Blocking buffers are used to prevent non specific binding within the assay. Proteins and other bio molecules may bind to unoccupied spaces on the surface of the wells which can be detrimental to the specificity and sensitivity of the assays results as less of the antigen of interest will be able to bind. Non specific binding can be diminished through the use of a blocking buffer. The two major classes of blocking reagents are proteins and detergents. the key aspects to consider when chosing a blocking reagent is making sure it exhibits no cross reactivity with other components of the kit, helps to stabilise biomolecules minimising the effects of denaturation that can be caused from assay transitions, exhibit low enzyme activity to prevent interference with the detection method.
This is one of the most commonly used blocking agents. Tween 20 is a non ionic detergent that is able to block areas on the surface that may be exposed. The benefit to these are they are inexpensive, extremely stable and can be used in washing solutions. The disadvantages are they are not permanent, they may disrupt non covalent bonds, they are only able to block hydrophobic interactions.
Is a type of protein blocker, unlike detergent blockers protein blockers are permanent and only need to be introduced into the wells once when the capture antibody has been added. Protein blockers block the unoccupied spaces on the surface and also stabilise the biomolecules bound to the surface helping to reduce denaturation. Other forms of protein blockers include non ft dry mil, whole natural serum and fish gelatin.
The type of blocker chosen is also dependent on the surface you are blocking.