Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

The molecular interaction of the Fab fragment of the human monoclonalantibody 3D6, directed against the transmembrane protein gp41of human immunodeficiency virus (HTV) 1, with its peptide epitopeis characterized by a panel of overlapping peptides, a peptideepitope library and molecular modeling techniques. The sequenceCSGKLICTTAVPW, corresponding to amino acids 605–617 ofgp41, was identified as the best binding peptide (K_D = 1x10^-8mol/1). This peptide served as a starting point to prepare acellulose-bound peptide epitope library in which each residueof the epitope is substituted by all L- and D-amino acids, resultingin 494 epitope peptide variants which were subsequently analyzedfor binding 3D6. The library was synthesized to identify residuescritical for binding and to obtain information about the molecularenvironment of the epitope peptide bound to 3D6. Both cysteineresidues, as well as isoleucine 6, threonine 8 and proline 12,of the epitope were highly sensitive to substitution. Usingthe data obtained from the epitope characterization, as wellas a low-resolution electron density map of a 3D6 Fab-peptidecomplex, a 3-D model of the Fab-peptide complex was generatedby molecular modeling. The modeling experiments predict bindingof the peptide, which is cyclized via the two cysteine residues,to a pocket formed dominantly by the hypervariable loops complementaritydetermining regions CDR3L, CDR2H and CDR3H.     

A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs

We constructed Fab libraries of bacteriophage-displayed H:CDR3mutants in the high-affinity anti-digoxin antibody 26-10 todetermine structural constraints on affinity and specificityfor digoxin. Libraries of mutant Fabs randomized at five or10 contiguous positions were panned against digoxin and threeC16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and16-acetylgitoxin. The sequence data from 83 different mutantFabs showed highly restricted consensus patterns at positionsH:100, 100a and 100b for binding to digoxin; these residuescontact digoxin in the 26-10:digoxin co-crystal structure. Severalmutant Fabs obtained following panning on digoxin-BSA showedincreased affinity for digoxin compared with 26-10 and retainedthe wild-type (wt) Trp at position 100. Those Fabs selectedfollowing panning on C16-substituted analogs showed enhancedbinding to the analogs. Replacement of H:Trp100 by Arg resultedin mutants that bound better to the analogs than to digoxin.This specificity change was unexpected, as C16 lies on the oppositeside of digoxin from H:CDR3. Substitution of wt Trp by Arg appearsto alter specificity by allowing the hapten to shift towardH:CDR3, thereby providing room for C16 substituents in the regionof H:CDR1.     

A V(L) single-domain antibody library shows a high-propensity to yield non-aggregating binders

A synthetic human V(L) phage display library, created by the randomization of all complementarity-determining regions (CDRs) in a V(L) scaffold, was panned against three test antigens to determine the propensity of the library to yield non-aggregating binders. A total of 22 binders were isolated against the test antigens and the majority (20) were monomeric. Thus, human V(L) repertoires provide an efficient source of non-aggregating binders and represent an attractive alternative to human V(H) repertoires, which are notorious for containing high proportions of aggregating species. Moreover, the solubility of V(L)s, in contrast to V(H)s, appears much less CDR dependent.     

Complementarity determining region residues aspartic acid at H55, serine at H95 and tyrosines at H97 and L96 play important roles in the B72.3 antibody-TAG72 antigen interaction

Structural analysis derived from the crystallographic studyof the chimeric B72.3 antibody illustrated some major atomicinteractions between complementarity determining region (CDR)residues. For example, hydrogen bonds are formed between H35/H95,L50/H97, H53/H55 and H96/L96 respectively. These CDR residuesmay play important roles in the B72.3–TAG72 (antibody-antigen)interaction either by direct interaction with the TAG72 antigenor by maintaining a CDR loop conformation through atomic interactionsbetween CDR residues. In order to confirm these assumptions,we altered these CDR residues by site-directed mutagenesis anddetermined binding affinities of these mutant chimeric antibodiesfor the TAG72 antigen in a solid-phase radioimmunoassay. Wefound that H55, H95, H97 and L96 are important CDR residuesfor the B72.3–TAG72 interaction. Single amino acid substitutionsof aspartic acid and serine by alanine at H55 of CDR2 and atH95 of CDR3 respectively and of tyrosine by phenylalanine atH97 and L96 of CDR3, significantly reduced the binding affinityfor the TAG72 antigen by 20-, 8-, 16- and 45-fold respectively.Therefore, this study reveals some of the requirements for maintainingthe integrity of the B72.3 antibody combining sites.     

Site‐Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne–Azide Cycloaddition Reactions

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine‐reactive probes that target surface‐exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)‐binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site‐specifically labeled product. Z₃₂BPA was synthesized by solid‐phase peptide synthesis and further functionalized to give alkyne‐Z₃₂BPA and azide‐Z₃₂BPA for Cu^I‐catalyzed cycloaddition, as well as DBCO‐Z₃₂BPA for Cu‐free strain‐promoted cycloaddition. The Z₃₂BPA variants were conjugated to the human IgG1 antibody trastuzumab and site‐specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2‐expressing cells in immunofluorescence microscopy.     

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.     

噬菌体展示抗乙型肝炎病毒表面抗原Fab抗体分子的筛选和序列分析

目的　从自建的免疫噬菌体展示Fab抗体库中获得抗乙型肝炎病毒表面抗原Fab抗体分子 ,并进行基因序列分析。方法　采用纯化的乙型肝炎病毒表面抗原作为包被抗原 ,对自建的 4× 10 5Fab噬菌体抗体库进行富集筛选 ,从阳性强的次级库中挑选单个克隆菌 ,分别进行噬菌体ELISA、PCR和限制性内切酶鉴定后 ,再选取三者均为阳性的克隆菌进行基因序列分析。结果　共挑选了 6 0个单克隆菌株 ,其中噬菌体ELISA阳性有 2 7个 ,阳性率为 4 5 % ;PCR鉴定均为相应大小片段 ;限制性内切酶鉴定表明 2 7个阳性克隆菌中有 13个具有约 15 0 0bp的片段 ,其余均为约 75 0bp大小 ;BstOI酶切鉴定表明各克隆菌的酶切方式不同 ;选取 5个含约 15 0 0bp片段和 2个含约75 0bp片段的克隆菌株 ,经测序均为人免疫球蛋白基因 ,且重链分别属于VH3、VH4、VH5家族 ,轻链分别属于L5、L6、L8和 0 12 /0 2家族。结论　从自建的免疫噬菌体展示Fab抗体库中成功地获得了抗乙型肝炎病毒表面抗原Fab抗体分子。表明所构建的抗乙型肝炎病毒表面抗原的Fab抗体库的抗体基因具有多样性。     

鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。     

1.85 A structure of anti-fluorescein 4-4-20 Fab

The crystal complex of fluorescein bound to the high-affinityanti-fluorescein 4-4-20 Fab {K_a = 10¹⁰ M^–1 at 2°C)has been determined at 1.85 Å. Isomorphous crystals oftwo isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. FunctGenet., 3, 155–160], have been grown. Both complexes crystallizewith one molecule in the asymmetric unit in space group P1,with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, = 95.15° , ß = 86.85° and = 98.01°.The final structure has an R value of 0.188 at 1.85 Åresolution. Interactions between bound fluorescein, the complementarity-determiningregions (CDRs) of the Fab and the active-site mutants of the4-4-20 single-chain Fv will be discussed. Differences were foundbetween the structure reported here and the previously reported2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins:Struct. Fund., 5, 271–280]. Our structure determinationwas based on 26 328 unique reflections — four times theamount of data used in the previous report. Differences in thetwo structures could be explained by differences in interpretingthe electron density maps at the various resolutions. The r.m.s.deviations between the variable and constant domains of thetwo structures were 0.77 and 1.54 Å, respectively. Fourregions of the light chain and four regions of the heavy chainhad r.m.s. backbone deviations of >4 Å. The most significantof these was the conformation of the light chain CDR 1.     

A new ligand for immunoglobulin g subdomains by screening of a synthetic peptide library

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.     

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 10⁹/L, respectively, to ≈13 days (both HR and RR) at 350 × 10⁹/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

Crystal structures of high affinity human T-cell receptors bound to peptide major histocompatibility complex reveal native diagonal binding geometry

Naturally selected T-cell receptors (TCRs) are characterised by low binding affinities, typically in the range 1-100 microM. Crystal structures of syngeneic TCRs bound to peptide major histocompatibility complex (pMHC) antigens exhibit a conserved mode of binding characterised by a distinct diagonal binding geometry, with poor shape complementarity (SC) between receptor and ligand. Here, we report the structures of three in vitro affinity enhanced TCRs that recognise the pMHC tumour epitope NY-ESO(157-165) (SLLMWITQC). These crystal structures reveal that the docking mode for the high affinity TCRs is identical to that reported for the parental wild-type TCR, with only subtle changes in the mutated complementarity determining regions (CDRs) that form contacts with pMHC; both CDR2 and CDR3 mutations act synergistically to improve the overall affinity. Comparison of free and bound TCR structures for both wild-type and a CDR3 mutant reveal an induced fit mechanism arising from restructuring of CDR3 loops which allows better peptide binding. Overall, an increased interface area, improved SC and additional H-bonding interactions are observed, accounting for the increase in affinity. Most notably, there is a marked increase in the SC for the central methionine and tryptophan peptide motif over the native TCR.     

Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.     

Platinum(II) as Bifunctional Linker in Antibody–Drug Conjugate Formation: Coupling of a 4‐Nitrobenzo‐2‐oxa‐1,3‐diazole Fluorophore to Trastuzumab as a Model

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD) fluorophore and trastuzumab (Herceptin?) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L‐NBD)Cl](NO₃) (en=ethylenediamine), with L=N‐heteroaromatic, N‐alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO₄ or Na₂SO₄ in tricine or HEPES buffer. Reaction of N‐coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5–2.9 that were stable in phosphate‐buffered saline at room temperature for at least 48 h. In contrast, thioether‐coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.     

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.     

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6–7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.     

Selection and characterization of HER2/neu-binding affibody ligands

Affibody® (affibody) ligands that are specific for the extracellulardomain of human epidermal growth factor receptor 2 (HER2/neu)have been selected by phage display technology from a combinatorialprotein library based on the 58 amino acid residue staphylococcalprotein A-derived Z domain. The predominant variants from thephage selection were produced in Escherichia coli, purifiedby affinity chromatography, and characterized by biosensor analyses.Two affibody variants were shown to selectively bind to theextracellular domain of HER2/neu (HER2-ECD), but not to controlproteins. One of the variants, denoted His₆-Z_HER2/neu:4, wasdemonstrated to bind with nanomolar affinity (     

人源性抗甲型肝炎病毒Fab噬菌体抗体分子的初步筛选

目的从自建的大容量非免疫Fab噬菌体抗体库中筛选具有抗甲型肝炎病毒(HAV)抗原活性的噬菌体抗体,并进行鉴定。方法采用纯化的甲型肝炎病毒作为包被抗原,对自建的容量为1.26×1011pfu/ml的Fab噬菌体抗体库进行筛选和富集,分别对原始库和筛选后的抗体库进行Phage-ELISA和活性检测,经酶切鉴定和DNA测序分析后进行诱导表达。结果经2轮筛选,抗-HAV抗原的噬菌体抗体得到了明显富集,ELISA检测证实其具有良好的抗甲肝病毒抗原的特异性,抑制率为38.61%。酶切鉴定表明抗体库重组较稳定。SDS-PAGE检测显示Fab抗体蛋白在E.coliDH5α中得到了表达。结论从自建的非免疫Fab噬菌体抗体库中成功获得了抗甲肝病毒噬菌体抗体,所构建的非免疫抗体库可用于人源性抗体的筛选。