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The avidity index (AI) measures the binding strength between the antibody and the antigen, reflecting the affinity maturation. It can be measured by a modified ELISA, adding a chaotropic agent to disrupt the antigen x antibody interaction. However, details of the protocols used affect the final results. We compared the AI of mice sera after a three-dose immunization with meningococcal antigens using different adjuvants. The AI was assessed using potassium thiocyanate (KSCN) and urea as chaotropic agents, incubated at 4 °C, room temperature (RT) and 37 °C. KSCN presented statistically different results when the incubation was set at 4 °C vs RT and 4 °C vs 37 °C, thus, the mean AI obtained were lower. For Urea, 4 °C vs 37 °C presented relevant differences. Using whole-cells suspensions or OMVs as coating antigen provided similar results in some protocols. Thus, the affinity maturation was assessed after each immunization dose and adjuvant use (aluminium hydroxide and dimethyldioctadecylammonium bromide) supported affinity maturation. It is important to study the AI as a functional parameter of humoral response, and both KSCN and Urea are suitable chaotropic agents, however, the protocols should be standardized considering the nature of the antigen, the chaotropic activity and overall laboratory conditions. Adjuvants are important tools to improve antibody avidity following immunization., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)