Construction of multiform scFv antibodies using linker peptide

Multiform single chain variable fragments (acFvs) including different length linker scFvs and bispecific seFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON seFv and antizearalenone (anti-ZEN) scFv was also constructed through eormection by a short peptide tinker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecifie acFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgertic plants.     

Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff.     

Detection of constitutive molecules on Histoplasma capsulatum yeasts through single chain variable antibody fragments displayed in M13 phages

A nonimmune library, containing single chain variable fragments (scFv) of immunoglobulin human genes displayed on the surface of M13 filamentous phages, was used to recognize molecules exposed on Histoplasma capsulatum yeasts' surface, during their growth in synthetic medium. The scFv clones were checked in their consistency by Dot-ELISA using HRP/anti-M13 conjugate, and they were tested to recognize molecules on H. Capsulatum yeasts' surface by ELISA in plates. Three out of 80 scFv cones (C2, C6, and C52) reacted consistently with H. capsulatum molecules, and they recognized molecules from both H. capsulatum morphologic phases. However, C6 and C52 clones reacted better with molecules on the surface of whole yeasts, with molecules from the yeasts' cell-wall extract, and with molecules released to the supernatant of the yeast culture. Mycelial supernatants from other fungi, as well as from a Mycobacterium filtrate, were not recognized by scFv phage monoclones. Monoclones C2, C6, and C52 recognized yeast molecules irrespective of the H. capsulatum strains used; the C6 clone revealed a specific immunohistochemistry reaction when tested against homologous and heterologous fungal infected tissues. The scFv clones isolated will be a useful toll to define the role of their target molecules in the host-parasite relationship of histoplasmosis.     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin Zearalenone

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T₀ leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T₁ leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T₂ seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.     

Transgenic Tobacco Plants Expressing a Dimeric Single-chain Variable Fragment (scFv) Antibody Against <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serotype Paratyphi B

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T₀) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 μg of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T₁ plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T₀ plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T₁ generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.     

曲霉菌半乳甘露聚糖单克隆抗体的制备及初步应用

目的:制备抗曲霉菌半乳甘露聚糖的单克隆抗体,并基于获得的抗体建立用于快速准确检测曲霉菌感染的双抗体夹心酶联免疫吸附法(ELISA),以期可用于侵袭性曲霉菌病的临床诊断。方法:提取曲霉菌半乳甘露聚糖后免疫BALB/c小鼠,筛选与制备抗曲霉菌半乳甘露聚糖的单克隆抗体,通过间接ELISA法与Western Blot方法开展单克隆抗体检测性能分析,使用获得的单克隆抗体建立双抗体夹心ELISA方法,并初步应用于曲霉菌感染血清检测。结果:获得抗曲霉菌半乳甘露聚糖单克隆抗体5株,均可特异性识别曲霉菌半乳甘露聚糖,以其中性能最佳的3C9抗体和辣根过氧化物酶标记的3C9抗体配对为基础,建立了双抗体夹心ELISA方法,通过初步评价确定该方法可应用于临床侵袭性曲霉菌病血清检测,并且该方法与现有商品化试剂盒相比检测背景值较低,可更有效区分曲霉菌感染阴阳性血清。结论:本研究筛选获得针对曲霉菌半乳甘露聚糖的特异性单克隆抗体,以该抗体为基础建立双抗体夹心ELISA方法具有潜在转化应用前景,可为侵袭性曲霉菌病的临床诊断提供支持。     

基于CdSe阳离子交换的玉米赤霉烯酮新型荧光免疫检测方法的建立及应用

作为镰刀属真菌的次级代谢产物,玉米赤霉烯酮(zearalenone,ZEN)具有强烈的生殖毒性和免疫毒性,严重威胁动物和人类健康。本研究通过采用羧基修饰的CdSe水溶性量子点(quantum dots,QDs)标记ZEN单克隆抗体,并基于CdSe阳离子交换信号增强原理,建立了ZEN新型荧光免疫检测方法(CdSe QDs-FLISA),检测下限(IC₁₀)和半数抑制率(IC₅₀)分别为0.006 ng/mL和0.17 ng/mL,检测区间(IC20–IC80)为0.01–0.45 ng/mL。与ZEN的结构类似物(α-zearalanol、zearalanone、α-zearalenol、β-zearalenol and β-zearalanol)交叉反应性依次为22.3%、13.1%、6.2%、1.6%和3.9%,与农产品中其他真菌毒素如黄曲霉毒素B₁(AFB₁)、赭曲霉毒素A(OTA)、呕吐毒素(DON)和伏马毒素B₁(FB₁)几乎不存在交叉反应。该方法...     

Occurrence of deoxynivalenol and zearalenone in brewing barley grains from Brazil

Barley (Hordeum vulgare L.) is an important cereal crop for food and represents one of the main ingredients in beer production. Considering the importance of barley and its derived products, the knowledge about the mycotoxin contamination in the barley production is essential in order to assess its safety. In this study, the levels of deoxynivalenol (DON) and zearalenone (ZEN) in brewing barley were determined using a LC-MS/MS method. A survey was conducted in 2015 to estimate the mycotoxin levels in these products (n?=?76) from four crop regions in Brazil. The results showed high levels of DON and ZEN in the analyzed samples, with contamination levels of 94 and 73.6%, respectively. The mean levels of DON and ZEN ranged from 1700 to 7500 μg/kg and from 300 to 630 μg/kg, respectively. Barley samples from regions 1 and 2 presented higher levels of ZEN and DON, respectively, and those from region 4 presented lower levels of both. Co-occurrence of DON and ZEN was seen in the majority of the barley grain samples, and the mycotoxin content was above the maximum levels established by the Brazilian and European regulations.     

Integrated microfluidic systems with an immunosensor modified with carbon nanotubes for detection of prostate specific antigen (PSA) in human serum samples

This paper describes the development of an immunosensor coupled to glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (CNT-GCE) integrated with microfluidic systems for rapid and sensitive quantification of prostate specific antigen (PSA) in human serum samples. Mouse monoclonal (5G6) to PSA antibodies were immobilized on a rotating disk. PSA in the serum sample are allowed to react immunologically with the immobilized anti-tPSA and horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to PSA. HRP, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on CNT-GCE at -0.15 V. The electrochemical detection can be done within 1 min and total assay time was 30 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.08 and 0.5 microg L(-1), respectively and the intra- and inter-assay coefficients of variation were below 4.5%. The electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows potential for detecting PSA in clinical diagnosis.     

Zearalenone production and growth in drinking water inoculated with <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Production of the mycotoxin zearalenone (ZEN) was examined in drinking water inoculated with Fusarium graminearum. The strain employed was isolated from a US water distribution system. ZEN was purified with an immunoaffinity column and quantified by high-performance liquid chromatography (HPLC) with fluorescence detection. The extracellular yield of ZEN was 15.0 ng l^?1. Visual growth was observed. Ergosterol was also indicative of growth and an average of 6.2 μg l^?1 was obtained. Other compounds were also detected although remain unidentified. There is no equivalent information available. More work is required on metabolite expression in water as mycotoxins have consequences for human and animal health. The levels detected in this study were low. Water needs to be accepted as a potential source as it attracts high quality demands in terms of purity.     

The effect of zearalenone on PSII photochemical activity and growth in wheat and soybean under salt (NaCl) stress

The effects of mycotoxin zearalenone (ZEN) on the photochemical activity of photosystem II (PSII) in wheat and soybean leaf discs incubated in ZEN solutions as well as the after-effects of pre-sowing soaking of seeds in solutions containing ZEN on the photochemical activity of PSII and on the seedlings growth under salt stress (NaCl solutions were investigated). The incubation of wheat leaf discs in ZEN solutions strongly inhibited the energy flux per cross section (CS) for absorption (ABS/CS), trapping (TRo/CS) and electron transport (ETo/CS), while the effects of ZEN action on soybean discs were opposite and the values of those parameters significantly increased with the increase in ZEN concentration. Incubation of seeds in a ZEN solution resulted in an increase in photochemical efficiency of PSII in soybean seedlings, but did not induce any response of PSII in those of wheat at medium illuminations. Only at the stronger illumination for both species did ZEN induce an increase in efficiency of excitation energy capture by open PSII reaction centers, photochemical quenching of chlorophyll a fluorescence and quantum yield of PSII electron transport. Pre-sowing soaking of seeds in a ZEN solution decreased the photoinhibitory injuries of PSII in wheat and soybean due to safe scattering of the excess excitation energy through an increase in energy-dependent quenching (qE) and state transition quenching (qT). ZEN when added to NaCl solutions during the period of germination contributed to reduction in the growth inhibition of wheat seedlings. The incubation of wheat leaf discs in ZEN solutions strongly inhibited CS, ABS/CS, TRo/CS and ETo/CS. Possible effects of ZEN on some physiological processes in plants have been discussed especially in the context with photochemical activity of PSII and a salt stress.     

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.

     

Zearalenone production and growth in drinking water inoculated with <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Production of the mycotoxin zearalenone (ZEN) was examined in drinking water inoculated with Fusarium graminearum. The strain employed was isolated from a US water distribution system. ZEN was purified with an immunoaffinity column and quantified by high-performance liquid chromatography (HPLC) with fluorescence detection. The extracellular yield of ZEN was 15.0 ng l⁻¹. Visual growth was observed. Ergosterol was also indicative of growth and an average of 6.2 μg l⁻¹ was obtained. Other compounds were also detected although remain unidentified. There is no equivalent information available. More work is required on metabolite expression in water as mycotoxins have consequences for human and animal health. The levels detected in this study were low. Water needs to be accepted as a potential source as it attracts high quality demands in terms of purity. An erratum to this article can be found at     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II)

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA-ITCBE-Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL(-1). The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.     

玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立

玉米赤霉烯酮具有较强的生物毒性,检测谷物中的玉米赤霉烯酮在食品和饲料安全中具有重要的作用.将玉米赤霉烯酮与牛血清白蛋白的偶联物免疫BALB/c小鼠制备单克隆抗体,并建立基于单克隆抗体的酶联免疫法作为检测玉米赤霉烯酮的方法.结果共筛选到4株抗玉米赤霉烯酮单克隆抗体,3株抗体亚类为IgG1,1株为IgG2b.选择其中的一株杂交瘤细胞2C9制备小鼠腹水,纯化后测定了抗体效价为1/40 000.以此单抗建立的间接竞争ELISA方法,其半数抑制率(IC50)为1.90 ng/mL,检测限(IC10)为0.051 ng/mL,检测区间(IC20-IC80)为0.115-13.900 ng/mL;且对玉米赤霉烯酮有很好的特异性.回收率检测在样品含1.46-93.80 μg/kg时回收率为96.5％-113.0％.本实验建立的检测方法可用于多种谷物及饲料样本中玉米赤霉烯酮的检测.     

Immunoassay based on carbon nanotubes-enhanced ELISA for Salmonella enterica serovar Typhimurium

Among the methods used to detect pathogenic bacteria, enzyme linked immunosorbent assay (ELISA) is one of the most widely used techniques in routine sample analysis. For Salmonella enterica serovar Typhimurium detection, a typical ELISA yields a sensitivity of 10(6)-10(7)CFU/ml. To enhance the detection sensitivity, single-walled carbon nanotubes (SWCNTs) was employed in this study as a labelling platform for antibody and horseradish peroxidase (HRP) co-immobilizing. With high proteins recovery after the coupling process, the resulting Ab/SWCNTs/HRP bioconjugate was used in the proof-of-concept ELISA experiments. Limit of detection (LOD) was found to be 10(3) and 10(4)CFU/ml for direct and sandwich ELISA, respectively, when Ab/HRP at 1:400 ratio was used. This figure accounts for 1000-time greater in detection sensitivity when compared to a commercial Ab-HRP conjugate. The Ab/SWCNTs/HRP bioconjugate was tested further in real samples and found a superior activity over the commercial Ab-HRP by showing 100-time greater detection limit.     

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1

Fumonisin B₁ (FMB₁) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB₁ (anti-FMB₁ mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB₁ (FMB₁-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB₁ mAb has about 10 ppb of minimum FMB₁ detection concentration and 220 ppb of 50% inhibition concentration (IC₅₀). Much lower cross-reactivity of anti-FMB₁ mAb on ochratoxin A, aflatoxin B₁ and deoxynivalenol provided that anti-FMB₁ mAb was specific for FMB₁. The gene coding single chain variable fragment against FMB₁ (anti-FMB₁ scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB₁ scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB₁ scFv on FMB₁-BSA was obtained in comparison with that of the parental mAb.     

Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.