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Newsletter 1

general blocking incubation additives

BACKGROUND SUPRESSION using AURION BSA-c

The AURION NEWSLETTER an immunogold users exchange platform for practical problem solving

gold reagents enhancement technical support

Jan L. M. Leunissen, Ph.D. Peter van de Plas

AURION Costerweg 5 6702 AA Wageningen The Netherlands

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 Reactions in Immuno Localizations In order to understand the nature of background it is necessary to take a close look at the nature of reactions in immuno localization studies. Reactions that may occur are: (i) the intended specific reactions, (ii) the unintended specific reactions and (iii) the unintended non-specific reactions. The intended specific reactions lead to the localization of the antigen(s) under investigation. They are biased by the quality of the specific binding agent on the one hand and on the preservation and availability of antigen(s) on the other hand. Fixation, denaturation and masking of antigens play a major role next to specificity, affinity and avidity of the specific antibodies or antibody/marker conjugates. The unintended but specific reactions may have one of the following causes: -- the specific binding agent (e.g. antibody) recognizes epitopes in molecules other than the antigen under investigation (cross-reactivity). Sometimes such reactions can be prevented by using e.g. antibodies that have been purified by adsorption. An alternative is using a different serum or monoclonal. Characterizing an antibody by immuno blotting offers possibilities to judge whether an antibody reacts with only one or with more Sodium Dodecyl Sulphate denatured proteins (Moeremans et al., J. Immunol. Methods 74, (1984), 353). -- in sera many clones of antibodies are present. Part of these will be directed to the specific antigen, and part may be directed to other antigens (that may have been present as impurities in the antigen preparation used for immunization). A test using pre-immune serum may elucidate this. Although these reactions are based on a specific interaction they are frequently considered as background. Strictly, background is caused by unintended non-specific reactions. They are governed by general physico chemical properties such as hydrophobic and charge determined forces. In the generation of background the hydrophobic and charge characteristics of both the specimen/substratum and of the specific binding agents (primary antibodies and secondary antibody/marker conjugates) have an equal influence. Next to this residual fixative activity in the specimen will give rise to co-valent binding of immuno reagents, more or less randomly distributed over the specimen. How to Discriminate between Specific and Non-specific reactions Background caused by primary antibodies is often difficult to characterize. Using pre-immune serum instead of the primary antibody does not necessarily discriminate between background and specific interactions as the specific fraction of an immune serum may give rise to background as well. Cross-adsorption will almost always reduce background and so does using high dilutions of the primary reagent (see also Newsletter 4). Background caused by immuno conjugates is more easily characterized as it occurs in specimens which were incubated without the specific primary antibody. Of course this background will also be present in those specimens which are incubated with the primary antibody. We have tried to evaluate the principles governing background and considered both hydrophobic and charge determined interactions. These types of interactions are the same for particulate markers, alkaline phosphatase, peroxidase and fluorescent markers. Because of our interest in immunogold

conjugates we have chosen these as a model system. Once more it should be noted that the physical chemical properties of both the reacting molecules (primary antibody and secondary antibody, protein A or G etc.) and the coupled marker have their part in background reactions. Immuno Conjugates based on Colloidal Gold Particles Colloidal gold particles have a negative surface charge next to hydrophobic characteristics. The expression of these characteristics is influenced by the conjugated protein and the post stabilization method. AURION reagents are prepared in such a way that the expression of these characteristics is minimized. Nevertheless this can never be completely overcome. This is in many cases easily achieved by adding BSA and/or gelatine (several types do exist and the effect on background is a.o. described by Behnke et al., Eur. J. Cell Biol. 41, (1986), 326 and Birrell et al., J. Histochem. Cytochem. 35, (1987), 843. Even with these additives, on occasion background may persist. Evidently in such cases the additives are not capable of an adequate suppression. Charge Determined Interactions The negative charges present on the gold particle surface structure the surrounding water dipoles in such a way that the charge is felt over distances away from the particle surface. Even upon stabilization of gold particles by protein (like an antibody) this is still the case. The spatially expressed charge causes gold conjugates to be bound to e.g. extremely high positively charged poly-l-lysine coated grids as is used in a positive sense to check the size and cluster characteristics of the conjugate by electron microscopy. The staining of proteins by colloidal gold particles at low pH as described by Moeremans et al. (1985) is a logical consequence of the same principle (Anal. Biochem. 145, 315). While the charge characteristics find a useful application in these cases, they will on the other hand give rise to background in immuno cytochemistry on those sites in the specimen where positive charges are abundant (for instance some proteins of the cytoskeletal network, the extracellular matrix and the nuclear histone proteins). Preventing Charge Determined Background Charge determined background can be dealt with by influencing the spatial charge distribution at the sites of interaction: close to the gold particle surface or close to the specimen area. There are several ways to achieve this: --by increasing the concentration of positively charged ions. Increasing the NaCl concentration from 150 mM to 600 mM has quite a strong background reducing effect. The Na+-ions disturb the structured orientation of the water dipoles surrounding the gold conjugate and the oppositely charged sites in the specimen. This chaotropic effect results in a decreased attraction and thus in decreased background levels. However, frequently the ultrastructure and specific protein activity suffer from high salt concentrations and gold conjugate clusters may be formed by reduced repulsion. --by adding proteins to the incubation mix which have a net positive charge at the pH of incubation (7.0 - 8.2). An example is gelatine or at least some of its components. Several authors observed a reduction of background as a consequence of the addition of gelatine. The mechanism of action may be explained by the attraction of the positively charged components to the gold conjugate, thereby (partly) neutral-

izing its charge and reducing background. Gelatine proves to be especially suited for the larger gold probes. It is a disadvantage if the addition of gelatine leads to an increase of the gold conjugate size and thus to a reduction of reaction kinetics. This is especially experienced with ultra small gold probes where an increasing concentration of gelatine (>0.1%) to the incubation mixture is coupled with a decreasing reactivity. --by adding proteins to the incubation mix which have a net negative charge at the pH of incubation (7.0 - 8.2). An example is BSA with an iso-electric point of approximately 5. At the pH of incubation BSA molecules compete with the gold conjugate for binding on positively charged sites in the specimen. Since BSA has only a weak net negative charge its background suppressing activity (as far as charge is concerned) is not extraordinary. An advantage of this procedure is that due to repulsion between gold conjugate and BSA molecules the conjugate is not increased in size. Increasing concentrations of BSA have little effect on the reaction kinetics. From this listing of possibilities to prevent charge determined background it may be obvious that addition of inert negatively charged complexes to the incubation medium is a preferred method of background prevention. It may also be obvious that increasing the concentration of positively combined with negatively charged protein additives to the incubation buffer not necessarily leads to decreasing background since the components have a charge determined affinity for each other as well. AURION BSA-c and Charge Determined Background AURION developed a chemically modified BSA with an increased net negative charge by acetylation of amino groups of basic amino acids (AURION BSA-c). The use of AURION BSA-c at a concentration as low as 0.01% in the gold conjugate solution inhibits binding of gold conjugates to poly-l-lysine coated grids almost completely. In practice we find concentrations of 0.1 - 0.2% in PBS pH 7.4 to be most effective in immuno cytochemical localization studies. Hydrophobic Interactions Part of the gold particle surface appears to have hydrophobic characteristics. In a hydrophilic environment (as during incubation) the binding force caused by hydrophobic interaction can be quite prominent. This means that once a bond has been formed by hydrophobic interaction it is difficult to break it up and separate the interacting components. Hydrophobic forces thus exist between gold conjugates too. However this does in general not lead to aggregation due to the prevailing charge determined repulsion (see above). In specimens hydrophobic areas are in general abundantly present (membranes, lipid droplets, embedding media, hydrophobic substrata like nitro-cellulose). Some of these areas carry a negative charge high enough for repulsion (for instance the negatively charged phospholipids in biological membranes). These areas do show little background if any. Background due mainly to hydrophobic interactions occurs in those areas which are both hydrophobic and uncharged (or which carry only a weak negative charge).

Preventing Background by Hydrophobic Interaction Hydrophobic interactions can be minimized in several ways: --by increasing the temperature during incubation. This has the effect that hydrophobic interactions do not as easily result in binding. Increasing the temperature to 37C during incubation often has a positive influence on background suppression. --by blocking the hydrophobic sites in the specimen before the immuno gold incubation. This can be done with e.g. BSA or Casein (possibly the component in skimmed milk powder responsible for background suppression). This is most effective at a pH-value close to the iso-electric point of the blocking agent. These conditions leave the blocking agent with the least net charge, thus favouring hydrophobic interaction. Once blocking of hydrophobic areas has occurred, this is not easily reversed under standard incubation conditions. A combination of the two indicated possibilities is also possible: blocking of the specimen at an initially elevated temperature so as to allow existing hydrophobic interacting areas to separate (e.g. between BSA molecules) and next allowing the temperature to drop (to room temperature or below room temperature) in order to promote hydrophobic interaction between the blocking agent and the specimen. AURION BSA-c and Background caused by Hydrophobic Interaction AURION BSA-c consists of acetylated BSA which has been (partly) linearized to facilitate the exposure of hydrophobic areas. The use of AURION BSA-c at a concentration as low as 0.05 - 0.1% as a blocking agent before the gold conjugate incubation almost completely inhibits binding of gold conjugates to hydrophobic substrata (e.g. many plastics used in electron microscopy, hydrophobic plastics in disposables). NOTE: the strong blocking capabilities of AURION BSA-c may cause antigens to become covered with BSA-c, especially when antigens are present in minor amounts on a hydrophobic substratum like nitro-cellulose. In such cases AURION BSA-c should only be used as an additive during the immuno gold incubation step. Blocking is then performed with unmodified BSA. In Summary: How to deal with background reactions. In principle: inactivate residual fixative in the specimen, make sure the specimen is rendered hydrophilic and eliminate attraction by charges. Step wise: 1. Inactivation of residual fixative activity: - Wash specimens after fixation to remove fixative residues - Inactivate aldehydes by using low molecular weight compounds that will react with aldehyde groups, such as hydroxyl amine, NaBH4 and glycine. 2. Make the specimen hydrophilic: - Use a block step before the immuno incubations with a highly soluble but still hydrophobic protein like BSA. During blocking the hydrophobic domains of the specimen will be forced to react with the hydrophobic domains in BSA molecules. As a consequence the hydrophilicity of the specimen is improved. 3. Eliminate charge determined interactions between specimen and reagents by using an incubation buffer with Aurion BSA-c.

 Presumed interaction between AURION BSA-c, specimen and immuno conjugate

An overview of Aurion Blocking Solutions, Incubation Buffer additives and Blocking and Incubation Buffer Compounds Blocking Solutions
For Protein A and G gold conjugates For Goat gold conjugates For Rabbit gold conjugates For Sheep gold conjugates For Donkey gold conjugates

Product Code
905.001 905.002 905.003 905.004 905.005

Incubation Buffer Additives

100 ml BSA-c concentrate (10%) 30 ml BSA-c concentrate (10%) 900.022 900.099

Compounds
BSA 25 grs CWFS gelatin 40% Tween-20 900.011 900.033 900.044

Normal Sera The surface properties of the specimen can be simplified by division into four compartments - negatively charged (polyanions, proteins, especially after aldehyde fixation, lipids), 0 neutral, + positively charged (histone proteins, polycations) and H hydrophobic (lipids, fat droplets, resins). After an appropriate blocking step these areas are covered with blocking compounds. In low ionic strength media negatively charged antibodies and gold conjugates are repulsed by negatively charged specimen areas which frequently may contain the antigens to be detected. Background does not likely occur in such areas. The positively charged areas attract antibodies and gold conjugates potentially leading to background. In a moderate ionic strength incubation solution, repulsion and attraction are diminished due to the presence of ions. The negatively charged BSA-c competes with the negatively charged antibodies and gold reagents for non-specific binding to the positively charged specimen compounds, thus reducing background to the greatest possible extent without interfering with antigen detection.
Normal Goat Serum Normal Rabbit Serum Normal Sheep Serum Normal Donkey Serum 900.077 900.066 900.111 900.122

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Costerweg 5 6702 AA Wageningen The Netherlands phone: (+31)-317-497676 fax: (+31)-317-415955 http://www.aurion.nl e-mail: info@aurion.nl

Newsletter 1

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