Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.     

The effects of postprandial glucose and insulin levels on postprandial endothelial function in subjects with normal glucose tolerance

ABSTRACT: BACKGROUND: Previous studies have demonstrated that postprandial hyperglycemia attenuates brachial artery flow-mediated dilation (FMD) in prediabetic patients, in diabetic patients, and even in normal subjects. We have previously reported that postprandial hyperinsulinemia also attenuates FMD. In the present study we evaluated the relationship between different degrees of postprandial attenuation of FMD induced by postprandial hyperglycemia and hyperinsulinemia and differences in ingested carbohydrate content in non-diabetic individuals. METHODS: Thirty-seven healthy subjects with no family history of diabetes were divided into 3 groups: a 75-g oral glucose loading group (OG group) (n = 14), a test meal group (TM group) (n = 12; 400 kcal, carbohydrate content 40.7 g), and a control group (n = 11). The FMD was measured at preload (FMD0) and at 60 minutes (FMD60) and 120 (FMD120) minutes after loading. Plasma glucose (PG) and immunoreactive insulin (IRI) levels were determined at preload (PG0, IRI0) and at 30 (PG30, IRI30), 60 (PG60, IRI60), and 120 (PG120, IRI120) minutes after loading.ResultPercentage decreases from FMD0 to FMD60 were significantly greater in the TM group ([MINUS SIGN]21.19 % [PLUS-MINUS SIGN] 17.90 %; P < 0.001) and the OG group ([MINUS SIGN]17.59 % [PLUS-MINUS SIGN] 26.64 %) than in the control group (6.46 % [PLUS-MINUS SIGN] 9.17 %; P < 0.01), whereas no significant difference was observed between the TM and OG groups. In contrast, the percentage decrease from FMD0 to FMD120 was significantly greater in the OG group ([MINUS SIGN]18.91 % [PLUS-MINUS SIGN] 16.58 %) than in the control group (6.78 % [PLUS-MINUS SIGN] 11.43 %; P < 0.001) or the TM group (5.22 % [PLUS-MINUS SIGN] 37.22 %; P < 0.05), but no significant difference was observed between the control and TM groups. The FMD60 was significantly correlated with HOMA-IR (r = [MINUS SIGN]0.389; P < 0.05). In contrast, FMD120 was significantly correlated with IRI60 (r = [MINUS SIGN]0.462; P < 0.05) and the AUC of IRI (r = [MINUS SIGN]0.468; P < 0.05). Furthermore, the percentage change from FMD0 to FMD120 was significantly correlated with the CV of PG (r = 0.404; P < 0.05), IRI60 (r = 0.401; p < 0.05) and the AUC of IRI (r = 0.427; P < 0.05). No significant correlation was observed between any other FMDs and glucose metabolic variables. CONCLUSION: Differences in the attenuation of postprandial FMD induced by different postprandial insulin levels may occur a long time postprandially but not shortly after a meal.     

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.     

Adipocytokine levels mark endothelial function in normotensive individuals

ABSTRACT: BACKGROUND: Endothelial dysfunction is an independent risk factor for cardiovascular events. Inflammatory mediators released by the adipose tissue can lead to local insulin resistance and endothelial dysfunction. This study addressed the relationship of adipocytokines with endothelial function and blood pressure. METHODS: In 92 newly diagnosed, drug-naive essential hypertensive patients (HT, mean age 49 yrs) without organ damage and 66 normotensive subjects (NT, mean age 47 yrs), by an automated system, we measured endothelium-dependent and -independent vasodilation as brachial artery flow-mediated dilation before and after administration of glyceryl-trinitrate. Retinol binding protein-4 (RBP4) and resistin levels were determined by ELISA and RIA, respectively. Oxidative stress was evaluated by measuring serum malondyaldehyde (MDA). RESULTS: Flow-mediated dilation was significantly (p = 0.03) lower in HT (5.3 [PLUS-MINUS SIGN] 2.6%) than NT (6.1 [PLUS-MINUS SIGN] 3.1%), while response to glyceryl-trinitrate (7.5 [PLUS-MINUS SIGN] 3.7% vs 7.9 [PLUS-MINUS SIGN] 3.4%) was similar. RBP4 (60.6 [PLUS-MINUS SIGN] 25.1 vs 61.3 [PLUS-MINUS SIGN] 25.9 mug/ml), resistin (18.8 [PLUS-MINUS SIGN] 5.3 vs 19.9 [PLUS-MINUS SIGN] 6.1 ng/ml) and MDA levels (2.39 [PLUS-MINUS SIGN] 1.26 vs 2.08 [PLUS-MINUS SIGN] 1.17 nmol/ml) were not different in HT and NT.RBP4 (r = [MINUS SIGN]0.25; p = 0.04) and resistin levels (r = [MINUS SIGN]0.29; p = 0.03) were related to flow-mediated dilation in NT, but not in HT (r = [MINUS SIGN]0.03 and r = [MINUS SIGN]0.10, respectively). In NT multivariate analysis, including RBP4 and confounders showed that only BMI or waist circumference remained related to flow- mediated dilation. In the multivariate model including resistin and confounders, BMI, age and resistin were significantly related to flow-mediated dilation, while only age significant correlated with this parameter when BMI was replaced by waist circumference. CONCLUSIONS: Adipocytokine levels may be independent predictors of endothelial dysfunction in the peripheral circulation of healthy subjects, providing a pathophysiological link between inflammation from adipose tissue and early vascular alterations.     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency.

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (VH) and variable light chain (VL), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of VH/VL domain-domain assembly with a site-directed mutagenesis study that probes a highly conserved VH/VL hydrogen bonding interaction. Gln168 of the S5 scFv (Kabat VH 39) is absolutely conserved in 95% of all VH, and Gln44 (Kabat VL 38) is found in 94% of all kappa VL (Glx in 95% of all lambda VL). These side chains form two hydrogen bonds in head-to-tail alignment across the VH/VL interface. Double mutant cycles at Gln168 and Gln44 were constructed to first investigate their contribution to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln168-Gln44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m(g) associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the VH/VL association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the VH/VL interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.     

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

ABSTRACT: BACKGROUND: Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15 % (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production. RESULTS: Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 [PLUS-MINUS SIGN] 3.1, 252 [PLUS-MINUS SIGN] 2.9 and 298 [PLUS-MINUS SIGN] 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at [MINUS SIGN]100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 [PLUS-MINUS SIGN] 3.1 and 252 [PLUS-MINUS SIGN] 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 [PLUS-MINUS SIGN] 1.3 and 120.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 [PLUS-MINUS SIGN] 0.4 and 17.0 [PLUS-MINUS SIGN] 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol was produced at 72 h when ORP was controlled at [MINUS SIGN]150 mV for the VHG fermentation with medium containing 298 [PLUS-MINUS SIGN] 3.8 g/L glucose, since yeast cell viability was improved more significantly. CONCLUSIONS: No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at [MINUS SIGN]150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at [MINUS SIGN]100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.     

Balancing selected medication costs with total number of daily injections: a preference analysis of GnRH-agonist and antagonist protocols by IVF patients

ABSTRACT: BACKGROUND: During in vitro fertilization (IVF), fertility patients are expected to self-administer many injections as part of this treatment. While newer medications have been developed to substantially reduce the number of these injections, such agents are typically much more expensive. Considering these differences in both cost and number of injections, this study compared patient preferences between GnRH-agonist and GnRH-antagonist based protocols in IVF. METHODS: Data were collected by voluntary, anonymous questionnaire at first consultation appointment. Patient opinion concerning total number of s.c. injections as a function of non-reimbursed patient cost associated with GnRH-agonist [A] and GnRH-antagonist [B] protocols in IVF was studied. RESULTS: Completed questionnaires (n = 71) revealed a mean +/[MINUS SIGN] SD patient age of 34 +/[MINUS SIGN] 4.1 yrs. Most (83.1 %) had no prior IVF experience; 2.8 % reported another medical condition requiring self-administration of subcutaneous medication(s). When out-of-pocket cost for [A] and [B] were identical, preference for [B] was registered by 50.7 % patients. The tendency to favor protocol [B] was weaker among patients with a health occupation. Estimated patient costs for [A] and [B] were $259.82 +/[MINUS SIGN] 11.75 and $654.55 +/[MINUS SIGN] 106.34, respectively (p < 0.005). Measured patient preference for [B] diminished as the cost difference increased. CONCLUSIONS: This investigation found consistently higher non-reimbursed direct medication costs for GnRH-antagonist IVF vs. GnRH-agonist IVF protocols. A conditional preference to minimize downregulation (using GnRH-antagonist) was noted among some, but not all, IVF patient sub-groups. Compared to IVF patients with a health occupation, the preference for GnRH-antagonist was weaker than for other patients. While reducing total number of injections by using GnRH-antagonist is a desirable goal, it appears this advantage is not perceived equally by all IVF patients and its utility is likely discounted heavily by patients when nonreimbursed medication costs reach a critical level.     

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.     

Scaling-up from an implementation trial to state-wide coverage: results from the preliminary Melbourne Diabetes Prevention Study

ABSTRACT: BACKGROUND: The successful Greater Green Triangle Diabetes Prevention Program (GGT DPP), a small implementation trial, has been scaled-up to the Victorian state-wide 'Life!' programme with over 10,000 individuals enrolled. The Melbourne Diabetes Prevention Study (MDPS) is an evaluation of the translation from the GGT DPP to the Life! programme. We report results from the preliminary phase (pMDPS) of this evaluation. METHODS: The pMDPS is a randomised controlled trial with 92 individuals aged 50 to 75 at high risk of developing type 2 diabetes randomised to Life! or usual care. Intervention consisted of six structured 90-minute group sessions: five fortnightly sessions and the final session at 8 months. Participants underwent anthropometric and laboratory tests at baseline and 12 months, and provided self-reported psychosocial, dietary, and physical activity measures. Intervention group participants additionally underwent these tests at 3 months. Paired t tests were used to analyse within-group changes over time. Chi-square tests were used to analyse differences between groups in goals met at 12 months. Differences between groups for changes over time were tested with generalised estimating equations and analysis of covariance. RESULTS: Intervention participants significantly improved at 12 months in mean body mass index ([MINUS SIGN]0.98 kg/m2, standard error (SE) = 0.26), weight ([MINUS SIGN]2.65 kg, SE = 0.72), waist circumference ([MINUS SIGN]7.45 cm, SE = 1.15), and systolic blood pressure ([MINUS SIGN]3.18 mmHg, SE = 1.26), increased high-density lipoprotein-cholesterol (0.07 mmol/l, SE = 0.03), reduced energy from total ([MINUS SIGN]2.00%, SE = 0.78) and saturated fat ([MINUS SIGN]1.54%, SE = 0.41), and increased fibre intake (1.98 g/1,000 kcal energy, SE = 0.47). In controls, oral glucose at 2 hours deteriorated (0.59 mmol/l, SE = 0.27). Only waist circumference reduced significantly ([MINUS SIGN]4.02 cm, SE = 0.95).Intervention participants significantly outperformed controls over 12 months for body mass index and fibre intake. After baseline adjustment, they also showed greater weight loss and reduced saturated fat versus total energy intake.At least 5% weight loss was achieved by 32% of intervention participants versus 0% controls. CONCLUSIONS: pMDPS results indicate that scaling-up from implementation trial to state-wide programme is possible. The system design for Life! was fit for purpose of scaling-up from efficacy to effectiveness.Trial registrationAustralian and New Zealand Clinical Trials Registry ACTRN12609000507280.     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïe phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

抗芽殖酵母端粒G4-DNA结构的单克隆抗体和基因工程单链抗体片段的制备和定性研究

大多数真核生物端粒3'末端由富含鸟苷酸的重复序列组成,并可以在体外形成四链G4- DNA结构。为了解这种结构是否在体内存在,本文中我们以芽殖酵母作为研究对象,将G4-DNA作为抗原免疫BALB／c小鼠,制备抗G4-DNA的单克隆抗体,结果显示该抗体能够特异性识别富含鸟苷酸重复序列DNA。为了提高抗体的特异性,我们通过基因工程制备抗体:利用RT-PCR的方法,得到抗体重链和轻链可变区的基因,然后克隆到载体pET22b中得到表达质粒pET22b-scFv,转入大肠杆菌进行表达。在细胞周质中我们检测并纯化到了目的基因的表达产物。另外,我们还利用该基因序列进行了初步的结构分析。基因工程抗体在大肠杆菌中的成功表达及结构分析为今后利用该抗体进行定点突变来研制高特异性和亲和力的抗G4-DNA抗体奠定了基础。     

Improving the productivity of single-chain Fv antibody against c-Met by rearranging the order of its variable domains

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., VL-linker-VH and VHlinker- VL), expressed them in the cytoplasm of E. coli trx/ gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results to the case of scFv antibodies.     

Molecular and immunochemical characteristics of monoclonal and recombinant antibodies specific to bisphenol A

Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA). Among these mabs, BBA-2187 was the most reactive towards bisphenol A. The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported. Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells. The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs. The most sensitive assay was achieved with BBA-2187scFv. Its cross-reactivity to the related compounds was similar to that of the parent mab. Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.