Production and specificity of polyclonal antibodies against soluble proteins from the arbuscular mycorrhizal fungus Glomus intraradices

Rabbit polyclonal antibodies were produced against a soluble protein fraction from a vesicle and spore mixture of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices. The protocol for isolation of vesicles and spores from plant roots was optimized to minimize debris contamination. Protein extract purification and preparation for immunization was adapted to increase protein content and immunogenicity. Active antisera were produced starting from the second boost immunization. Antibodies obtained were specific for surface antigens of AMF and revealed different patterns of soluble protein antigens in G. intraradices, G. constrictum and an unidentified Glomus species. Accepted: 6 December 2000     

Antibodies against synthetic peptides used to determine the topology and site of glycosylation of the cGMP-gated channel from bovine rod photoreceptors.

Peptides corresponding to amino acids 321-339 (peptide GS21) and 416-431 (peptide GS31) of the cGMP-gated channel from bovine rod photoreceptors were synthesized and used as antigens for the preparation of polyclonal antibodies. After affinity purification, both antipeptide antibodies were found to bind specifically to the channel protein after Western blotting, but only the antibody against GS21 gave satisfactory results on enzyme-linked immunosorbent assay and electron microscopy. Using immunocytochemistry, we were able to localize amino acids 321-339 to the extracellular side of the rod photoreceptor plasma membrane. By synthesizing heptapeptides corresponding to amino acids 324-330 (peptide GS2s) and 420-426 (peptide GS3s), we were able to affinity purify antibodies specific for two N-glycosylation consensus sites in the channel protein. As assessed by Western blotting, antibodies against GS3s were found to bind to both the glycosylated and deglycosylated channel proteins, whereas antibodies against GS2s only bound to the channel protein after enzymatic deglycosylation. Together, these results allow the refinement of folding models for the cGMP-gated channel and implicate Asn-327 as being the sole site of N-glycosylation.     

抗欧亚活血丹凝集素特异性多克隆抗体的制备

欧亚活血丹外源凝集素(Gleheda)是分离自欧亚活血丹 (Glechoma hederacea) 叶片中的一种糖基化植物新蛋白. 如同其他糖基化蛋白,通过免疫学方法探测 Gleheda 的过程中通常受到一些不相干糖蛋白的妨碍,为此制定了抗 Gleheda 特异性多克隆抗体的纯化方案. 免疫血清蛋白经硫酸铵选择性沉淀后,分别以 Gleheda 和刺槐外源凝集蛋白 (RPA) 结合在 Sepharose 4B作为亲和配体,采用亲和层析法连续纯化 2 次,然后进一步采用离子交换层析 Q Fast Flow 提纯. 经每一步骤提纯得到的抗体组分对 Gleheda 的特异性,均同时采用双向免疫扩散检验和 Western blot 分析. 结果表明,以 Gleheda 为配体,亲和纯化制备得到的抗体组分对叶片粗提物中的许多植物 (糖) 蛋白仍然表现交叉反应. 为除去由植物糖蛋白中的聚糖所引起这些非特异性交叉反应抗体,接着以 RPA 为配体再次进行亲和纯化,Western blot 分析显示,抗体的特异性得到提高但并非除去了所有非特异性交叉反应的抗体. 最后进一步采用离子交换层析制备得到仅抗 Gleheda 蛋白的特异性抗体组分,此抗体组分适用于免疫探测研究. 该抗体纯化制备程序简易而高效,而且不需要昂贵的设备.     

树鼩IgG纯化鉴定及其多克隆抗体制备和检测

目的:比较两种抗体纯化方法在分离纯化树鼩IgG抗体的应用,制备抗IgG的多克隆抗体及检测。方法:采用两种商品化IgG抗体纯化试剂盒分离树鼩血清IgG抗体,采用SDS-PAGE和蛋白定量测定提纯IgG。以树鼩IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合皮下注射免疫兔,对分离血清进行多克隆抗体纯化及Western Blot检测及定量分析。结果:两种方法均能有效分离纯化树鼩IgG,在经过Montage PROSEP-A试剂纯化后的IgG在纯度和含量方面均优于Protein A/G Matrix试剂。通过纯化后的树鼩IgG免疫兔制备的抗IgG抗体能有效识别树鼩IgG。结论:纯化的树鼩IgG具有良好免疫原性,由此制备的抗体具有高度特异性。研究结果为利用树鼩作为实验动物提供了必要的实验基础。     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.     

Identification of two epitopes in the carboxyterminal 15 amino acids of the E1 glycoprotein of mouse hepatitis virus A59 by using hybrid proteins.

cDNA fragments coding for the carboxy terminus of the E1 envelope glycoprotein from mouse hepatitis virus A59, a coronavirus, were cloned into the bacterial expression vector pEX. Clones expressing E1 antigenic determinants were selected with a polyclonal anti-E1 antibody and used for immunization of rabbits and for affinity purification of existing polyclonal antisera. Immunofluorescence testing and immunoperoxidase labeling of coronavirus-infected cells showed that these reagents were monospecific for E1. In addition, by using hybrid proteins containing different lengths of the E1 carboxy terminus to affinity-purify a polyclonal antiserum against E1, we have been able to define two epitopes within the last 15 amino acid residues of the protein. These epitope-specific antibodies bind to E1 in Golgi and perinuclear membranes as well as to budding viruses; they do not, however, label the plasma membrane or the membranes of post-Golgi vesicles transporting virions to the cell surface.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.     

Fluorescent IgG fusion proteins made in E. coli

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.     

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。     

Expression and purification of SARS coronavirus proteins using SUMO-fusions

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.     

Preparation of monospecific antisera to IgA in laboratory animals using cascade immunization without preliminary isolation of the antigen

The proposed method of "cascade" immunization for the preparation of monospecific antisera to IgA of the experimental animals (mice, rats, guinea-pigs) requires no isolation and purification of an antigen, since it is based on using precipitation lines containing IgA. The subsequent steps of the method are the following: 1) preparation of a polyspecific antiserum against one of the secretions or IgA-containing serum fraction; 2) preparation of precipitation lines using this antiserum and some other secretions; 3) immunization of other rabbits with these precipitation lines to obtain monospecific anti-IgA antiserum. The antisera obtained do not require any additional absorption except for the removal of anti-IgA antibodies. The method can be used for the preparation of monospecific antisera to any other serum or secretion proteins.     

The transparent accessory reproductive gland secretes a polypeptide into the hemolymph of male Rhodnius prolixus

The transparent accessory reproductive gland of Rhodnius prolixus synthesizes and accumulates a variety of polypeptides. Ouchterlony immunodiffusion demonstrates that the hemolymph contains proteins which react with polyclonal antibodies against extracts of transparent accessory glands. Accessory glands and hemolymph contain a 170 kDa polypeptide with similar mobility on SDS-polyacrylamide gel electrophoresis. This polypeptide reacts with antibodies against extracts of accessory glands. Surgical removal of the accessory glands prevents the appearance of the 170 kDa polypeptide in the hemolymph. In vivo labeling of accessory gland proteins with a mixture of [¹⁴C]amino acids demonstrates that the newly synthesized TARG polypeptide appears in the hemolymph between days 2 and 3 after feeding. It is concluded that a specific polypeptide which is synthesized in the transparent accessory gland is exported to the hemolymph.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Purification of antibodies using the synthetic affinity ligand absorbent MAbsorbent A2P

A protocol for the purification of polyclonal antibodies from ovine serum using the synthetic protein A absorbent MAbsorbent A2P is described. Clarified serum is loaded directly onto the affinity column without prior adjustment and albumin and unwanted serum components are washed from the column using a sodium octanoate buffer before elution of bound antibodies. MAbsorbent A2P was shown to bind approximately 27 mg ml(-1) of polyclonal immunoglobulin under overloading conditions, with eluted IgG purities of >90% and minor levels of albumin (approximately 1%). The anticipated time required to complete the purification protocol is 6-7 h. Although the protocol is similar to methods utilized for antibody purification using chromatography with protein A derived from the cell wall of the microorganism Staphylococcus aureus or protein G from Streptococcus as the affinity ligands, affinity absorbents based on synthetic ligands offer a number of advantages to compounds derived from biological sources, in particular robustness, relatively low cost, ease of sanitization and, in principle, lack of biological contamination.     

Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells

Monoclonal antibodies against the highly conserved ubiquitous calcium- binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.     

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33?kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.