Letter to the editor: "hook" (prozone) effect in sars-cov-2 anti-spike binding antibody levels following vaccination, infection, or monoclonal antibody in solid organ transplant recipients.

Anti-spike binding antibody (Ab) levels are used by some providers to inform COVID-19 risk assessment for solid organ transplant recipients (SOTRs). As has been observed with other Ab assays, in the setting of high binding Ab, quantitative results may demonstrate artifactually low values (i.e., “hook” or prozone effect). Within two studies of SARS-CoV-2 vaccination of SOTRs (an observational cohort and a single-center trial), Ab levels were assessed using the semiquantitative Roche Elecsys anti-SARS-CoV-2 S assay. In the observational cohort, we flagged 9 samples with either a paradoxical decrease or weak (<10x) rise after Tixagevimab/Cilgavimab (T/C) administration. This prompted retesting with up-front 1:50 dilution, with serial dilution performed until returning two results within expected assay variation. Subsequently, all post-vaccination clinical trial samples were retested. Hook effect was suspected if retest level was both ≥15% and ≥200U/mL higher than original level. From the observational cohort, all 9 flagged samples demonstrated a hook effect. Of 377 clinical trial samples (all rerun), 34/377 (9%) demonstrated a hook effect. Among the hook effect samples (n=43), the original median (IQR) titer was 1950 (650 – 4390) U/mL, and upon retesting this increased to 5685 (2981 – 9853) U/mL representing a 1.6 (1.3–6.0)-fold increase (p=0.03). Marked hook effect (>700x increase) was observed in two participants with recent vaccination plus breakthrough infection. Hook effect was observed in SOTRs tested using a SARS-CoV-2 clinical Ab assay in the setting of high analyte. Laboratories and clinicians should be aware of this artifact and consider serial dilution to confirm accurate quantitative results.