Figure 4

Thermal stability assay. Antibodies were heated at 85°C for various time points up to 60 min and cooled down prior to measuring binding affinity and specificity using luminex direct binding assays. Each antibody at a concentration of maximal binding mentioned in Fig. 3 was used for this thermal stability assay. Mab-Ric07AG1, and ricin binder, P4RA7-1, retained 60% and 40% of the original activity, respectively; while Cy3-Rab anti-Ricin had only 10% of remaining activity upon 5 min of heating (A). During the heating, P4RA7-1 binding to irrelevant targets, CT and SEB, was insignificant. (B). After 5 min of the heating, P4BH8 and P4BF7-1 had 130% and 60% of the untreated activity, while Rab anti-BoNT/A retained 20% of the untreated activity. Upon 60 min of heating, both retained 60%–80% of untreated activity, while Rab anti-BoNT/A lost almost 100% of the initial activity within the first 10 min of heating. (C). P4BH8 decreased 75% of the initial non-specific binding to ricin after 15 min of heating (D). P4BF7-1 decreased 50–60% of the initial non-specific binding to irrelevant target(s), ricin and CT, after 10 min of heating (E). Both P1SD3-3 and P2SC8 retained about 55% of the initial activity after 5 min of heating, while two conventional antibodies, Mab 2b3a, and Mab 6b, lost about 90–95% of the initial activity (F).