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 multi-site 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.     

Immobilization of monoclonal antibodies for affinity chromatography using a chelating peptide.

A procedure was developed for oriented immobilization of monoclonal antibodies on a solid support. The technique involves the specific oligosaccharide-directed covalent modification of the monoclonal antibody (mAb) with the chelating peptide, Lys-Gly-(His)6, in conjunction with immobilized metal ion affinity chromatography. Chelating peptide-mAb conjugates with a molar ratio of 2.2 retained full antigen binding activity. On immobilization of the modified antibodies on a nickel affinity resin, the molar antigen binding ratio was 1.4. The high antigen binding capacity is indicative of oriented immobilization providing maximum access for the antigen. The described method can be used for the preparation of high-capacity immunosorbents for affinity chromatography and it is applicable for all immunoglobulin classes.     

Development of immunoaffinity columns for pyraclostrobin extraction from fruit juices and analysis by liquid chromatography with UV detection

Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose-CNBr gel took place with high yield (over 90%), whereas the immunosorbent efficacy to retain the analyte (from 28 to 68%) was shown to depend on the amount and type of antibody immobilized on the support. As a matter of fact, columns prepared with the monoclonal antibody PYs5#14 were able to selectively bound up to 53 μg of pyraclostrobin per gram of beads. Acetonitrile solutions were preferred over methanolic ones for analyte elution, and some immunosorbents could be reused at least 4-6 times provided that the amount of pyraclostrobin and the volume of sample did not overload the column. Effectiveness of the selected immunoaffinity column was evidenced by the development of an extraction procedure for pyraclostrobin residues from fruit juices and further determination by high-performance liquid chromatography with UV detection. A concentration factor of 50 times was achieved with the developed immunoaffinity column, which eventually resulted in a limit of quantification of 0.01 mg L(-1). Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with pyraclostrobin from 0.01 to 1 mg L(-1).     

Preparation of immobilized tannins for protein adsorption

Preparation and adsorption specificity of tannins immobilized by covalent binding on water-insoluble matrices were investigated. Immobilized tannins were prepared by condensing cyanogen bromide activated tannins with aminohexyl derivatives of several kinds of matrices. The most suitable matrix for the immobilization of tannin was alkali-treated cellulose powder. The concentration of sodium hydroxide solution for alkali treatment influenced the adsorption capacity of immobilized tannin for a protein, but temperature and time for alkali treatment did not. Immobilized tannins having spacers of long chain length exhibited high adsorption capacity for a protein. Chinese gallotannin was the most favorable ligand for protein adsorption. The immobilization of tannin on aminohexyl matrices was also possible by using epichlorohydrin instead of cyanogen bromide. The maximum adsorption capacity of the immobilized tannin for a protein was about 50 mg/ml of the absorbent. Immobilized tannin adsorbed proteins specifically but did not absorb low molecular weight compounds.     

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.     

Immunoaffinity Chromatography of s-Triazines

Abstract

Immunoaffinity chromatography (IAC) provides a selective method for sample preparation prior to high-performance liquid chromatography (HPLC) or gas chromatography (GC). Five different support materials were tested for their suitability with regard to IAC of herbicides (s-triazines). Three gels showing low (0-10 %) nonspecific s-triazine adsorption were used as affinity supports (Beaded cellulose ONB-carbonate A, LiChroprep hydrazide tentacle gel and LiChroprep azlactone gel). A monoclonal antibody (K4E7), directed against atrazine, was covalently coupled to the gels. High antibody immobilization rates (100 %) were obtained with the beaded cellulose gel in comparison to the other gels (60-70 %). Subsequently the IAC columns were loaded with triazine solutions. Glycine-HCl 0.2 mol/L, pH 2.2 yielded the best results for the elution of the bound triazines from the beaded cellulose IAC columns (70-100 %). Both the hydrazide tentacle and the azlactone IAC columns showed lower elution rates (60-80 %).     

Immobilization of antibodies on glass surfaces through sugar residues

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.     

Immobilization of protein on aldehyde-containing gels—II. Activation of pyridine rings with cyanogen bromide

Cyanogen bromide was used to convert pyridine rings in polymers to polyaldehyde. By reaction with NH₂-containing substances, the rings are rebuilt, resulting in a pyridinium compound. Thus proteins and other NH₂-containing substances can be covalently bound. This method provides a new means for a immobilization technique. Pyridine-gels based on polysaccharide and polyacrylamide matrices, as well as pyridine glass beads, were synthesized and used to study the conditions necessary for coupling. Trypsin and — chymotrypsin were used as test substances for immobilization of proteins. Some properties of the bound protein were studied and compared to native enzyme. Some general results on the applicability of these gels for affinity chromatography are also presented.     

Orientation of Antibodies on a 3-Aminopropyltriethoxylsilane-Modified Silicon Wafer Surface

An antibody can be specifically oxidized with periodate (NaIO₄) on the carbohydrate side chains at its C-terminal. Rabbit anti-hepatitis B surface antigen (anti-HBsAg) IgG antibodies were bound to the silicon wafer surface by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibodies and the reactive amine groups of 3-aminopropyltriethoxylsilane(APTES)-modified silicon wafer surfaces. A control experiment was also performed by direct attachment of antibodies to glutaraldehyde-treated silicon surfaces. Two different coupling antibody strategies were investigated in this paper. Atomic force microscopy was used to observe the orientation of the site-directed and random attachment of rabbit anti-HBsAg IgG antibodies and the conservation of their antigen-binding capacity (AgBC) was assessed using an enzyme immunoassay (EIA).     

Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues

The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier. The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40–120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.     

免疫亲和质谱法研究β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法, 对β2-微球蛋白抗原表位进行了系统研究. 完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后, 用Endoproteinase Glu-C, Trypsin, Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子, 并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究. 结果表明: β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位, 即为SFYLLYY. 通过合成肽段的分析, 证明了SFYLLYY即为抗原表位, 与亲和质谱方法分析结果一致.     

Immunosorbents: A new tool for pesticide sample handling in environmental analysis

 Immunoaffinity techniques have been widely used for the determination of different analytes in the medical field. However the use of antibodies immobilized in an appropriate support material to preconcentrate pesticides from environmental samples is only recent. The production of antibodies, election of supports, antibody immobilization procedures, elution of analytes from immunosorbents and the more recent applications in the field of pesticide analysis are reviewed. The present review concludes that immunosorbents have great potential and discusses the present limitations and expected future trends. Received: 29 July 1996 / Accepted: 14 August 1996     

Direct introduction of a hydrazide group into a quartz crystal microbalance surface with dodecanoic hydrazide embedded in a hybrid bilayer membrane

Hydrazide group has a potential of immobilizing an antibody on a sensor surface in a way that ensures an optimal orientation and efficiency of the antibody. However, a multi-step chemical process, required for the preparation of a hydrazide group, is a barrier to its extensive application. This paper describes a new method to introduce a hydrazide group to a sensor surface by a one-step process using dodecanoic hydrazide. The method is based on an ability of the dodecanoic hydrazide to be incorporated into a hybrid bilayer membrane (HBM) layer, thereby presenting its hydrazide group to the surface. Liposome containing dodecanoic hydrazide was added to a hydrophobic self-assembled monolayer surface of a quartz crystal microbalance for the formation of a HBM. Then, the hydrazide group, presented in the surface of the HBM layer, was utilized for the oriented immobilization of an antibody via its carbohydrate moiety which was partially oxidized prior to the conjugation reaction. Activity and stable status of the incorporated dodecanoic hydrazide was revealed by the efficiency and reproducibility of the resulting immunosensor chip.     

Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection

The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Sol-gel entrapment of monoclonal anti-atrazine antibodies

We report the successful doping of a sol-gel matrix with an antibody, retaining its ability to bind free antigen from an aqueous solution. The particular system described is monoclonal anti-atrazine mouse antibody which was entrapped in SiO₂ sol-gel matrices, prepared from tetramethoxysilane by several methods. Atrazine was selected as a model compound for this study, within the framework of the development of immunochemical-based methods for monitoring pesticide residues and other organo-synthetic environmental contaminants. Nanogram quantities of atrazine were applied on SiO₂ sol-gel columns doped with this antibody, and the amount of eluted antigen was determined by Enzyme Linked ImmunoSorbent Assay (ELISA). Under appropriate sol-gel-forming conditions, high amounts of atrazine were bound to the sol-gels, ranging between 60% and 91% of the amount applied to the column. The combination of the properties of the sol-gel matrix (e.g., stability, inertness, high porosity, high surface area and optical clarity), together with the selectivity and sensitivity of the antibodies, enable extension of this feasibility study to development of a novel group of immunosensors which could be used for purification, concentration and monitoring of a variety of residues from different sources.Contribution from the Agricultural Research Organization (ARO), Bet Dagan, Israel. No., 1697-E, 1995 series.     

Biophysical characterization of the molecular orientation of an antibody-immobilized layer using secondary ion mass spectrometry

The molecular orientation of antibody layers formed on separate solid matrices (e.g., gold-coated glass substrate) was characterized by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS) in static mode. For comparison, three different antibody species, IgG, F(ab')(2), and Fab, were prepared, biotinylated in random and site-directed fashions, and immobilized on distinct streptavidin-coated surfaces. ToF-SIMS analyses of each antibody layer revealed that the secondary ion intensity peaks measured at the mass-to-charge (m/z) ratio 253, 325, and 647 were unique to the site-directly immobilized antibodies. The ions in the three peaks were detected neither from the streptavidin layer nor from the randomly prepared antibody, indicating that the insolubilized antibody layers constructed in the two different manners had distinct molecular arrangements. The antibody preparations were further tested for their binding characteristics in sandwich-type immunoassays, which showed that the site-directed antibodies consistently enhanced the detection capability comparing to those randomly prepared. Based on the analytical results of both the ToF-SIMS analysis and sandwich-type immunoassays, the site-directed antibody species were immobilized on the surfaces in a more orientated manner, with their antigen binding sites exposed to the bulk solution, than when random immobilization was used.     

Recent advances in immobilization methods of antibodies on solid supports

Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody-antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures.     

Oriented immobilization of periodateoxidized monoclonal antibodies on amino and hydrazide derivatives of eupergit C

Amino and hydrazyno derivatives of Eupergit C were prepared by reaction of the beads with hexamethylene diamine (HMD) and adipic acid dihydrazide (ADH), respectively. Monoclonal antibodies (mAbs) against carboxypeptidase A (CPA) and horse radish peroxidase (HRP) were prepared, and those that did not inhibit the respective enzymatic activities were selected. The carbohydrate moieties of these antibodies were oxidized by reaction with sodium periodate and then coupled onto the modified beads. The oxidation and coupling reactions were optimized to achieve highly active matrix-conjugated antibodies. Thus, antibody-matrix conjugates that possessed antigen-binding activities close to the theoretical value of 2 mol antigen bound/mol immobilized antibody were obtained.     

Immunosensing based on site-directed immobilization of antibody fragments and polymers that reduce nonspecific binding

Antibody Fab'-fragments can be directly coupled onto gold, and the space between the fragments can be filled with protein repellent disulfide bearing polymers. Coupling of the antibody Fab'-fragments, and thus both the amount of nonspecific binding and antigen binding but also the ability to regenerate the layer, is dependent on the immobilization procedure. First, the immobilization has taken place by coupling the Fab'-fragments to the surface and thereafter attaching the polymer in the remaining space between the antibodies. Second, the Fab'-fragments have been added after the surface has been coated by polymer. Third, the Fab'-fragments and polymer have been added onto the surface from the same solution. Up to 80% of the antigen could be removed during regeneration, if proper concentrations of polymer and Fab'-fragments were immobilized onto the gold surface. Only about 60% of the antigen could be removed, when the fragments were coupled directly onto a clean Au surface before the polymer or if low concentrations of polymer were attached onto gold before the Fab'-fragments. The first immobilization method, however, showed the highest response to antigen.