Direct and homogeneous immunoassay for IgG analyses

A concept involving the use of antibody conjugated with reporter molecules for direct sensing of subtle changes in the local electrostatic environment of an antigen-antibody complex due to the antigen binding is presented. The proposed direct sensing mechanism is studied using the protein A-immunoglobulin G (IgG) complex as the model system. The change in the local electrostatic potential and pH around the protein A-IgG complex is observed by measuring the fluorescence intensity of fluorescein molecules conjugated with protein A before and after the introduction of IgG. Factors affecting the efficiency of our direct sensing mechanism including the conjugation ratio of fluorescein per protein A molecule, Solution conditions, and the amount of protein A-fluorescein conjugate used in the experiment are investigated. The IgG(2A) concentrations in the cell supernatants are analyzed by using our direct sensing method and then compared with the results obtained from enzyme-linked immunosorbent assay (ELISA). The comparisons between our homogeneous assay and heterogeneous ELISA in the analysis time, analysis procedures, cost, and sensitivity are reported and discussed. (c) 1992 John Wiley & Sons, Inc.     

Immunochemical characterization of an IgG-binding protein of Streptococcus suis

Several bacterial species express surface proteins with affinity for the constant region (F_c) of immunoglobulin (Ig) of different animal species. Previous studies from our group have reported the presence of an IgG-binding protein in various serotypes of Streptococcus suis . This molecule was also shown to bind in a non-immune fashion chicken IgY and to our knowledge this characteristic is unique. In the present study, by dot-blotting, we showed that the native protein, obtained by affinity chromatography, reacted more strongly with IgG from various animal species than the denatured material. Using a competitive enzyme-linked immunosorbent assay the affinity of the native 60-kDa protein (previously identified as a 52-kDa protein) towards IgG of various animal species was compared to pig IgG. Bovine, goat and human IgG were able to compete effectively with pig IgG whereas chicken IgY constituted a poor competitor. Peptide mapping analysis using denatured protein indicated that pig and bovine IgG recognized the same proteolytic fragment whereas chicken IgY did not. The smallest proteolytic fragment that retained the binding activity towards the IgG of the different animal species tested had a molecular mass of approximately 40 kDa. Fragments with M _r<40 kDa showed specific binding activities. That is, the smallest fragment binding pig and bovine IgG had a M _r of 30 kDa whereas for goat and human IgG a fragment of less than 16 kDa still showed binding activity. Finally, we observed that antisera raised against a heat-shock protein of Pseudomonas aeruginosa reacted with the 60-kDa S. suis protein indicating that the S. suis 60-kDa protein is a member of the 60-kDa hsp family that possesses the characteristic of binding in a non-immune way mammalian IgG and chicken IgY.     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.     

CTLA4Ig融合蛋白在CHO细胞中的表达

CTLA4Ig是人CTLA4胞外区与人免疫球蛋白铰链区、CH2区、CH3区组成的融合蛋白,可以与B7结合,通过阻断B7与CD28的结合,从而阻断B7介导的T细胞活化必需的共刺激信号,可作为免疫抑制剂用于器官移植。将CTLA4Ig融合分子克隆到真核表达载体pCI-dhfr,并用脂质体方法转染到COS7和CHO-dhfr-细胞中,用氨甲喋呤筛选转染的CHO-dhfr-细胞。用RT-PCR、ELISA、细胞免疫荧光染色和Western-blot鉴定重组蛋白的表达。采用A蛋白纯化重组蛋白。     

Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris

We constructed a fusion protein ZZ-EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ-EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ-EGFP/l was obtained. The resulting ZZ-EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ-EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ-EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Abstract Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG₁, IgG₃ and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

Generation and application of a fluorescein-specific single chain antibody

A recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.     

hsRAGE-IgG Fc融合蛋白的表达及其生物学活性

将编码人可溶性晚期糖基化终产物受体-免疫球蛋白G Fc段(hsRAGE-IgG Fc)融合蛋白的DNA片段克隆到大肠杆菌表达载体pET-20b中,构建重组表达质粒,转化大肠杆菌Rosetta(DE3)并表达。SDS-PAGE分析表明其表达形式为包涵体,相对分子质量约为66kDa,表达量占菌体总蛋白的38.4%。经复性后,获得纯度为96.6%的融合蛋白,得率约为29.5mg/L。经Western印迹法鉴定,该融合蛋白可与sRAGE抗体产生阳性反应。同时,hsRAGE-IgG Fc融合蛋白可以显著抑制晚期糖基化终产物(AGE)引起的ECV-304细胞NF-κB p65表达的上调,其活性与hsRAGE相似。     

Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase.

While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis.     

尿激酶抗人交联纤维蛋白-D-二聚体单抗化学偶合体的制备

利用双功能试剂Ｎ－琥珀酰亚胺－３（２－二硫吡啶）丙酸酯（ＳＰＤＰ）作交联剂,合成了尿激酶（ＵＫ）－抗人交联纤维蛋白降解物Ｄ－二聚体单抗（ＭＡ－ＨＩＤ１）化学偶合体（ＵＫＭＡ－ＨＩＤ１）,并用苯甲脒－Ｓｅｐｈａｒｏｓｅ６Ｂ及人交联纤维蛋白降解物Ｄ－二聚体－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,获得偶合体产物．ＳＤＳ－ＰＡＧＥ呈现一条带,其分子量约为２０００００．纤维蛋白平板法测活结果显示,偶合体中酶比活为５３０００ＩＵ／ｍｇ尿激酶蛋白,与偶联前的５４３００ＩＵ／ｍｇ蛋白相仿．ＥＬＩＳＡ测试显示,偶合体对人交联纤维蛋白降解物Ｄ－二聚体有免疫反应性,并且与偶联前的抗Ｄ－二聚体单抗对此抗原的反应性相当     

Vsig4 和免疫球蛋白Fc段融合蛋白的真核表达纯化

目的:本项目将通过构建中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)真核表达系统获取小鼠Vsig4膜外端和免疫球蛋白Ig G3a-Fc段的融合蛋白,鉴定Vsig4-Fc和Vsig4纳米抗体的相互作用。方法:采用重合延伸PCR法融合小鼠Ig G3a-Fc和Vsig4胞外段的基因序列,将该融合基因插入真核表达载体中并转染CHO细胞。Western blotting鉴定转染细胞上清中的目标蛋白,通过连续两次亚克隆筛选,获得高表达小鼠Vsig4-Fc融合蛋白的单克隆,之后大量培养增殖转染细胞并收集细胞培养上清,选择Protein A柱纯化方法纯化Vsig4-Fc蛋白,最后经ELISA法鉴定Vsig4-Fc和纳米抗体的结合能力。结果:在CHO细胞中成功构建了小鼠Vsig4-Fc真核表达稳转系,并且在真核表达体系中获得可表达15 mg/L的双分子结构Vsig4-Fc的稳定转染细胞系。经鉴定小鼠Vsig4-Fc融合蛋白能与Vsig4纳米抗体结合。结论:重合延伸PCR法使得Vsig4和Fc基因片段的融合更为高效,两次亚克隆筛选优势细胞系大幅提高了真核蛋白的表达量,为进一步研究Vsig4的生物学功能奠定重要基础。     

Thermodynamic Stability and Functional Activity of Tumor-Associated Antibodies

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2–CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.     

Detection of Protein A Produced by Staphylococcus aureus with a Fiber-optic-based Biosensor

Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.     

Application of a trifunctional reactive linker for the construction of antibody-drug hybrid conjugates

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin alpha(4)beta(1)-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin alpha(4)beta(1). The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.     

Dissection of the protein G B1 domain binding site for human IgG Fc fragment.

The contribution to the free energy of binding of each of the residues forming the binding site for a human IgG Fc fragment on the surface of the B1 domain of protein G was determined by alanine-scanning mutagenesis. The interface between these two proteins is atypical in that it is smaller than usual, polar in character, and involves two well-defined "knobs-into-holes" interactions. The bulk of the free energy of binding is contributed by three central residues, which make hydrogen bonds across the interface. Of these, the most critical interaction is formed by Glu27, which acts as a charged knob on the surface of the B1 domain, inserting into a polar hole on the Fc fragment. A single alanine mutation of this residue virtually abolishes stable complex formation. Formation of a stable interface between these two proteins is therefore dominated by a small, polar "hot spot."     

Fluorescent biorecognition of gold nanoparticle-IgG conjugates self-assembled on E-beam patterns

A new concept for line patterning of immunoglobulin G (IgG) in nanometer scale using gold nanoparticles (AuNPs) self-assembled in a nanochannel written with an electron beam is proposed and demonstrated. AuNPs are synthesized by reducing KAuCl4 with NaBH4, producing AuNPs 40-70 nm in size, where Cl- ions are capping AuNPs thus making them negatively charged and subsequently stabilized. IgG is conjugated to these AuNPs by simple adsorption. Single or multiple nanochannels are written with an electron beam using a scanning electron microscope (SEM) in a layer of poly(methyl methacrylate) (PMMA), which is spin-coated on a p-doped Si wafer. AuNPs bind into the etched nanochannel where the Si surface is exposed, while the relatively hydrophobic PMMA area repels the particles. The particles with a diameter larger than the channel width are not able to go inside of it. Anti-IgG, conjugated with fluorescein isothiocyanate (FITC), is then exposed to the patterned surface, binding specifically to the IgG-AuNP conjugates within the line patterns. These antibody-antigen bindings can be visualized with a fluorescent microscope, showing the fluorescent signal only along with the nanometer line pattern. These initial steps will lead to the formation of complex protein nanoarrays, based on the size-dependent self-assembly of AuNPs within variously sized nanopatterns.     

抗人卵巢癌单域抗体V_H 基因的克隆、表达及放射免疫显像

 为研究小分子抗体作为导向载体的作用 ,制备抗人卵巢癌单克隆抗体COC1 83B2 单域抗体(重链可变区 ,VH) ,并进行放射免疫显像 (RII) .利用PCR技术从COC1 83B2 杂交瘤细胞中扩增COC1 83B2 的VH 基因片段 ,并将其克隆入高效表达载体pTHA90中 .重组子转化大肠杆菌TOP10 ,IPTG诱导表达VH 融合蛋白 ,经改进氯化亚锡法进行锝 ( 99mTc)的标记 ,在卵巢癌裸鼠皮下瘤模型上进行RII.结果表明 :( 1 )VH 融合蛋白以包涵体形式获高效表达 ;( 2 )改进氯化亚锡法成功进行VH 融合蛋白99mTc的标记 ,标记率达 96%以上 ;( 3)用99mTc标记VH 融合蛋白进行RII,肿瘤显像早 ,图像清晰 .说明99mTc可用于小分子抗体标记 ,应用单域抗体VH 可改善RII     

Simultaneous interaction of monoclonal antibody-targeted liposomes with two receptors on K562 cells

We have investigated the interaction of targeted liposomes with human erythrocytes, and K562 cells, a human leukemic line which expresses both glycophorin A and Fc receptors. Liposomes conjugated to monoclonal anti-human glycophorin A bind to human erythrocytes in 80-fold greater amounts than liposomes conjugated to a non-specific monoclonal antibody. Binding is inhibited by soluble anti-glycophorin but not by its Fab fragment. In contrast, binding of antibody-conjugated liposomes to K562 cells is very high irrespective of the specificity of the antibody. Liposomes conjugated to a nonspecific monoclonal antibody interact with K562 cells via an Fc receptor, and binding is inhibited by soluble human IgG. Liposomes conjugated to anti-human glycophorin A interact with K562 cells via an Fc receptor and glycophorin A. Binding is not inhibited by either human IgG or anti-glycophorin Fab alone. Binding is only partially inhibited by anti-glycophorin, or by human IgG in the presence of anti-glycophorin Fab, and completely inhibited only by human IgG in the presence of anti-glycophorin. Simultaneous binding of targeted liposomes to two cell membrane antigens is therefore partially resistant to inhibition by single soluble ligands even when they are present in large excess. We conclude that simultaneous binding to more than one receptor may be of considerable advantage for in vivo applications of targeted liposomes.     

A smart membrane based on an antigen-responsive hydrogel

Hydrogel membranes have been fabricated that incorporate antibody/antigen moieties. The permeability of large solutes through these membranes is dependent on the presence of soluble antigen that can compete with the internal interactions between antibody and antigen leading to an increase in gel mesh size. Specifically, the membrane's structure is based on a dextran backbone grafted with a fluorescein isothiocyanate (FITC) antigen and a sheep anti-FITC IgG antibody. The backbone is covalently cross-linked by conjugated divinyl sulfone (DVS) groups. The gel structure is additionally stabilized by affinity crosslinks formed by biospecific interactions between the bound IgG and FITC. FTIR spectra of the gel are consistent with formation of covalent bonds between cysteine groups in the IgG and DVS groups in the dextran. Results obtained using isothermal titration calorimetry (ITC) confirmed the competitive interaction binding between IgG-FITC-dextran and free sodium fluorescein at pH 5.0. Scanning electron microscopy (SEM) of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate with free fluorescein-dependent structural changes in the gel. Three-dimensional images obtained from confocal laser scanning microscopy show that these changes occur throughout the gel and indicate that SEM results are not artifacts of sample preparation. The permeability of these gels, as shown by blue-dextran (12 kDa) diffusion, increases in response to the presence of free fluorescein of the external medium, which causes competitive displacement of the affinity cross-links. Sequential addition and removal of sodium fluorescein showed that these permeability changes are reversible.