New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA

Carmen Muñoz, Alberto Nieto, Antoni Gayá*, Jorge Martínez, Jordi Vives

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

26 Citations (Scopus)


A peroxidase saturation technique for the determination of coating antigen concentration necessary to saturate polystyrene plates with a wide range of different antigens is described. The same technique has also been used to compare the stability of antigen-polystyrene bonds for native and denaturated antigens. Furthermore, the inappropriate selection of solid-phase antigen concentration and its influence on ELISA results is analyzed and an experimental criterion to select the optimum antigen concentration is proposed. Two different antigens, BSA-Ar and hydatid antigen, were used for ELISA determination of specific antibodies as a model system. Optimum solid-phase antigen concentration wasdetermined by two different methods: (1) peroxidase saturation, in which binding of peroxidase to non-antigen-occupied polystyrene surface sites was used to evaluate the degree of coating by antigen; (2) chequer-board titration, using several immune sera of different affinity. Optimum antigen concentration selected by chequer-board titration using low affinity sera was similar to that selected with peroxidase saturation. On the other hand, lower antigen concentration would be selected by chequer-board titration using high affinity sera. For this reason, the concentrations of low affinity antibodies would be underestimated using the chequer-board titration. These results indicate that peroxidase saturation should be used to avoid avidity-dependent artifacts in ELISA.

Original languageAmerican English
Pages (from-to)137-144
Number of pages8
JournalJournal of Immunological Methods
Issue number1-2
Publication statusPublished - 20 Nov 1986


  • Chequer-board titration
  • Peroxidase saturation technique


Dive into the research topics of 'New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA'. Together they form a unique fingerprint.

Cite this