96.5 A, beta = 118.3 degrees. 相似文献
5.
We report the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragment from a heteroclitic murine (BALB/c) monoclonal anti-hen egg-white lysozyme antibody complexed with a heterologous antigen, pheasant lysozyme. The complex between the heterologous antigen and the antibody has been crystallized from polyethylene glycol 8000 solutions in a form suitable for X-ray crystallographic studies. The crystals are monoclinic, space group C2 with a = 158.2 A, b = 49.1 A, c = 177.6 A, beta = 92.0 degrees (1 A = 0.1 nm). 相似文献
6.
Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen 总被引:1,自引:0,他引:1
R A Mariuzza D L Jankovi? G Boulot A G Amit P Saludjian A Le Guern J C Mazié R J Poljak 《Journal of molecular biology》1983,170(4):1055-1058
Preliminary crystallographic data are given for the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen. This crystalline complex was found by screening a number of Fab-lysozyme complexes prepared from monoclonal anti-lysozyme antibodies produced by hybrids of BALB/c immune spleen cells with a non-secreting mouse hybrid myeloma line. The complex crystallizes in the monoclinic space group P21 with a = 55.5 (+/- 0.1) A, b = 143.5 (+/- 0.3) A, c = 49.1 (+/- 0.1) A, beta = 120 degrees 20' (+/- 10'). X-ray photographs show reflections extending to a resolution of 2.7 A. The crystals are suitable for high-resolution X-ray diffraction studies. 相似文献
7.
I. M. Petyaev 《Redox report : communications in free radical research》2013,18(6):365-372
SummaryUsing various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab’ fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity.Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2–6.6 which is unlike Cu-Zn SOD (pH 7.8–8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity.Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD. 相似文献
8.
Yonghong Zhao Lester Gutshall Haiyan Jiang Audrey Baker Eric Beil Galina Obmolova Jill Carton Susann Taudte Bernard Amegadzie 《Protein expression and purification》2009,67(2):182-189
Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis. 相似文献
9.
T Bizebard Y Mauguen F Petek P Rigolet J J Skehel M Knossow 《Journal of molecular biology》1990,216(3):513-514
Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins. 相似文献
10.
Suzuki M Takemura H Suzuki H Sumida T 《Biochemical and biophysical research communications》2000,271(1):240-243
We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies. 相似文献
11.
人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达 总被引:2,自引:2,他引:2
运用噬菌体表面呈现(phage display)技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。 相似文献
12.
Zakharov AV Smirnov IV Serebriakova MV Dronina MA Kaznacheeva AV Kurkova IN Belogurov AA Friboulet A Ponomarenko NA Gabibov AG Bobik TV 《Molekuliarnaia biologiia》2011,45(1):86-95
Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively. 相似文献
13.
G Boulot C Rojas G A Bentley R J Poljak E Barbier C Le Guern P A Cazenave 《Journal of molecular biology》1987,194(3):577-579
An anti-lysozyme antibody, D1.3, was used as immunogen to obtain syngeneic (Balb/c) monoclonal anti-idiotopic antibodies. The complex between Fab D1.3 and the Fab fragment from the anti-idiotopic antibody E225 has been crystallized. The crystals are monoclinic, space group P2(1), with a = 75.7 A, b = 77.4 A, c = 97.2 A, beta = 111.90 degrees and one molecule of the complex in the asymmetric unit. X-ray photographs show reflections extending to a resolution of about 3 A. Although twinning occurs frequently in the large crystals obtained, this material is suitable for high-resolution X-ray analysis. 相似文献
14.
A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. 总被引:26,自引:0,他引:26
H J de Haard N van Neer A Reurs S E Hufton R C Roovers P Henderikx A P de Bru?ne J W Arends H R Hoogenboom 《The Journal of biological chemistry》1999,274(26):18218-18230
We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics. 相似文献
15.
The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (VH) and variable light domains (VL) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy. 相似文献
16.
Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3 总被引:7,自引:0,他引:7
Rauchenberger R Borges E Thomassen-Wolf E Rom E Adar R Yaniv Y Malka M Chumakov I Kotzer S Resnitzky D Knappik A Reiffert S Prassler J Jury K Waldherr D Bauer S Kretzschmar T Yayon A Rothe C 《The Journal of biological chemistry》2003,278(40):38194-38205
The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., W?lle, J., Plückthun, A., and Virnek?s, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications. 相似文献
17.
Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli 总被引:2,自引:0,他引:2
Expression in Escherichia coli of recombinant genes coding for the kappa-chain and the Fd fragment of an antibody directed against carcinoembryonic antigen gives rise to Fab dimers. These Fab fragments possess antibody activity, as demonstrated by enzyme-linked immunosorbent assay as well as by ligand competition assay. Effective production of soluble Fab in Escherichia coli was achieved by a decrease in the growth temperature. Following a one step purification by anion exchange chromatography, the bacterially-produced Fab retains its activity at 4 degrees C for at least two months. The relatively simple methodology described in this study should be useful for the design and production of antibodies in bacteria. 相似文献
18.
A.F.S.A. Habeeb 《Archives of biochemistry and biophysics》1981,212(2):618-628
Pigs immunized with lysozyme responded by producing only nonprecipitating antibody throughout the immunization period. Fig antilysozyme antibodies were found to be resistant to papain fragmentation, only 33% of the antibodies were fragmented with papain. From the binding of fluorescein labeled or 14C-labeled lysozyme to antilysozyme antibodies it was concluded that the antibodies elicited in pigs recognized only two antigenic determinants of lysozyme. These results were confirmed from the binding of Fab fragments to 14C-lysozyme. Fab fragments prepared from precipitating rabbit antilysozyme antibody bound 14C-lysozyme at a molar ratio of Fab/lysozyme = 3. Therefore nonprecipitating antibodies are the outcome of recognition of only two antigenic determinants on lysozyme and inability to form a lattice structure when antibody and antigen interact. This work emphasizes the limitations of using antibodies as a biological reagent for delineating the antigenic determinants on proteins. 相似文献
19.
Benny Chain Jack Arnold Samia Akthar Michael Brandt David Davis Mahdad Noursadeghi Thabo Lapp Changhua Ji Surya Sankuratri Yanjing Zhang Lata Govada Emmanuel Saridakis Naomi Chayen 《PloS one》2015,10(6)
The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution. 相似文献
20.
Jos Tormo Nuria B. Centeno Emilia Fontana Thomas Bubendorfer Ignacio Fita Dieter Blaas 《Proteins》1995,23(4):491-501
The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods.1 The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known.2 The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 Å from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone3 indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding,4 might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating. © 1995 Wiley-Liss, Inc. 相似文献