A blocking agent and a blocking step are not needed in ELISA, immunostaining dot-blots and western blots.

The effect of blocking and non-blocking was compared in ELISA, immunostaining dot-blots and western blots using phosphate-buffered saline with and without the addition of Tween 20 for washes and dilutions. The results indicated that when Tween 20 was included in phosphate-buffered saline, a blocking agent and a blocking step were not required in immunoassays. Moreover, the use of protein-based blocking agents was found to somewhat lower the reactivity between the antibody and the antigen, possibly due to steric hindrance.