Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column

Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits’ antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.     

人copineV蛋白多克隆抗体的制备

目的：制备兔抗人copineV多克隆抗体。方法：将copineV N端423bp（626-1048bp）构建到原核表达载体pET28a（＋）,转化大肠杆菌BL21（DE3）,在IPTG诱导下进行蛋白表达;以镍柱纯化后的蛋白为抗原,与等体积佐剂混合后免疫家免3次;用ELISA和Western印迹检测抗血清,用（NH4）2SO4沉淀法初步纯化抗体。结果：表达并纯化了copineV N端蛋白,ELISA检测表明抗血清具有高亲和性,Western印迹检测表明抗体能特异性识别内源性和过表达的copineV。结论：制备了具有高亲和性和特异性的抗人copineV多克隆抗体。     

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.     

Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples

An immunoaffinity column (IAC) was developed by covalently coupling polyclonal antibodies against estrogenic bisphenols to CNBr-activated Sepharose 4B. The IAC showed high affinity for bisphenol A, while phenol was barely retained. Proteins in the sample matrix showed little nonspecific adsorption on the column. The best binding solvent for bisphenol A was found to be 0.01 mol l(-1) phosphate-buffered saline (PBS) and the optimal operating temperature was 4 degrees C. The bound bisphenol A could be quantitatively recovered by 1 ml of methanol-water (80:20) with an average recovery of 91.8% and a relative standard deviation of 7.1% (n=6). The immunoaffinity column has been successfully used for the isolation and purification of bisphenol A from serum samples.     

Improved clean-up method for the enkephalins in plasma using immunoaffinity chromatography

A rapid and selective method of sample clean-up using immunoaffinity chromatography (IAC) was developed to isolate enkephalins from plasma. The enkephalin antibodies were produced utilizing novel protein carriers. Two antibodies, LE4H8 and 33FC6, were selected because of their moderate binding affinity and different epitopes. Enkephalin-spiked plasma was loaded onto the immunoaffinity column and eluted with acidic pH buffer. The eluate was derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA-CN), and the enkephalins were separated using reversed-phase liquid chromatography (RPLC). IAC sample clean-up of enkephalin-spiked plasma was compared to the existing solid-phase extraction method. The limit of detection for IAC was 30 pmol. The recovery of the enkephalins from plasma was 90% with a variance ranging from 2 to 9%. The immunoaffinity column was used for approximately 70 samples without any deterioration in performance.     

禽白血病病毒p27基因在原核细胞的表达及生物学特性的初步分析

将禽白血病病毒(ALV)的p27基因克隆人表达性载体pET28a,在大肠杆菌以His Tag融合蛋白的形式获得了高效表达。以表达产物免疫家兔,制备了抗ALV p27的多克隆抗体,经亲和纯化后,用此抗体建立了对ALV抗原的双抗体夹心法ELISA,并对疑似病料进行了实验室诊断。检测结果与IDEXX的禽白血病抗原检测试剂盒符合率达到98．6％,证明表达产物保留了天然p27蛋白的相关抗原性。交叉试验和群特异性试验证明,此方法具有良好的特异件,并且可以检测出A、B、J亚群的禽白血病病毒p27抗原。用此ALV抗体和所建立的ELISA方法成功地进行了禽白血病病毒抗原的实验室诊断。     

微孔板蛋白质芯片技术应用于单克隆抗体分型研究

设计与构建了可用于单克隆抗体分型鉴定的微孔板蛋白质芯片,利用该芯片进行了12株单克隆抗体和2种多克隆抗体的分型鉴定,并与ELISA方法进行了对比。结果表明,蛋白质芯片方法对单克隆抗体和多克隆抗体进行鉴定的结果,与ELISA方法进行鉴定的结果一致;与ELISA方法相比,蛋白质芯片的方法降低了试剂与样品的用量,缩短了工作时间,提高了工作效率。对于高通量的单克隆抗体制备体系,单克隆抗体分型蛋白质芯片是一种敏感、快捷的分型鉴定工具。     

青杄FKBP12基因原核表达和多克隆抗体的制备

目的:制备青杄FKBP12基因的多克隆抗体,为进一步分析FKBP12的蛋白定位、表达等提供基础。方法:采用PCR方法扩增FKBP12基因得到其全长cDNA序列,并将其克隆至原核表达载体pET-48b中,转化入BL21菌株。经IPTG诱导,表达了分子量约为33kD的重组蛋白,SDS-PAGE和Western blotting检测鉴定表达产物。此蛋白经亲和纯化后,作为抗原注射新西兰兔,进行抗体制备。结果:成功获取了多克隆抗体,制备的FKBP12兔抗血清效价在1∶729 000以上,ELISA结果表明融合蛋白具良好的免疫原性。间接ELISA法检测纯化后抗体效价,表明纯化后抗FKBP蛋白兔多克隆抗体效价高,检测灵敏度为16ng/mL。结论:所制备的抗体能满足后续试验要求的效价值,为进一步研究提供基础。     

O-GlcNAcase抗原片段的选择、优化表达和多克隆抗体的制备

为了探讨O-GlcNAc修饰的生物学作用和相关疾病的发病机理,需制备高效、专一的O-GlcNAcase (OGA) 抗体。通过对人源OGA蛋白进行序列分析发现,氨基端1~350 aa片段 (sOGA) 抗原性和亲水性较强,将该片段构建至原核表达载体pET-28a,并在大肠杆菌Escherichia coli BL21(DE3) 中进行诱导表达,通过优化IPTG浓度 (0.05 mmol/L) 和诱导时间 (10 h) 获得了高可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析和分子筛层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小 (45 kDa) 和纯度 (95％以上)。以4-MU-O-GlcNAc为荧光底物,检测到sOGA的糖苷酶活性为106 nmol/(min·mg),表明该片段是OGA糖苷酶的活性区域。以此片段作为抗原免疫新西兰大白兔,以CNBr活化Sepharose 4B微珠纯化抗血清制备OGA特异性多克隆抗体。Western blotting和ELISA检测表明,该抗体可以特异识别含有OGA糖苷酶活性区域的多种变体,检测灵敏度为0.11 ng/mL (效价为1∶80 000),可应用于O-GlcNAcase生物功能研究。     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

Development and characterization of a chitosan-supported immunoaffinity chromatography column for the selective extraction of methandrostenolone from food and feed samples

The development of a chitosan-supported immunoaffinity chromatography (IAC) column and its application to the selective extraction of methandrostenolone (MA) from food and feed samples were described in this paper. Using hybridoma technique, a monoclonal antibody (mAb) against MA was produced. The IAC column was prepared by coupling the produced antibody with crosslinked chitosan. Scanning electron microscopy and IR spectroscopy was used to characterize the chitosan crosslinking and antibody coupling. 2% and 90% methanol were respectively selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was 1790 ng/mL gel. The extraction recoveries of the column for MA at three different spiked concentrations ranged from 83.7 to 98.5%. After 2 cycles of usage, the column capacity and extraction recovery still remained 84.6% and 80.5%. To further verify the effect of matrix on the IAC cleanup, MA-fortified food and feed samples were extracted using the prepared IAC column, and MA recovery rates were found to be 86.2% and 70.4%, respectively.     

Selective sample cleanup by immunoaffinity chromatography for determination of fenvalerate in vegetables

This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000 ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC-ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7-96.5% and 2.5-5.2%, respectively, and the calculated limit of detection of the whole method was 0.008-0.012 ng g(-1).     

A direct dot-enzyme immunoassay to detect human ovulation.

This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.     

A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.     

NPR1多肽抗体的制备和应用

根据NCBI GenBank中报道的NPR1一级结构信息,采用Blastn、Blastx、ExPASy和Protean等软件进行序列同源性和抗原性指数分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和LC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达88%、目的多肽分子量为1.92234 kD。采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原Pep-KLH,并将其免疫新西兰大白兔以获得抗血清和多克隆抗体,采用ELISA和Western blotting测定其效价和特异性,经ELISA检测表明抗血清和多克隆抗体可与Pep发生特异性免疫反应,经Western blotting试验表明抗血清和多克隆抗体可识别烟草叶片特异性条带,其相对分子量为65 kD,与预测分子量相符,表明利用该方法制备的NPR1多肽抗体具有较高特异性和灵敏度。     

Purification of human chorionic gonadotrophin from urine by membrane filtration affinity chromatography with a positively charged membrane.

A new method of affinity chromatography, termed membrane filtration affinity chromatography (MFAC), has been developed and applied to purify HCG from urine. By filtrating urine through ZBM (HCG in urine would bind to the antibody on ZBM) and by dissociating the HCG from the antibody on ZBM in purified form, we developed the MFAC and purified HCG from urine of pregnant women by MFAC. The purified HCG showed a single band in polyacrylamide gel electrophoresis. ZBM (1 cm(2)) could harvest 90.3 microg HCG, which showed immunoactivity of 8554 IU/mg. The rate of recovery was 87%. CONCLUSION: MFAC with ZBM is an effective method, which is much easier and cheaper than conventional affinity chromatography for purification of proteins from solution, especially from highly diluted solution.     

肺炎链球菌SpxA蛋白的原核表达、纯化及多克隆抗体的制备

目的 构建肺炎链球菌SpxA蛋白的原核表达系统,制备其多克隆抗体.方法 设计引物,利用PCR技术扩增肺炎链球菌D39菌株的spxA基因,并插入表达载体pET-28a（＋）内,测序鉴定.重组质粒转化至大肠埃希菌BL21（DE3）中,以IPTG诱导表达含6个组氨酸标签的SpxA重组蛋白,经Ni-NTA亲和层析柱纯化后,以其为抗原免疫BALB/c小鼠制备多克隆抗体.用ELISA及Western印迹方法分别检测多克隆抗体的效价及特异性.结果 从大肠埃希菌中诱导出高表达的SpxA重组蛋白,纯化后免疫小鼠获得抗血清,ELISA测定其效价可达1：2 560 000以上,Western印迹结果显示其能特异性地作用于肺炎链球菌SpxA.结论 成功构建了pET-28a（＋）-spxA原核表达质粒,获得了高纯度的目的 蛋白和高滴度、高特异性的多克隆抗体.     

Immunodetection of pneumolysin in human urine by ELISA

An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.