Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin–avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

The Synthesis of the Immobilization Antigen in Paramecium aurelia: In vitro Localization of Antigen in Ribosomal Cell Fractions

SYNOPSIS. The distribution of a cell surface protein, the immobilization antigen, has been studied in homogenates of Paramecium aurelia stock 168, syngen 1. Two fractions, a 9,000 g supernatant fluid and a 25,000 g supernatant fluid, have been investigated for their ability to bind ¹²⁵I-labelled antibodies specific against immobilization antigen. These 2 fractions have been characterized by electron microscopy. Antigen was detected in a pure microsome fraction and in the polysome fraction. Sucrose gradient analysis of the polysome fractions indicates that antigen is associated specifically with polysomes of 228S and 311S, the latter being located primarily in the microsome fraction. These results correlate well with the structure of the immobilization antigen proposed by Steers. A possible mechanism for the synthesis of this protein is outlined.     

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.     

Polymer nanoparticles covered with phosphorylcholine groups and immobilized with antibody for high-affinity separation of proteins

Novel polymer nanoparticles were prepared for the selective capture of a specific protein from a mixture with high effectiveness. The nanoparticle surface was covered with hydrophilic phosphorylcholine groups and active ester groups for easy immobilization of antibodies. Phospholipid polymers (PMBN) composed of 2-methacryloyloxyethyl phosphorylcholine, n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate, were synthesized for the surface modification of poly( l-lactic acid) nanoparticles. Surface analysis of the nanoparticles using laser-Doppler electrophoresis and X-ray photoelectron spectroscopy revealed that the surface of nanoparticles was covered with PMBN. Protein adsorption was evaluated with regard to the nonspecific adsorption on the nanoparticles that was effectively suppressed by the phosphorylcholine groups. The immobilization of antibodies on nanoparticles was carried out under physiological conditions to ensure specific binding of antigens. The antibody immobilized on the nanoparticles exhibited high activity and strong affinity for the antigen similar to that exhibited by an antibody in a solution. The selective binding of a specific protein as an antigen from a protein mixture was relatively high compared to that observed with conventional antibody-immobilized polymer nanoparticles. In conclusion, nanoparticles having both phosphorylcholine and active ester groups for antibody immobilization have strong potential for use in highly selective separation based on the biological affinities between biomolecules.     

Optimization of methods for immobilizing antibodies against the carcino-embryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of the immobilized antibodies with the antigen

To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.     

Characteristics of Bioreactors Made with Urease and Nad Glycohydrolase Reversibly Bound to Immobilized Antibodies

The characteristics of urease and NAD glycohydrolase, immobilized on different matrices through linkage with polyclonal antibodies, were studied for their use as enzyme reactors. The stability of the derivatives was enhanced by immobilization but some leakage of activity was found, due to the equilibrium between the free and bound enzyme. The release of the enzyme to the solution depends on the affinity constant of the antibodies and the concentration of the substrate circulating in the reactor. On the other hand the main advantage of this easy method of immobilization is the low cost replacement of the inactive enzyme with fresh catalyst.     

Immobilization of the glutathione by the complementary coordination binding

A simple method for immobilization of biologically imporant molecules with many functional fragments by selective binding of their thiogroups with the surface caroxyl groups by cadmium ions was proposed. Biofunctional properties of these structures were studied by surface plasmon resonance method on the model of the glutathione (GSH), which was immobilized by means of mixed (a:b form 1:100 o 1:700) thiol monolayers with terminal groups of the methyl/hydroxyl (b) and carboxyl (a) type. The maintenance of the biofunctional conformation ofglutathione-S-transferase (GST) after its interaction with GSH was checked by the use of specific anti-GST antibodies. It was shown that CH3 matrix has considerable non-specific binding and is not suitable for the formation of the biofunctional GST layer. At the same time OH-based structures demonstrate specific interaction GST-anti-GST, the stoichiometry of which corresponds to the bidentate binding. Considered simple method of the immobilization can be used to create the functional surface architectures in the analyticasl biochemistry and chemical analysis.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

Comparative study of horseradish peroxidase immobilization on modification of a gold surface using a surface plasmon resonance method

Horseradish peroxidase is immobilized by a periodate method on the gold surfaces previously modified with 16-mercapto-hexadecanoic acid or with hydrogen disulfide and soybean trypsin inhibitor. The effect of gold surface modification conditions on the immobilization of the enzyme as well as on the properties of the immobilized glycoprotein are studied using surface plasmon resonance technique. Restoration of the ability to bind specific antibodies is demonstrated for the immobilized enzyme. The low level of non-specific antibody binding to the immobilized glycoprotein is also shown.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

A high-capacity streptavidin-coated microtitration plate

A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). The maximum immobilization capacity of the modified plate was up to 2.5 times higher than that of the control plate. The higher binding capacity was especially emphasized with small-size molecules. The modified high capacity plate increased the linear ranges of the immunoassays and thus delayed the high-dose hook effect. At high antigen concentrations the signal increased up to 59%, and at the conventional linear ranges of the assays, the increase was up to 29%. We conclude that the modified coating method will be valuable for the future miniaturized systems, where high immobilization capacity is needed at limited areas.     

Stabilization of yeast invertase by insoluble immunocomplex formation using unfractionated glycosyl-specific and peptide specific polyclonal antiinvertase antibodies

Summary A strategy for the production of non-inhibitory polyclonal antisera for the immobilization of glycoenzymes is described. Insoluble complexes of invertase prepared using antiinvertase glycosyl antisera were more resistant to heat and urea induced denaturation than those prepared using antiinvertase antisera from which the glycosyl specific antibodies were removed. Unfractionated antiinvertase antibodies however gave most stable immunocomplexes with invertase.     

Immobilization of glutathione by complementary coordination binding

A simple method was proposed for the immobilization of biologically important molecules consisting of many functional fragments, by means of the selective binding of their thiol groups to the surface carboxyl groups with participation of cadmium ions. Biofunctional properties of these structures were studied by the surface plasmon resonance method, with the example of glutathione (GSH), which was immobilized onto mixing thiol monolayers containing terminal groups of the carboxyl (a) and methyl/hydroxyl (b) types (a: b, from 1: 100 to 1: 700). The maintenance of the biofunctional conformation of glutathione-S-transferase (GST) after its interaction with GSH was monitored using specific anti-GST antibodies. The CH3 matrix was shown to be capable of considerable nonspecific binding and not suitable to form the biofunctional GST layer. At the same time, the OH-based structures demonstrated the specific GST-anti-GST interaction, with stoichiometry corresponding to the bidentate binding. The above simple method of the immobilization can be used to create functional surface architectures in analytical biochemistry and chemical analysis.     

抗体固定化方法研究进展

在免疫分析和生物芯片中,抗原-抗体特异性结合被广泛应用,其中抗体的固定化是研发高效诊断和分离工具的关键环节。生物分子工程、材料化学与交联剂化学的进步极大地促进了抗体固定化技术的发展。 抗体可以通过物理吸附、共价偶联和亲和相互作用固定到不同类型的固相表面。 抗体固定化的目标是以一种正确的空间取向将抗体固定到固相表面,在完全保留抗体构象和活性的同时最大化抗原的结合能力,这对固相化抗体的分析性能至关重要。 对固定抗体到固相载体表面的各种最新方法进行了阐述,包括物理吸附法,通过羧基、氨基、巯基、糖基和点击化学的共价结合法以及基于生物亲和作用的固定法,并对固定化抗体的表征方法进行了归纳,最后对抗体固定化方法的发展方向进行了展望。     

Direct ring conjugation of catecholamines and their immunological interactions

Catecholamine derivatives were synthesized with potential applications as coating antigens in biosensors or in the raising of specific antibodies. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine, and epinephrine that attach carboxylic acid functionalities directly to the aromatic ring via an easily incremented linker chain were synthesized by an electrochemical method. These derivatives were purified by convenient ion-exchange chromatography, exact positions of conjugation determined by NMR, and a dopamine derivative immobilized in situ in a BIAcore surface plasmon resonance (SPR) biosensor and its antibody binding studied in comparison with immobilization via the catecholamine primary amine. Binding of an antibody raised to an amine-conjugated protein conjugate showed clear distinction between conjugations at different positions on the catecholamine, illustrating the importance of rational conjugate design in immunosensing of the catecholamines.     

Immobilization of murine lymphocytes by gel entrapment

Summary Murine non-transformed lymphocytes were immobilized by alginate and agarose entrapment. After lipopolysaccharide activation, immunoglobulin production was followed as a criterion of viability of the cells. In alginate beads, diffusion limitations result in cell death. In agarose, the production level of specific antibodies is 40% lower than with suspended cells while immobilization does not alter polyclonal antibody production.