The history of the pneumococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) is characterized by a continuous search for increased specificity. A third-generation ELISA that uses 22F polysaccharide inhibition has increased the specificity of the assay, particularly at low antibody concentrations. The present work compared various 22F ELISAs and non-22F ELISAs. The comparisons involved three different laboratories, including a WHO reference laboratory, and included sera from subjects from different geographic areas immunized with different pneumococcal conjugate vaccines, including the licensed 7-valent Prevenar vaccine and the 10-valent Synflorix vaccine. All comparisons led to the same conclusion that the threshold defined as 0.35 microg/ml for the WHO non-22F ELISA is lower when any 22F ELISA is used. The use of highly purified polysaccharides for coating further improved the specificity of the assay. In conclusion, we confirm that the 22F ELISA can be recommended as a reference method for the determination of antibodies against pneumococcal polysaccharides.