Introduction The actual prevalence of a SARS-CoV-2 infection and the individual assessment of being or having been infected may differ. Facing the great uncertainty - especially at the beginning of the pandemic - and the possibility of asymptomatic or mildly symptomatic, subclinical infections, we evaluate the experience of SARS-CoV-2 antibody screening at a tertiary clinical setting. Methods and analysis All employees of a tertiary eye centre and a research institute of ophthalmology were offered antibody testing in May 2020, using a sequential combination of different validated assays/antigens and point-of-care (POC) testing for a subset (NCT04446338). Before taking blood, a systematic inquiry into past symptoms, known contacts and a subjective self-assessment was documented. The correlations between serostatus, patient contacts and demographic characteristics were analysed. Different tests were compared by Kappa statistics. Results Among 318 participants, SARS-CoV-2 antibodies were detected in 9 employees. Chemiluminescence assays (chemiluminescence immunoassay and electrochemiluminescence) showed superior specificity and high reproducibility, compared with ELISA and POC results. In contrast to the low seropositivity (2.8%) of healthcare workers, higher than that of the other departments of the hospital, a large proportion mistakenly assumed that they might have already been infected. Antiviral antibody titres increased and remained on a plateau for at least 3 months. Conclusions The great demand and acceptance confirmed the benefit of highly sensitive testing methods in the early phase of the pandemic. The coincidence of low seroprevalence and anxious employees may have contributed to internalising the need of hygiene measures.