Effect of storage time and lighting conditions on the stability of anti-Leishmania infantum antibody titers in canine serum

Abstract Immunofluorescence is a widely used technique for the detection and characterization of antibodies or antigens, based on the emission of fluorescence following binding to specifically labeled molecules. However, fluorescence may be influenced by factors such as exposure to light and time until observation. The study aimed to evaluate the effects of different lighting conditions (natural, artificial indoor light, and darkness) and storage time prior to microscopical examination in assessing antibody titers against Leishmania infantum in canine sera examined by indirect immunofluorescence assay (IFA). In addition, inter-observer agreement during microscopic assessment was evaluated. In total, 40 canine sera were used for the assessment of the antibody titer against L. infantum . Each serum sample was distributed onto three immunofluorescence slides and stored under three different lighting conditions. The assessment was performed by two observers (a third observer was involved in case of disagreement) at multiple time points ranging from 0 to 300 min after preparation. There was an almost perfect inter-observer agreement. The lighting conditions had no significant effect on the observed titers. There was a significant decrease in titers after 120 min, and there was a significant interaction between lighting and time. The antibody titers remain stable for up to 120 min, regardless of lighting conditions. Exposure to natural daylight results in a faster reduction in fluorescent intensity, and consequently in antibody titers, compared to other lighting conditions.