Short Communication Regular monitoring of susceptible animal species for specific antibodies is essential to achieve or to maintain disease free status for a country. The absence of certain disease in a country for many decades would yield expectation that collected animal serums would be negative for the presence of specific antibodies. However, large-scale tests often dismiss singlereactor findings as poor sample quality. The current study aimed to investigate the effect of storage conditions of negative serum samples and the specificity of ELISA kits on the test results, focusing on two key livestock diseases: foot-and-mouth disease (FMD) and peste des petits ruminants (PPR). Serum samples from bovine and ovine sources were stored at varying temperatures and durations, were subjected to freeze-thaw cycles, and were retested. Results were compared with zero-day tests which were considered to be truly accurate and negative. The quality of ELISA test results is less significantly affected by serum samples quality (affected by temperature, storage time, and freeze-thaw cycles) and occurrence of false positive single reactors, than the diagnostic specificity of different ELISA lots. This study challenges the conventional justification for single-reactor findings and underscores the importance of ELISA kit quality. https://macvetrev.mk/LoadArticlePdf/361 2025-03-15 101 107 https://doi.org/10.2478/macvetrev-2025-0019 ELISA foot and mouth disease sample quality peste des petites ruminants diagnostic specifity Ljubiša Veljović ljubisa.veljovic@nivs.rs false 1 Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia LEAD_AUTHOR Dimitrije Glišić false 2 Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia AUTHOR Zorana Zurovac Sapundžić false 3 Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia AUTHOR Sofija Šolaja false 4 Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia AUTHOR Vesna Milićević false 5 Institute of Veterinary Medicine of Serbia, Janisa Janulisa 14, 11000 Belgrade, Serbia AUTHOR Minić, R., Živković, I. (2020). 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