New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants

Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20mM sodium phosphate buffer pH 7.5, containing 0.1M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn(2+)-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.     

CdSe quantum dots as labels for sensitive immunoassay of cancer biomarker proteins by electrogenerated chemiluminescence

A sensitive and specific immunoassay method for detecting α-fetoprotein (AFP) based on electrogenerated chemiluminescence (ECL) was described. ECL could perform detection for a series of different concentrations of AFP. CdSe quantum dots (QDs) were used as labels and were linked to AFP antibody (anti-AFP, the secondary antibody, Ab2*). Immunoassay was carried out on a modified electrode using a sandwich assay approach, where anti-AFP (Ab1) was covalently bound to the surface of an Au electrode to be allowed to capture AFP specifically. Afterwards, Ab2* was allowed to bind selectively to the captured AFP. The non-specific adsorption was negligible. In the presence of H(2)O(2), the ECL intensity increased with the increase of AFP, which indicated that an immunosensor for AFP was constructed. The detection of AFP based on measuring the ECL intensity of CdSe without the enzyme and mediator can promote the stability of the immunosensor. The linear range of the AFP assay was from 0.002 to 32 ng mL(-1). Furthermore, the immunosensor showed high sensitivity, good precision, stability, and reproducibility and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method. The strategy was successfully demonstrated as a simple, cost-effective, specific, and potential method to detect AFP in practical samples.     

An ELISA-based approach to optimize elution conditions for obtaining hapten-specific antibodies

The correct choice of the elution conditions to break an affinity interaction is important for the successful purification of biomolecules. The optimal elution buffer liberates the bound substance in a minimum volume and maintains the activity of the purified material. The present study demonstrates an enzyme-linked immunosorbent assay (ELISA)-based approach for selection of specific elution conditions for eluting antibodies against a small molecule (atrazine) from pooled sera. Six different elution conditions were tried for the removal of antibodies from the complex. Large-scale purification of anti-atrazine antibodies from the sera was done with a hapten-specific column using an amino-terminal crosslinked agarose gel. Efficacy in terms of total amount of recovery and binding affinities of eluted antibodies from the column were further investigated by ELISA. Results indicate that the ELISA-based elution approach is ideal for the selection of suitable elution buffer that can subsequently be utilized for affinity purification applications.     

An immunoaffinity column for the determination of peanut protein in chocolate.

An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2-3.2 micrograms/g of peanut protein averaged 77% (range, 72-84%), and the minimum detection limit was 0.1 microgram/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.     

Evaluation of a mixed immunosorbent for selective trace enrichment of phenylurea herbicides from plant material

A mixed immunosorbent, composed of anti-isoproturon and anti-chlortoluron antibodies immobilized on aldehyde-activated silica was evaluated for the trace enrichment and clean-up of several phenylurea herbicides from potatoes, carrots and peas. As the immunosorbent exhibited non-specific interactions with the matrix components, a previous clean-up step based on gel filtration (Sephadex G-25 PD-10 column) or anion-exchange (Power 1 × 8 Dionex column) was necessary. The coupling of gel filtration and immunosorbent with subsequent HPLC/UV determination allowed the determination of selected herbicides in potatoes in the 30 ng/g range with high precision, but for more complex samples (carrots and peas) the combination of anion-exchange resin and immunosorbent was required. With 30 ng/g spiked samples recovery rates for isoproturon, chlortoluron, metobromuron, linuron and chlorbromuron averaged 80 ± 20% (n = 5 in two consecutive days) for the whole procedure and the estimated detection limits ranged from 5 to 20 ng/g.     

Ultrasonic energy as a new tool for fast isotopic 18O labeling of proteins for mass spectrometry-based techniques: preliminary results

Preliminary results regarding fast isotopic labeling of proteins with (18)O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar (16)O/(18)O isotopic labeling ratios were found for the overnight procedure (12h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and alpha-lactalbumin proteins. The procedure, however, failed to promote double (18)O isotopic labeling for the proteins, ovalbumin and alpha-lactalbumin. Two different sonication frequencies, 35 and 130 kHz, were studied at two different sonication times of 15 and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic (18)O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 degrees C.     

Evaluation of a mixed immunosorbent for selective trace enrichment of phenylurea herbicides from plant material

A mixed immunosorbent, composed of anti-isoproturon and anti-chlortoluron antibodies immobilized on aldehyde-activated silica was evaluated for the trace enrichment and clean-up of several phenylurea herbicides from potatoes, carrots and peas. As the immunosorbent exhibited non-specific interactions with the matrix components, a previous clean-up step based on gel filtration (Sephadex G-25 PD-10 column) or anion-exchange (Power 1 × 8 Dionex column) was necessary. The coupling of gel filtration and immunosorbent with subsequent HPLC/UV determination allowed the determination of selected herbicides in potatoes in the 30 ng/g range with high precision, but for more complex samples (carrots and peas) the combination of anion-exchange resin and immunosorbent was required. With 30 ng/g spiked samples recovery rates for isoproturon, chlortoluron, metobromuron, linuron and chlorbromuron averaged 80 ± 20% (n = 5 in two consecutive days) for the whole procedure and the estimated detection limits ranged from 5 to 20 ng/g. Received: 29 January 1998 / Revised: 4 May 1998 / Accepted: 30 May 1998     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Polyclonal-based ELISA for the identification of cyclohexanedione analogs that inhibit maize acetyl coenzyme-A carboxylase

Cyclohexanedione herbicides inhibit monocotyledonous acetyl coenzyme-A carboxylase (ACCase; E.C. 6.4.1.2.), which catalyzes the first committed step in fatty acid biosynthesis. Although the target site has been identified, little is known about the mechanisms involved in herbicide binding. An immunological study was undertaken to create a model to better characterize the herbicide-enzyme interaction. Cyclohexanedione-specific antiserum was raised in New Zealand white rabbits by immunizing them with a cyclohexanedione analog-bovine serum albumin conjugate. Two indirect enzyme-linked immunosorbent assays (ELISA) were developed using 2 different cyclohexanedione analogs conjugated to ovalbumin as coating conjugates. Nineteen cyclohexanedione analogs, 13 active ACCase inhibitors, and 6 inactive analogs were tested for their ability to compete with both coating conjugates for antiserum binding. All active ACCase inhibitors were observed to compete with both coating conjugates, whereas all inactive analogs failed to compete with at least one coating conjugate. On the basis of these results, the immunological model could be used to distinguish all active ACCase inhibitors from inactive analogs using the 2 ELISAs sequentially.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法

建立了转Bt基因棉花中Cry杀虫蛋白的提取、样品前处理以及酶联免疫(ELISA)定量分析方法,并使用凝胶电泳、普通聚合酶链式反应(PCR)和实时荧光定量PCR等分子生物学手段对转基因棉花中的Bt基因进行定性和定量检测.所建立的苏云金芽孢杆菌杀虫晶体蛋白(Cry1Ab蛋白和Cry1Ac蛋白)标准曲线线性关系良好,相关系数r2均大于0.999,相对标准偏差RSD均小于2.0%.方法简单、快速、重现性和精密度好,可为农业食品行业和环境领域科研人员提供一种简便快速地从转基因棉花中检测Bt毒蛋白的分析方法.     

Development of immunoaffinity columns for pyraclostrobin extraction from fruit juices and analysis by liquid chromatography with UV detection

Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose-CNBr gel took place with high yield (over 90%), whereas the immunosorbent efficacy to retain the analyte (from 28 to 68%) was shown to depend on the amount and type of antibody immobilized on the support. As a matter of fact, columns prepared with the monoclonal antibody PYs5#14 were able to selectively bound up to 53 μg of pyraclostrobin per gram of beads. Acetonitrile solutions were preferred over methanolic ones for analyte elution, and some immunosorbents could be reused at least 4-6 times provided that the amount of pyraclostrobin and the volume of sample did not overload the column. Effectiveness of the selected immunoaffinity column was evidenced by the development of an extraction procedure for pyraclostrobin residues from fruit juices and further determination by high-performance liquid chromatography with UV detection. A concentration factor of 50 times was achieved with the developed immunoaffinity column, which eventually resulted in a limit of quantification of 0.01 mg L(-1). Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with pyraclostrobin from 0.01 to 1 mg L(-1).     

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser-induced fluorescence detector

A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na?B?O?/NaH?PO? buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.     

Rapid Detection of Antibody in Biological Fluids on a Bioarray Chip

A bioarray chip was developed for the rapid and sensitive detection of the antibody (Ab) in nanoliter volumes of biological fluids. The chip was used to detect the anti-hemagglutinin (anti-HA) Ab from mouse ascites fluids. On the bioarray chip, lanes of antigens (i.e., a 13-amino acid peptide, HA, that is bound by anti-HA Ab) were printed in one dimension on the chip surface. The antigens probed the protein samples (i.e., mouse anti-HA) obtained from biological fluids flowing in channels arranged in the perpendicular dimension. To determine if the detection sensitivity was increased by using different fluorescent labeling methods, two types of labeling reagents were compared, namely AlexaFluor 647-labeled goat anti-mouse Ab (AxIgG), and biotin-labeled anti-mouse Ab (BioIgG) followed by cyanine 5-tagged streptavidin (SA-Cy5). In addition, the assay procedure was shortened by omitting the reduction and blocking steps, while maintaining the sensitivity. In conclusion, a bioarray chip was adopted to detect as low as 10?pM mouse monoclonal antibody (anti-HA) in 500 nL of biological fluids (i.e., corresponding to 5 attomoles of antibody). Our results demonstrate that on-chip detection using this bioarray format is more sensitive and less time-consuming than traditional analyses.     

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, the 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex (Phos-tag), which is linked to a highly cross-linked 4% (w/v) agarose. The affinity gel (Phos-tag agarose) was prepared by the quantitative reaction of N-hydroxysuccinimide-activated Sepharose and a Phos-tag derivative having a 2-aminoethylcarbamoyl group in dry CH3CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos-tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, alpha(s1)-casein, and beta-casein at physiological pH.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract

In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

克百威的免疫亲和色谱分析研究

Sepharose CL-4B经碳酰二咪唑(CDI)活化后与纯化的克百威抗体共价偶联,合成了免疫亲和色谱(IAC)固定相,并用其制备了对克百威具有特异性亲和力的IAC柱。对IAC条件进行了优化,选择0.02 mol/L pH 7.2磷酸盐缓冲液(PB)作为吸附与平衡介质,60%（体积分数）甲醇水溶液作为洗脱剂。结果表明:在优化条件下,IAC柱对克百威的动态柱容量达1.58 mg/L。当标样溶液中克百威质量浓度低于 2　μg/L时,经IAC柱富集的效率高于167倍。在河水中按0.1 mg/L水平添加克百威标准品,经IAC柱分离富集,洗脱液采用包被抗体直接竞争酶联免疫吸附分析(ELISA)法检测,5次重复测定的平均回收率为89.8%,相对标准偏差为4.8%。同时采用高效液相色谱(HPLC)法测定洗脱液,与ELISA法测定结果基本一致。