The use of magnetic beads in IVD is not new. Recent developments –as the described in the next chapters- promise easier and better coating procedures where the orientation and the availability of the captured molecule can be controlled. However, most of the current applications are still using the classical surfaces.
The way of couple your protein or antibody to the surface of the magnetic bead depends basically on its nature and the assay you are working on.
The simpler alternative for coating your beads is the plain surface. Your antibody is coated to the beads surface by passive adsorption. This sort of attachment typically relies on hydrophobic interactions to bind the molecule to the bead. It allows for very little control over the final orientation of the attached molecule. It is not uncommon, as a consequence, to have multiple layers of a capture molecule bound to a particle. Problems with specificity and stability of the capture molecule may arise as a result.
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Modified surface (Carboxyl, amino, hydroxyl and sulfates)
To minimize non-specifity problems and reduce the background, you may covalently and stably couple your antibody to the magnetic beads. The typical groups added to the surface are carboxyl, amino or hydroxyl.
Carboxylated particles require activation adding carbodiimide (EDC), N-Hydroxysuccinimide (NHS) or sulfo-NHS and ethyl (dimethylaminopropyl). This activation yield intermediate esters that will then bind to the amino groups in the protein being conjugated.
Magnetic beads functionalized with surface amino groups require activation of the carboxyl groups on the protein to be attached. Utilizing cross-linkers can serve as spacers between the bound protein and the particle, or they can serve to expand the repertoire of molecules capable of being conjugated to the particle.
Particles containing surface hydroxyl groups are hydrophilic due to their inherent negative charge, and show less aggregation and fewer non-specific binding. These particles are more complicated to coat and require activation in non-aqueous solution to avoid hydrolization of any intermediates. Once activated, however, they can bind a number of different groups, making these particles quite versatile.
Some problems can occur such as aggregation and nonspecific binding. If these problems are an issue, you may need to use pre-activated surfaces.
Pre-activated surface (tosyl, epoxy and chloromethyl groups)
There are a number of commercial magnetic beads pre-activated or functionalized with different chemical groups on their surfaces, such as tosyl, epoxy, or chloromethyl groups. The main advantage of using this type of beads is that coating the beads covalently with the capture protein is more straightforward than with the typical groups mentioned in the previous point. There is no preliminary activation that needs to be carried out prior to attaching a molecule to the bead.
These surfaces allow for very stable covalent coupling of antibody to the beads. The coupling is usually simple: it just needs incubating the pre-activated beads in the correct buffer, pH and temperature.
The Tosyl groups will bind to amino or to sulfhydryl groups in a protein depending on the pH during the coating process. Neutral pH is used for sulfhydryl groups, whereas a more basic pH is used for binding amino groups. Chapter 3 will discuss in detail this pre-activated surface.
Epoxy groups can bind a number of different groups, again depending on the pH of the binding reaction. If the pH is slightly basic, epoxy groups will bind thiol groups. At higher pH conditions, the epoxy group will bind to amino groups. Finally, at very high alkaline conditions, epoxy groups can bind to hydroxyl-containing ligands.
The third group, the chloromethyl, is probably the easiest one to work with: at room temperature and neutral pH it will bind amino groups.
The binding, however, might occur with a lesser degree of specificity than covalent bonding functional groups that require activation, such as carboxyl, amino, or hydroxyl groups.
Bio-activated surface (protein A, protein G, streptavidin, biotin)
A third family of surfaces are the bio-activated ones. This is an expensive but highly effective option. The beads contain a surface biolink such as streptavidin, biotin, protein A/G or others. These biolinks have unique and specific properties that govern their use, rendering beads coated with these types of groups suitable for a number of different applications.
Unlike surface functional groups that bind covalently to a protein, biolinks attach molecules in a non-covalent manner that is governed by their affinity for said molecule. Protein A and protein G, for instance, are small proteins originally derived from bacteria. These proteins bind certain immunoglobulins subtypes with a very high degree of affinity. Although each of these two proteins has a unique antibody binding profile, there is some degree of overlap in the antibody fractions that are recognized and bound.
Streptavidin is another small bacteria-derived protein that is utilized as a biolink on bead coatings. Streptavidin has an extraordinarily high affinity for biotin. The strength of binding between streptavidin and biotin is such that it can withstand high temperatures, a wide range of pH values, variations in buffer salts and the presence of detergents. As such, these links are ideal to use in cases where a sample might require extreme conditions. The biotin-streptavidin link can be disassociated with a short 70ºC incubation without denaturing the streptavidin.
The use of these biolinks might result with issues of non-specific binding. For instance, biotin, is a naturally occurring molecule and, as such, the highly circulating endogenous biotin present in samples may interfere with an assay. Consequently, when working with beads containing surface biolinks, it is important to modify the protocol to minimize the effects of any non-specific binding.
- Magnetic bead coatings: Today and tomorrow
- Rapid biofunctionalization of magnetic beads with function-spacer-lipid constructs
- Surface Attenuation for High Sensitivity Assays