Optimization of biotin labeling of antibodies using mouse IgG and goat anti-mouse IgG-conjugated fluorescent beads and their application as capture probes on protein chip (original) (raw)
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Journal of Histochemistry & Cytochemistry, 1986
The capacity of the avidin-biotin system for post-embedding cytochemical detection of a biotin-labeled proteinaceous tracer was investigated. By testing modifications of the fixatives, the epoxy embedding medium, and various etching solutions, a procedure was developed to localize specifically the biotinylated tracer on 1-micron thick and thin sections. By use of this technique, a systemically administered IgG tracer was demonstrated after 24 hr throughout the endomysium of mouse skeletal muscle. In the adjoining sciatic nerve, the tracer IgG occurred at the perineurium and within endoneurial blood vessels, although the endoneurium itself was spared because of the presence of the blood-nerve barrier. Because of the small size of the biotin ligand and the non-denaturing method of labeling proteins, our mode of application of the avidin-biotin system appears suitable for tracer studies.
Journal of Immunological Methods, 2009
Microtiter wells are commonly used for bioassays. The sensitivity of such an assay depends on several instrumental and biochemical parameters such as the signal-to-background ratio and nonspecific binding of the label molecules. In this study, we have investigated the possible effects of well surface defects, well edges and denaturation of capture antibodies on the assay sensitivity. We used internally dyed Eu(III) chelate polystyrene nanoparticles as high specific activity labels in a thyroid stimulating hormone (TSH) sandwich-type model assay. The nanoparticle labels provide a high signal-to-background ratio in assays but the major limiting factor of the assay sensitivity is nonspecific binding of the labels. In our model assay the capture monoclonal antibodies were immobilized on microtiter wells passively or through streptavidin (SA)-biotin linkage. At first, commercially manufactured microtiter well surfaces were probed with an atomic force microscopy and significant structural inhomogeneities were found. The nonspecific binding of the nanoparticle conjugates did not appear to follow any of the microtiter well surface defect patterns in a number of experiments. In addition, the microtiter well edges did not increase the nonspecific binding. Denaturation of capture antibodies on solid surfaces has been proposed to expose amino acid sequences promoting nonspecific binding. This was studied by intentionally denaturing the surface capture antibodies by heat, detergent or acid treatment prior to the assay. Although specific signal was almost entirely lost no significant effect on nonspecific binding was observed. The passively adsorbed antibodies denatured at lower temperatures than those captured through streptavidin-biotin linkage. Evidently, the additional protein (SA) layer protected the capture antibody from denaturation whereas the solid surface appeared to act as a "catalyst" making the passively adsorbed antibody more susceptible to denaturation.
A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
Journal of Immunological Methods, 2007
Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for highthroughput applications.
Journal of Immunological Methods, 2012
Synthetic peptides are widely used in indirect ELISA to detect and characterize specific antibodies in biological samples. Small peptides are not efficiently immobilized on plastic surfaces by simple adsorption, and the conjugation to carrier proteins with different binding techniques is the method of choice. Common techniques to conjugate peptide antigens to carrier proteins and to subsequently purify such complexes are time consuming, expensive, and occasionally abrogate immunogenicity of peptides. In this report we describe a simple, fast and inexpensive alternative protocol to immobilize synthetic peptides to plastic surfaces for standard ELISA. The technique is based on use of maleimide-activated bovine serum albumin or keyhole limpet hemocyanin as a protein anchor adsorbed on the polystyrene surface of the microtiter plate. Following adsorption of the carrier protein, sulfhydryl-containing peptides are cross-linked with an in-well reaction, allowing their correct orientation and availability to antibody binding, avoiding the time consuming steps needed to purify the hapten-carrier complexes. The immunoreactivity of peptides was tested by using both monoclonal and polyclonal antibodies in standard ELISA assays, and compared with established coating methods.
Journal of Immunological Methods, 1993
Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.
Biomedical Microdevices, 2009
A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO 2 laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antibody-antigen interaction as a sandwich immunoassay with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the intensity of fluorescence was measured onchip to determine the concentration of the target analyte. The present immunosensor chip showed a linear response range for horse IgG between 1 μg/ml and 80 μg/ml (r=0.971, n=3). The detection limit was found to be 0.71 μg/ml. The developed microfluidic system can be extended for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.
Biosensors and Bioelectronics, 2013
The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS Biotin) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1 J/cm 2 yielded an average conjugation efficiency of ∼1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBS Biotin method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH 3 + surface conjugation, NHS-Biotin, and direct physical adsorption. Taken together, the UV-NBS Biotin method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.
Fluorescent-labeled antibodies: Balancing functionality and degree of labeling
Analytical Biochemistry, 2010
A critical assumption in utilizing labeled antibodies is that the conjugation reaction has no deleterious effects on antibody avidity. This study demonstrates that this assumption need not hold true and presents a methodology to quantitatively determine the degree of inactivation and/or changes in antibody-antigen binding that can occur with conjugation. Fluorescein isothiocyanate, FITC, was conjugated to a mouse monoclonal antibody (Fc125) against hemagluttinin (HA) using varying fluorophore:protein (F:P) labeling ratios. Antibody binding, as a function of the F:P labeling ratio, was evaluated using a kinetic ELISA assay and analyzed using global fitting. A two parameter adjustment of the antibody concentration and the maximum rate were sufficient to describe the rate changes. The concentration parameter dominated the rate changes consistent with the hypothesis that the coupling reaction inactivated an increasing fraction of the antibody population with a smaller change (~15 % at the highest F:P ratio) in antibody-antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be utilized to prepare functional, labeled antibody reagents with defined activity and can aid in quantitative applications in which the stoichiometry and functionality of the labeled antibody is critical.
Sensors, 2014
Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.