Fig. 3: Titration of antibodies in the agglutination assay.

A Eighteen plasma samples from mild cases were compared for titration in the HAT with 1:40 O−ve whole blood from a seronegative donor and endpoint titre in an RBD ELISA³². Four samples were negative in both assays. The data point marked with an arrow on the graph (plasma 2 on the plate, Fig. 3B) was checked with a reagent composed of IH4 without RBD and shown to be dependent on antibodies to the RBD. This sample did score positive for antibodies to full length spike in an ELISA (endpoint titre 1:1123). These results were confirmed in a repeat assay. B An example of titration: positive agglutination endpoints (loss of teardrop) are marked with a black solid-line circle, partial teardrops are marked with a dotted-line circle. C Titration of mAb CR3022 and a high titre serum from a COVID-19 patient show that agglutination in the HAT detected by eye correlates with antibody binding to RBCs, as revealed by FACS analysis. Standard HAT titration was performed by double dilutions of RBC suspension containing 1 µg/mL of the IH4-RBD reagent, bar the first and last well. After the HAT assay, the RBCs were stained for FACS analysis by performing three washes before incubation for 60 min at 4 °C with FITC-labelled goat–anti-human IgG. RBCs were then washed twice before analysis by flow cytometry. Green numbers in the upper right corners of the histograms correspond to the geometric mean fluorescence intensity. Original graphs are provided as Supplementary Fig. 4. Similar results were obtained in three other experiments.