Application of the avidin-biotin system for post-embedding cytochemical demonstration of a biotin-labeled IgG tracer (original) (raw)
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Acta Neuropathologica, 1985
To study the permeability of the bloodbrain barrier (BBB) and the blood-nerve barrier (BNB) for immunoglobulin G (IgG) we adapted the avidin-biotin system for postembedding demonstration of the tracer IgG in the central and peripheral nervous system (CNS, PNS). Normal mouse and human IgG were biotinylated and injected daily into the intraperitoneal (i.p.) space of adult BDF1 mice. After 24 h, IgG was detected in blood vessels and in the interstitium of various organs, but staining was restricted to the dura mater in the CNS, to the spinal ganglia, and to the perineurium of peripheral nerves. After 4 days, IgG was also present in the endoneurial connective tissue of peripheral nerves, while the brain, spinal cord, and spinal roots remained free of IgG. Our results show a partial permeability of the normal mouse BNB for homologous and heterologous IgG.
Journal of Immunological Methods, 2010
This study shows the optimization of biotin labeling to antibodies using mouse IgG. Several parameters of the biotin labeling, including the molar ratio of biotin to antibody, the coupling time and the dialysis time, were studied to optimum conditions. The biotin-tagged mouse IgGs were immobilized on avidin-coated PMMA (Polymethyl Methacrylate) plates via a biotinavidin linkage. The immobilization of the IgG to the chip was quantified using goat anti-mouse IgG bound fluorescent beads. It was found that the binding of the fluorescent beads saturated when a 10-fold or higher molar ratio of biotin to antibody was used. In biotin coupling time tests, sixty minutes was sufficient for the capture probes to bind to the surface. However, the results from the dialysis experiments showed no difference, indicating that 2 hours was sufficient to remove any unbound biotin. Finally, to prove the universality of this protocol using mouse antibodies, the optimum conditions were successfully applied in sandwich immunoassays designed to detect troponin I (TnI) and N-terminal probrain natriuretic peptide (NT-proBNP).
Journal of Microscopy, 1999
A super-sensitive epitope-detection technique based on gold±silver intensi®cation was adapted for pre-embedding immunolabelling of titin ®laments in vertebrate striated muscle. Indirect immunoelectron microscopy of titin ®laments was performed with monoclonal titin antibodies as primary antibodies and F ab anti-mouse IgG conjugated with 1´4 nm gold particles as secondary antibodies. The secondary antibodies penetrated easily into the tissue owing to their reduced size and the very small gold particles. After the labelling procedure, the tissue was ®xed in glutaraldehyde. Since the gold particles were not visible by conventional transmission electron microscopy, they were intensi®ed with a silver developing system. Although the particle size varied nonlinearly with the developing time, very ®ne grain size was achievable. The technique provided super-sensitive detection with excellent contrast and demonstrated epitopes with both strong and weak af®nities.
Protein A, avidin, and biotin in immunohistochemistry
Journal of Histochemistry & Cytochemistry, 1981
Conjugated and unlabeled peroxidase antibody methods have proven to be quite satisfactory in localizing sites of antigen-antibody reaction. The use of avidin-biotin-peroxidase complex (ABC), as well as protein A, can contribute significantly to the field of immunohistochemistry. The sensitivity and specificity of several immunohistochemical methods is compared. In general, the ABC method produced the most intense staining and the least background staining of any method tested. The unlabeled antibody (peroxidase-antiperoxidase: PAP) method also yielded satisfactory results, but it was less intense than the ABC method. In comparison to the PAP method, the indirect conjugated method presented slightly inferior staining intensities and significantly higher background staining. Protein A techniques produced a range of staining sensitivities similar to or inferior to the PAP technique. The main disadvantage in using protein A is that it reacts with intrinsic immunoglobulin (Ig) G, thus pr...
AvidinOX™ for tissue targeted delivery of biotinylated cells
International Journal of Immunopathology and Pharmacology, 2012
AvidinOX®, a product containing aldehyde groups, generated by ligand-assisted sugar oxidation of avidin by sodium periodate, maintains the capacity to bind biotin with very high affinity and exhibits the property to chemically link cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks. In recent studies, we have shown that AvidinOX exhibits much higher persistency in the skeletal muscle than native avidin. The aim of the present study is to evaluate whether AvidinOXbiotin interaction might be exploited to target biotinylated cells to an AvidinOX pre-treated muscle. To accomplish this we performed the following experiments: 1) The proliferation and differentiation properties of biotinylated C2C12 myoblasts were tested in vitro upon linkage to AvidinOX; 2) Bone marrow-derived cells (BMDC) were isolated from GFP positive transgenic mice [strain C57 BL/6-tg (UBC-GFP)] and after biotinylation (bBMDC) were intravenously administered to naive and MAVA+ (Mouse anti Avidin Antibody) C57/B6 mice previously injected with AvidinOX in a tibial muscle (TM). Localization efficiency of GFP+ bBMDC was evaluated on serial sections of-the AvidinOX-and vehicletreated (contra lateral limb) TM, 5 days after transplantation. Results show that biotinylated C2C12 cells, once linked to AvidinOX, maintain their proliferation and differentiation capacity, in vitro. Intravenous injection ofbiotinylated GFP+ bone marrow-derived cells leads to their specific and efficient localization in the AvidinOX-pre-treated, but not contra lateral muscle of both naive and MAVA+mice. The present data suggest a potential use of AvidinOX to improve tissue targeted delivery of biotinylated cells.
Immunochemical detection of proteins biotinylated on nitrocellulose replicas
Journal of Immunological Methods, 1986
A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Application of the enzyme substrate, alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization and is inhibited by biotinylated bovine serum albumin or 10 nM biotin. The procedure permits detection of less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.
European journal of biochemistry / FEBS, 1990
We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.
Journal of Histochemistry & Cytochemistry, 1992
The effect of fixation technique and post mortem-to-fixation interval in immersion-fixed tissue from the central nervous system on immunocytochemical staining for the presence of an immunoglobulin was determined in mice. Immersion-fixed tissue was found to be inferior to perfusion-fixed tissue for immunocytochemical staining of this serum protein. Unlike what has been observed for other antigens, the quality of staining for IgG in immersion-fixed tissue decreased to unacceptable levels if the post mortem-to-fixation interval was increased to more than a few hours. This effect may be secondary to the rapid post-mortem disintegration of the blood-brain barrier and a resulting diffusion of serum proteins into surrounding tissue from the vasculature.
Journal of …, 2002
A highly sensitive avidin-biotin optical immunoassay (AB-OIA) has been developed for the detection of beta-bungarotoxin (b-BuTx), a neurotoxin from the venom of Bungarus multicinctus, in whole blood, plasma, and urine. Affinity purified rabbit IgG anti-b-BuTx antibody was immobilized on an optically active silicon surface (SILIASk wafer). The test sample was incubated and the antigen-antibody reaction was monitored by the addition of a biotinylated monoclonal antibody (mAb 15) specific to the toxin, avidin-horseradish peroxidase (HRP) and tetramethylbenzidine substrate. The silicon assay surface technology enables us to directly visualize a physical change in the optical thickness of the antibody thin film. The change in thickness is due to the specific capture of the toxin on the surface and when the substrate is added, the binding event is amplified, which then alters the reflected light path and a change in colour is visualized. The assay could detect b-BuTx levels as low as 16 pg/ml in sample buffer and 100 pg/ml in whole blood or plasma. The AB-OIA is simple, requires only 40 ml of biological fluid and can be performed without specialized equipment. The efficacy of the test for detection of b-BuTx in blood or plasma obtained from mice during experimental envenomation with B. multicinctus venom was demonstrated. The AB-OIA was also used to quantitate the postmortem level of b-BuTx in various organs such as brain, liver, and kidney, as well as the tissue at the site of injection. Development of a simple, rapid snake toxin detection kit based on AB-OIA technique potentially applicable in the clinics as well as in the field is discussed.