Colloidal gold as an electrochemical label of streptavidin-biotin interaction

A new electrochemical method to monitor biotin-streptavidin interaction, based on the use of colloidal gold as an electrochemical label, is investigated. Biotinylated albumin is adsorbed on the pretreated surface of a carbon paste electrode (CPE). This modified electrode is immersed in colloidal gold-streptavidin labelled solutions. Adsorptive voltammetry is used to monitor colloidal gold bound to streptavidin, obtaining a good reproducibility of the analytical signal (R.S.D. = 3.3%). A linear relationship between peak current and streptavidin concentration from 2.5 x 10(-9) to 2.5 x 10(-5) M is obtained when a sequential competitive assay between streptavidin and colloidal gold-labelled streptavidin is carried out. On the other hand, the adsorption of streptavidin on the electrode surface was performed, followed by the reaction with biotinylated albumin labelled with colloidal gold. In this way, a linear relationship between peak current and colloidal gold labelled biotinylated albumin concentration is achieved with a limit of detection of 7.3 x 10(9) gold particles per ml (5.29 x 10(-9) M in biotin).     

Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

Summary Enterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 m in length and 1–2 m in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.     

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.     

Distribution and stability of membrane proteins in lipid membranes on solid supports

Bacteriorhodopsin and the nicotinic acetylcholine receptor were biotinylated and reconstituted in lipidic membranes on silicon supports by fusion with proteoliposomes. The presence and distribution of the proteins were studied by binding with streptavidin. Radio-labelled streptavidin was employed for quantifying the amounts of protein remaining in the supported membranes after storage in buffer. The proteins within the membranes remained bound to the surface for weeks. The biological activity of reconstituted unlabelled receptor upon storage showed stability in membranes formed on silicon supports and a reduced stability when formed onto lipid monolayer covered supports. Atomic force microscopy studies on preparations in liquid showed bilayer structures but also attached, partly fused liposomes and membrane particles. In air, the surface was smoother and contained less of liposomes and more of stacked lipid layers. Preparations labelled with streptavidin conjugated to colloidal gold and imaged in air showed the proteins individually distributed, with no protein-rich patches or protein aggregates.     

Semiquantitative, bar code version of immunochromatographic assay system for human serum albumin as model analyte.

An immunochromatographic assay system was devised that can express the concentration ranges of analyte (e.g., urinary human serum albumin) as distinct numbers of the ladder bar (bar coding) for semiquantitation. We constructed a model system consisting of five membrane pad strips partially superimposed in a length. Upon wicking of sample from the bottom, the medium dissolved two different biotinylated species, antibody to the analyte and conjugates of the antibody with colloidal gold, and antigen-antibody reactions took place in the hollow space of the glass fiber membrane. After eliminating unreacted biotinylated molecules at the next strip with an immobilized albumin, the immune complexes were transferred to the pad with streptavidin immobilized in a ladder bar pattern. Analytical conditions here were set for competition between the two biotinylated species for the streptavidin binding sites. The degree of such competition was proportional to the analyte concentration and, consequently, the bar signal number was elevated as the concentration increased. Under optimal conditions for sensitivity, the analytical system responded to the analyte doses at between 30 and 120 mg/dL by producing different bar codes within 5 min.     

Double lectin and immunolabelling for transmission electron microscopy: pre- and post-embedding application using the biotin-streptavidin system and colloidal gold-silver staining

Summary Pre- and post-embedding methods are described that can be used for consecutive localization of two intracellular cytoplasmic binding sites in cells and tissues embedded in acrylic plastic for transmission electron microscopy. Both applications make use of the biotin-streptavidin system with colloidal gold detector particles and involve silver staining of the first gold signal to a predetermined size. Silver augmentation effectively masked any free binding sites on the biotinylated molecule and on the streptavidin complex of the first labelling reaction, thereby allowing a second cycle with the same detection system. Excellent ultrastructural localization was obtained with silver lactate as the silver ion donor in the developing solution, and the enhancement treatment did not destroy or even visibly reduce target site reactivity for the subsequently applied probe. Using these methods it was possible to achieve specific double lectin and immunological labelling; they could, however, be adapted to dual or multiple-labelling procedures with any biotinylated molecules.     

Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG,IgM and IgA using beads covalently coupled to the nucleocapsid protein

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5–96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.     

Functionalization of poly(oligo(ethylene glycol) methacrylate) films on gold and Si/SiO2 for immobilization of proteins and cells: SPR and QCM studies

Thin films of a biocompatible and nonbiofouling poly(oligo(ethylene glycol) methacrylate) ( pOEGMA) with various thicknesses were formed on gold and Si/SiO 2 substrates by a combination of the formation of self-assembled monolayers (SAMs) terminating in bromoester-an initiator of atom transfer radical polymerization (ATRP)-and surface-initiated ATRP. After the formation of the pOEGMA films, terminal hydroxyl groups of side chains divergent from the methacrylate backbones were activated with N, N'-disuccinimidyl carbonate (DSC), and the DSC-activated pOEGMA films were reacted with (+)-biotinyl-3,6,9-trioxaundecanediamine (Biotin-NH 2) to form biotinylated pOEGMA films. By surface plasmon resonance experiments with the target protein (streptavidin) and model proteins (fibrinogen and lysozyme), we verified that the resulting films showed the enhanced signal-to-noise ratio ( approximately 10-fold enhancement) for the biospecific binding of streptavidin compared with the biotinylated substrate prepared from carboxylic acid-terminated SAMs. Quartz crystal microbalance measurements were also carried out to obtain the surface coverage of streptavidin and fibrinogen adsorbed onto the biotinylated pOEGMA films with various thicknesses and to investigate the effect of film thicknesses on the biospecific binding of streptavidin. Both the binding capacity of streptavidin and the signal-to-noise ratio of streptavidin/fibrinogen were found to be saturated at the 20 nm thick pOEGMA film. In addition, to demonstrate a wide applicability of the pOEGMA films, we constructed micropatterns of streptavidin and cells by microcontact-printing biotin-NH 2 and poly- l-lysine onto the DSC-activated pOEGMA films, respectively.     

Immunogold signal amplification: Application of the CARD approach to electron microscopy.

Catalyzed reporter deposition (CARD) is a technique that allows amplification of routine immunolabeling in light microscopy. This procedure takes advantage of the horseradish peroxidase (HRP) from an HRP-avidin complex to catalyze the accumulation of reporter-conjugated tyramine (a phenolic compound) onto a surface displaying biotinylated antigen-antibody complexes. The large amount of labeled tyramine deposited allows the detection of an antigenic site with multiple reporter molecules. In this study we modified this amplification protocol to combine it with the immunogold technique for the ultrastructural localization of antigens in electron microscopy. We constructed various tyramide conjugates that permit the combination of this amplification method with a particulate colloidal gold marker. The new probes yield results of high specificity and enhanced intensity. Assessment of the level of resolution of the labeling has demonstrated that, in spite of the amplification, the resolution remains very good. Therefore, once associated, the immunogold and the CARD techniques lead to specific, high-resolution, sensitive and amplified signals that exhibit the advantages of both approaches.(J Histochem Cytochem 47:421-429, 1999)     

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.     

Immuno-Light and Immuno-Electron Microscopic Examination of Freeze-Dried Kidney Tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Ultrastructural localization of α-galactose-containing glycoconjugates in the rat vomeronasal organ

Binding sites of Griffonia simplicifolia I-B₄ isolectin (GS-I-B₄), which recognizes terminal α-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B₄ and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal α-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the α-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal α-galactose.     

Counting Chromosomes in Root Tips from a Large Population of Plants

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Immuno-light and immuno-electron microscopic examination of freeze-dried kidney tissue

Correlative morphology of kidney tissue can be accomplished by a combination of immunofluorescent staining and light microscopy using a pre-embedding technique. Further development of this method has been elaborated by freeze-drying and chopping of the tissue, which then is incubated in biotinylated immunoglobulins. Fluorochromes and colloidal gold can be coupled to the biotinylated immunoglobulins in a second step. This technique is an alternative to postembedding immunogold staining for immunoelectron microscopy, and permits a correlative immunohistochemical and detailed morphological study of kidney tissue.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin–streptavidin interaction monitoring

A new electrochemical method to monitor biotin–streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin–streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at +0.08 V was recorded in NH₃ 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H₂SO₄ 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at −0.18 V for 2 min and anodic stripping voltammetry were carried out in NH₃ 1.0 M containing 2.0×10⁻⁵ M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25×10⁻¹⁵ to 2.24×10⁻¹² M and a limit of detection of 2.0×10⁻¹⁵ M were obtained.     

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.     

Immunosensor for Mycobacterium tuberculosis on screen-printed carbon electrodes

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.