小鼠frataxin抗体的制备及应用

弗里德赖希共济失调(Friedreich ataxia,FRDA)是一种常染色体隐性遗传性疾病,由frataxin(FXN)第一个内含子中GAA重复扩增导致其表达量减少所致。FXN是一种线粒体蛋白,可以调节细胞中铁的代谢,参与铁硫簇和血红素的合成,去除氧化应激等。前期研究发现FRDA病人受累组织有特异性表达的FXN亚型蛋白。为了检测小鼠不同组织中Fxn亚型蛋白的表达,首先要获得具有高度特异性和灵敏性的小鼠Fxn抗体。利用PCR技术扩增小鼠Fxn基因,通过酶切、连接、转化等常规分子克隆方法构建重组原核表达质粒pET24(+)-mFxn,经转化大肠杆菌BL21(DE3),表达可溶性含有his6标记的融合蛋白。该蛋白不含Fxn氨基端77个氨基酸的信号肽,含有130个氨基酸,理论分子量为14.38 kDa。表达的蛋白经Ni-NTA柱和连续梯度离心纯化,获得目的蛋白作为抗原;免疫新西兰大白兔制备抗血清并用硫酸铵沉淀初步纯化得到多克隆抗体。经Western blotting和免疫荧光分析测试,所获得的抗体能够特异性识别细胞内源Fxn,也可应用于组织的免疫沉淀和免疫荧光。这是首次报道利用鼠源Fxn作为免疫原制备的具有高度特异性和灵敏性的Fxn抗体,为深入研究小鼠frataxin亚型蛋白的存在和功能奠定了基础。     

Production and characterization of polyclonal antibody against recombinant ORF 049L of rock bream (Oplegnathus fasciatus) iridovirus

Rock bream iridovirus (RBIV) is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. The structure of the isolated RBIV was observed by an electron microscope, and the virus particles were icosahedral and 120–130 nm in diameter. From the complete genomic DNA sequence of RBIV, the protein encoded in ORF 049L (RBIV-049L) was selected and the property of protein was evaluated with the transmembrane sequence TMHMM 2.0 tool. The ORF 049L gene of RBIV (RBIV-049L) was cloned into pGEX-4T-1 expression vector. The recombinant RBIV-049L was overexpressed in Escherichia coli BL21 (DE3) as a fusion protein (GST-049L, 42 kDa) with a glutathione S-transferase. Antiserum against this recombinant GST-049L protein was prepared in mouse. Dot blot analysis was carried out to identify the reaction abilities and sensitivity of anti-RBIV-049L polyclonal antibody to RBIV-infected rock bream with enzyme linked immunosorbent assay (ELISA) and one-step PCR. These novel RBIV-049L protein and anti-RBIV-049L polyclonal antibody will facilitate the development of more specific and standardized diagnostic techniques.     

Characterization of a rabbit polyclonal antibody against threonine-AMPylation

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.     

Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology

Metallothionein 2A(MT2A)is a small stress response protein that can be induced by exposureto toxic metals.It is highly expressed in breast cancer cells.In this study,the cDNA encoding the humanMT2A protein was expressed as glutathione S-transferase(GST)fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separatedby electrophoresis,the recombinant protein was visualized by Coomassie blue staining and the 33 kDarecombinant GST-MT2A fusion protein band was cut out from the gel.The gel slice was minced and used togenerate polyclonal antisera.Immunization of rabbit against MT2A protein allowed the production of hightiter polyclonal antiserum.This new polyclonal antibody recognized recombinant MT2A protein in Westernblot analysis.This low-cost antibody will be useful for detection in various immuno-assays.     

梅花鹿重组Galectin-1的表达及多克隆抗体的制备与鉴定

半乳糖凝集素-1（Galectin-1）是最先被报道的哺乳动物半乳糖凝集素,存在于多种组织和细胞内,参与细胞的粘附、增殖、凋亡和炎症反应等多种生理病理过程,并且与免疫系统的调节和肿瘤的发生发展密切相关。鹿茸是哺乳动物中罕见的能够周期性的脱落和再生的附属器官,作为研究哺乳动物器官再生的新模型受到关注。鹿茸再生是一个基于干细胞的过程,定位于角柄骨膜的干细胞是鹿茸再生的基础,Galectin-1在角柄骨膜细胞（pedicle periosteum cell, PPC）中高度表达,提示其在鹿茸再生中发挥着重要的作用。由于尚没有商用的鹿Galectin-1蛋白及其抗体,为进一步研究Galectin-1在鹿茸再生中的生物学功能,需要制备相应的蛋白和抗体,本实验将梅花鹿Galectin-1基因与pET28a连接,并将重组质粒pET28a-Galectin-1转入大肠杆菌（Escherichia coli）BL21(DE3)中诱导表达。用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔子制备多克隆抗体。酶联免疫吸附（enzyme-linked immunosorbent assay, ELISA）法检测抗体效价、Western blot 检测抗体特异性、细胞免疫荧光检测Galectin-1在角柄骨膜细胞中的表达情况。结果表明,本实验成功诱导重组原核表达载体pET28a-Galectin-1在BL21(DE3)中表达,通过Ni纯化获得融合蛋白。ELISA结果显示,抗体效价达到1:64000,Western blot结果表明该抗体特异性良好,细胞免疫荧光显示Galectin-1在PPC全细胞中表达。本实验获得了纯化的鹿Galectin-1蛋白和特异性较好的多克隆抗体,为揭示Galectin-1在鹿茸再生调控中的作用提供了重要的实验材料。     

Production and application of LPA polyclonal antibody

By using colloidal gold as a hapten carrier, a kind of antibody against lysophosphatidic acid (LPA) was developed and used to successfully detect 500 ng/mL LPA in dot immunogold filtration assay. Such application of the LPA antibody could offer us a way to diagnose ovarian cancer at its early stage.     

Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.     

Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid

Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO(3)-3Gal-ceramide) and seminolipid (SO(3)-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.     

Schistosoma mansoni: detection of circulating antigens in murine schistosomiasis by antigen-capture sandwich ELISA using a monoclonal antibody

A monoclonal antibody (MAb) 5H11/B1 that reacts with a repeating epitope on an excretory-secretory (E + S) antigen of adult worms of Schistosoma mansoni was used in the detection of circulating antigen (CA) in sera from S. mansoni-infected mice using an antigen-capture sandwich ELISA. Trichloroacetic acid (TCA) pretreatment of sera from mice infected for 8 or 16 weeks precipitated immune complexes and/or dissociated CA and allowed its detection. Sera obtained 8 weeks after infection contained high levels of CA. Upon treatment with praziquantel (100 mg/kg body wt), this level was significantly less within 1 week. A strong correlation was found between the worm count determined by perfusion and the level of antigenemia detected by the 5H11/B1 assay in light and heavy infection (r = 0.80). Based on the results of both TCA pretreatment and sodium periodate treatment, the 5H11/B1 sandwich ELISA assay detects a repeating carbohydrate epitope on an E + S antigen. This system appears to be a sensitive assay for the detection of schistosomal antigenemia in murine schistosomiasis. Studies on the detection of antigenemia in human schistosomiasis using this assay are in progress.     

Production of a monoclonal antibody directed against the recombinant SEL1L protein

SEL1L, highly similar to the C elegans sel-1 gene, is a recently cloned human gene whose function is under investigation. SEL1L is differentially expressed in tumors and normal tissues and seems to play a role in tumor growth and aggressiveness. We used the recombinant N-terminus of the SEL1L protein to immunize a Balb/c mouse and produce a monoclonal antibody. A hybridoma secreting an antibody specifically reacting on the SEL1L recombinant fragment was selected. This monoclonal antibody, named MSel1, recognizes the SEL1L protein by Western blotting, immunofluorescence and immunohistochemistry on normal and tumor cells. MSel1 is able to recognize SEL1L even on archival tumor specimens and is therefore particularly appropriate to study SEL1L involvement in tumor progression.     

Production and characterization of polyclonal antibody against Arabidopsis GIGANTEA,a circadian clock controlled flowering time regulator

Arabidopsis GIGANTEA (GI) is encoded by a single gene and highly conserved among vascular plants and its mutants display pleiotropic phenotypes involved in diverse biological processes such as light signaling, circadian clock, and sucrose metabolism as well as abiotic stress responses. However, molecular mechanisms of GI are largely unknown due to the lack of useful antibody. To date, the epitope tags have been widely used to detect GI in plants, but it needs to generate the transgenic plants which take a few months. Here, we produced polyclonal α-GI antibody using truncated variants of GI having amino-terminal (1–858 aa) and carboxyl-terminal (920–1173) regions as antigens. Both recombinant His-GI^1-858 and His-GI^920–1173 proteins were individually and successfully expressed in E. coli and immunized into rabbit. Anti-serum was purified by antigenspecific affinity purification method using both recombinant His-GI^1–858 and His-GI^920–1173 proteins. Purified polyclonal α-GI antibody not only detected endogenous GI proteins in wild-type Arabidopsis plants, but also reenacted its diel oscillations. Furthermore, the antibody showed cross-reactivity with the GI orthologs in other plants such as Chinese cabbage, rape and tomato. Our polyclonal GI antibody could help to determine the molecular mechanisms of GI involved in largely unknown pleiotropic responses in plants.     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

Production and validation of a polyclonal serum against bovine FSH receptor

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.     

Production of target-specific recombinant human polyclonal antibodies in mammalian cells

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.     

Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55–75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG₁ and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.     

Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification

AIMS: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. METHODS AND RESULTS: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae. CONCLUSIONS: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: This MAb could be used to detect Aer. hydrophila in human stool samples.