The effect of avidin-biotin interactions in detection systems for in situ hybridization.

The effect of avidin-biotin interactions in several detection systems for the non-radioactive in situ hybridization (ISH) technique was studied in a model system using a transitional cell carcinoma line and a biotinylated DNA probe. We performed fluorescence ISH to unravel the individual steps in a sensitive and frequently used amplification method which makes use of the alternating cytochemical detection layers of fluorescein isothiocyanate-conjugated avidin (AvFITC) and biotinylated goat anti-avidin (BioGAA) antibodies to detect the hybridized and biotinylated probe. Our experiments revealed that BioGAA antibodies bind with their antigen binding sites and not with their biotin moieties to avidin molecules that have already interacted with the DNA probe. The probable working mechanism of this amplification method is presented in a model. Furthermore, we used a peroxidase staining technique to compare with each other the sensitivity of several other detection systems in which avidin-biotin interactions play an important role, e.g., the avidin-biotinylated peroxidase complex (ABC) system. The experiments show that avidin molecules can not be efficiently used to interconnect two biotinylated molecular layers, since their introduction leads to firmly closed cytochemical networks. Such a closed network is already formed between the hybridized and biotinylated DNA probe and a first detection layer of avidin molecules, as appears from the finding that biotinylated molecules could hardly be coupled to these avidin molecules in a following detection layer. Therefore, the results presented here provide us with new insight into the molecular basis of cytochemical network formation. This will enable us to choose the proper procedures for increasing the sensitivity of ISH detection systems.     

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures

The use of avidin-biotin interaction in immunoenzymatic techniques provides a simple and sensitive method to localize antigens in formalin-fixed tissues. Among the several staining procedures available, the ABC method, which involves an application of biotin-labeled secondary antibody followed by the addition of avidin-biotin-peroxidase complex, gives a superior result when compared to the unlabeled antibody method. The availability of biotin-binding sites in the complex is created by the incubation of a relative excess of avidin with biotin-labeled peroxidase. During formation of the complex, avidin acts as a bridge between biotin-labeled peroxidase molecules; and biotin-labeled peroxidase molecules, which contains several biotin moieties, serve as a link between the avidin molecules. Consequently, a "lattice" complex containing several peroxidase molecules is likely formed. Binding of this complex to the biotin moieties associated with secondary antibody results in a high staining intensity.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies.

In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP--gold) in the avidin-biotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3'-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP--gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP--gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP--gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP--gold complexes.     

The use of avidin-biotin interaction in immunoenzymatic techniques.

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

Summary The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes nonspecific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidinbiotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.     

Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies

Summary In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP — gold) in the avidinbiotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP — gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP — gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP — gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP — gold complexes.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H2O2 generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-fetoprotein in liver and tissue polypeptide antigen in mammary gland served as models. The immobilized two-step enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

Enhanced Polymer One-Step Staining for Proliferating Cell Nuclear Antigen in Squamous Cell Carcinoma of the Tongue

A rapid enhanced polymer one-step staining for proliferating cell nuclear antigen [EPOS-PCNA) in formalin fixed, paraffin embedded sections of tongue squamous cell carcinoma is described. EPOS-PCNA was compared with PCNA immunohistochemistry using the avidin-biotin complex (ABC) method, with or without autoclave pretreatment. Significant correlation of PCNA labeling index (percentage of labeled cells/total cells counted) was observed between EPOS-PCNA and the immunohistochemical protocol using autoclave pretreatment. We consider this method a reliable, timesaving technique that would be useful in quantitative histopatbology and in performing double immunostaining.     

The use of avidin-gold complex for light microscopic localization of lectin receptors

In the present study, we have investigated the use of avidin-gold complex (AG) as a possible cytochemical marker for visualizing and identifying lectin receptors in deparaffinized tissue sections. Monodispersed gold sols of 15 nm average diameter were prepared by sodium citrate reduction. The AG complex was prepared with highly purified egg-white avidin (avidin-D). Deparaffinized sections of cat duodenum were labeled with five different biotinylated lectins, then were washed and stained for 1-2 h with AG. Intensification of the gold staining was achieved by a modification of the silver-enhancement method. For each lectin, the labeling properties of the avidin-gold-silver (AGS) were compared with those of the avidin-biotin-peroxidase (ABC) and the lectin-gold (LG) methods. We found the lectin binding pattern demonstrated by the AG method to be similar to that of the ABC. The AG localization of the carbohydrate residues is more precise, compared to the peroxidase reaction due to lack of diffusion of the gold marker. Labeling with AGS resulted in improved staining over the AG method, similar to the staining intensity of the ABC. In addition, the two-step AG method provided more intense staining than the direct one-step procedure of the lectin-gold labeling. In conclusion, the use of the AGS method for histochemical visualization of lectin receptors requires a simple two-step procedure which allows highly accurate localization of tissue glycoconjugates. It entails using only a single gold-ligand complex applicable to any biotinylated lectin regardless of its biochemical nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.     

The use of avidin-gold complex for light microscopic localization of lectin receptors

Summary In the present study, we have investigated the use of avidin-gold complex (AG) as a possible cytochemical marker for visualizing and identifying lectin receptors in deparaffinized tissue sections. Monodispersed gold sols of 15 nm average diameter were prepared by sodium citrate reduction. The AG complex was prepared with highly purified egg-white avidin (avidin-D).Deparaffinized sections of cat duodenum were labeled with five different biotinylated lectins, then were washed and stained for 1–2 h with AG. Intensification of the gold staining was achieved by a modification of the silver-enhancement method. For each lectin, the labeling properties of the avidin-gold-silver (AGS) were compared with those of the avidin-biotin-peroxidase (ABC) and the lectin-gold (LG) methods. We found the lectin binding pattern demonstrated by the AG method to be similar to that of the ABC. The AG localization of the carbohydrate residues is more precise, compared to the peroxidase reaction due to lack of diffusion of the gold marker. Labeling with AGS resulted in improved staining over the AG method, similar to the staining intensity of the ABC. In addition, the two-step AG method provided more intense staining than the direct one-step procedure of the lectin-gold labeling.In conclusion, the use of the AGS method for histochemical visualization of lectin receptors requires a simple two-step procedure which allows highly accurate localization of tissue glycoconjugates. It entails using only a single gold-ligand complex applicable to any biotinylated lectin regardless of its biochemical nature. It can also be easily adapted for use with other biotinylated ligands such as antibodies, hormones, toxins, etc.     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique.     

A nonradioactive assay for type IV collagen degradation

A sensitive assay for type IV collagen degradation using an avidin-biotin sandwich technique is described. Biotinylated type IV collagen is allowed to bind to an avidin-coated microtiter plate. The solution to be assayed is incubated with the biotinylated collagen bound to the avidin plate. Collagen degraded by the solution is released into the supernatant and transferred to a second plate coated with avidin. By addition of biotinylated horseradish peroxidase to this second plate, the amount of collagen degraded is determined. Our assay requires only 0.5 microgram of type IV collagen per microtiter plate and detects nanogram quantities of bacterial collagenase activity.     

Conjugated avidin binds to mast cell granules

The glycoprotein, avidin, conjugated either to the enzyme horseradish peroxidase, or to the fluorochrome dyes, fluorescein or rhodamine, identifies the granules of mast cells in both tissues and cell suspensions. In the absence of prior fixation, mast cells were not identified with conjugated avidin; however, granules released from these cells were stained with this labeled glycoprotein. The specificity of avidin for mast cells was confirmed by the absence of conjugated avidin-positive cells in the skin of mice (S1/S1d) deficient in mature dermal mast cells. Electron microscopic studies confirmed that avidin binds specifically to individual mast cell granules rather than to other cellular structures. Rodent and human mast cells were readily stained with avidin conjugated to horseradish peroxidase or to either of the fluorochrome dyes. The conjugated avidin staining technique is a reliable and simple method for identifying rodent and human mast cells, one that is useful as both an investigative and a clinical tool.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

Recombinant avidin and avidin-fusion proteins

Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin-biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin-biotin complex. 3. This reassociation still occurred in 3.5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin-biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin-biotin complex do not readily lose their biotin, even in 3.5m-guanidinium chloride, and that monomeric biotin-binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3.0m and 3.5m.