Non-specific background and auto-aggregation in chemiluminescent immunoassays are often caused by the presence of exposed hydrophobic surfaces on the magnetic beads. The use of a blocking reagents combined with gentle homogenous bio-magnetic separation will help reduce background and auto-aggregation of your coated beads.
This post is about using magnetic beads, such as streptavidin beads, in Chemiluminescent immunoassays. If you are interested in this topic, download our free ebook The Basic Guide for the use of Magnetic Bead in Chemiluminescent immunoassays:
A blocking step is standard in all immunoassays, regardless you were using covalent coupling (carboxil, amine, tosyl...) or bioactivated beads (Biotin or streptavidin magnetic beads). For CLIA beads, blocking reagents are often coated onto the microspheres using adsorption. This step occurs after the coupling reaction to the antibody. Blocking reagent concentrations as high as 0.1% are recommended in order to saturate all of the exposed hydrophobic surfaces.
After the beads are processed, it is important to store the beads in a standard blocking buffer with a concentration of 0.05%, so that blocking will be maintained throughout the storage life of the reagents.
Common blocking reagents
The most commonly used blocking reagents are:
Bovine Serum Albumin (BSA): BSA is used either alone or in combination with a surfactant.
Tween-20 and Triton X-100: These two are non-ionic surfactants that are used at a concentration of 0.05% in combination with 1% BSA.
Casein/Pepticase: These are cheap proteins that contain free biotin. Therefore, these blocking reagents are not suitable to use in a system that involves biotin.
‘Irrelevant’ IgG: This blocking reagent is used when coupling a very specific IgG to the microspheres. It is important to always test that this type of blocking reagent is truly non-reactive with the analyte.
Fish Skin Gelatin (FSG): This is a type of pure gelatin/hydrolysate. Other types of gelatins are also used as blocking reagents, depending on your preferences.
Polyethylene glycol (PEG): This is a very versatile blocking reagent because it comes in a wide variety of molecular weights and charges.
Sera: Sera from horse or fish are often used as a blocking reagent since they are typically inert when testing for cross-reactivity with many types of antibodies.
Commercial blockers: Since most commercial blocking reagents are proprietary and, therefore, have an unknown composition, they are not recommended for use in IVD reagent production.
Without the proper blocking reagent, it is unlikely that the end user will be able to achieve the sensitivity they require for their assays, especially if their analyte exists in very low concentrations. Blocking reagent choice, therefore, is extremely important to the quality of the final product.
Don't forget to check these posts from our blog in order to get a deeper insight into Chemiluminescent immunoassays:
- Five Things to Consider about Magnetic Bead IVD Kit Stability Studies
- The Five Most Popular Magnetic Microsphere Currently for Chemiluminescent Immunoassays
- How to Select the Best Surface for Your Chemiluminescent Immunoassays