Magnetic beads for immunoassays
Magnetic beads provide better efficacy for target detection, pull-down, protein-protein and protein-DNA interaction studies.
Magnetic beads are often blocked by the addition of BSA, skimmed milk, sperm DNA, gelatin, PEG or sera, to remove unwanted non-specific background, but ready to use Streptavidin blocked beads are available that do not require these additions. If a blocker is used, equilibrate the beads with lysis buffer and a low concentration of the blocker and incorporate the same blocker into the wash buffer to prevent non-specific binding. It is also recommended that you “clean” the solution containing your target first by mixing it with the beads in the absence of the antibody to reduce non-specific binding.
Antibody selection is a very important step in immunoassays. Optimize the antibody using standard immunoblotting to identify non-specificity, if any. Selecting a specific antibody and a high-affinity bead can greatly enhance the sensitivity of an experiment. Optimize your antigen with different tags and antibodies and select the one that yields the best result.
Excess antibody can remain unbound and result in background signals, while insufficient concentrations will not coat the beads uniformly and can reduce target binding capacity. Contaminations from light and heavy chains of antibodies can be eliminated using magnetic bead-antibody crosslinking and elution in a low pH and non-reducing buffer.
Antigen-antibody binding is determined by the level of antigen expression. This critical step needs to be standardized for different antigens against specific antibody concentrations.
Here are some hints and tips to keep in mind when selecting your beads and designing your experiment:
- For scarce antigens, increase binding time (this can also increase non-specific binding) and concentrate the samples.
- For abundant targets, increase antibody concentration or bead surface area.
- Binding the antigen to the bead-antibody complex is more specific than binding the antigen-antibody complex to the bead.
- Incorporate detergent in binding buffer to reduce non-specific binding.
- While binding at room temperature for 10 minutes is sufficient, slow binding at 4˚C for an optimal time of 4 hours might help in certain cases.
- Unless stated otherwise, all steps of the experiment can be performed on ice to reduce non-specificity.
- Elute at 70˚C for 10 minutes instead of 95˚C, to prevent the antibody from being released from the beads.
For sandwich immunoassays, donor and acceptor beads need to be carefully designed so they don’t bind to each other. Select beads that distinctly differ in their excitation and emission wavelengths, binding specificity for the respective antigen, and are not influenced by sample or buffer-specific interferences. High biomolecular concentrations should be avoided as they can overload the beads and reduce donor-acceptor signals.
Molecular diagnostic assays that use specific oligos bound to beads for sequence capture
Magnetic beads bound to specific sequence probes are used to detect single nucleotide polymorphisms and genetic biomarkers underlying diseases in molecular diagnostic assays. This method is also used to examine cell-free DNA in urine, plasma, breast milk and serum, and bacterial or viral infections with complementary probes. Beads bound to desired probes can be customized from the manufacturer, or they can be prepared in the lab.
As diagnostic samples can be from various sources, the most common issues are
- Variable quantity of DNA/RNA
- Presence of inhibitors
It is important to ensure that the sample to be detected is intact and that samples such as blood, urine, and plasma are processed immediately for better recovery of nucleic acids.
Specificity and efficiency of the beads in diagnostics is determined by the selected probe, incubation time and sample abundance. Highly specific and non-overlapping sequence oligo probes need to be designed for best results. Nucleic acid-bead incubation times should be standardized to allow efficient binding and sensitive detection. In case of low abundance samples, PCR amplification or sample concentration can enhance detection efficiency. Magnetic beads bound to capture probes and a surface-enhanced Raman scattering (SERS) nanoplatform is a useful method that provides faster results in molecular diagnostics.