Thermoblock

Inactivation was performed in triplicate using 140 µl aliquots of SARS-CoV-2 isolates (1775, 2018, and 2310; passage 3 from Vero cells). Chemical inactivation was performed following manufacturers’ recommendation and included: (i) adding 560 µl viral lysis buffer (AVL buffer including carrier RNA; AVL buffer) from the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) for 10 min at room temperature according to the manufacturer’s recommendations; (ii) 200 µl inactivating sample buffer (GeneReach, Taichung City, Taiwan) containing 50 % guanidinium thiocyanate (GITC) and 6 % t-Octylphenoxypolyethoxyethanol (Triton X-100) for 15 min at room temperature; or (iii) 140 µl 4 % Formaldehyde in PBS (General Drugs House, Bangkok, Thailand; end concentration 2 %) for 15 min at room temperature. Thermal inactivation similarly performed on 140 µl aliquots of fresh virus culture: (iv) 56 °C for 30 min; (v) 56 °C for 60 min; and (vi) 98 °C for 2 min in a thermo-block (Eppendorf, Hamburg, Germany). Sterile DMEM treated in the similar methods served as negative controls, and untreated SARS-CoV-2 isolates as positive controls. All samples and controls were handled in 1.5 ml PP reaction tubs (Greiner bio-one, Kremsmünster, Austria) during the treatment.

The High-Capacity RNA-to-cDNA Reagent Kit (4387406, Applied Biosystems, Foster City, CA, USA) was used to transcribe mRNA into complementary DNA by reverse transcription. A 10:1 mixture of reaction buffer and reverse transcriptase was prepared and 5.5 μL of the mixture was added to each sample containing 0.5 μg of RNA in 4.5 μL RNase-free water. The solution was placed in a thermoblock (Eppendorf), and the reaction was carried out at 37 °C for one hour. The temperature was then increased to 95 °C for 5 min. The contents of the tubes were cooled to 4 °C, then frozen at −20 °C and stored until RT-qPCR analysis.

Bivalent, bispecific and trispecific complexes were formed upon mixing of SpyTagged, SnoopTagged and/or SdyTagged VHHs with ELP scaffolds with C-terminally, N-terminally and/or internal Spy-, Snoop- or SdyCatchers. Briefly, scaffold and VHH solutions were diluted in PBS to concentrations between 5–32 µM. Subsequently, solutions were mixed at a 1:3-1:4 molar ratio (scaffold:VHH) under agitation (300 rpm) in a thermoblock (Eppendorf, Hamburg, Germany) for 3 hr at 20°C. Of note, for the trispecific complex the SdyTagged VHHs were incubated 3 hr at 20°C before the Spy- and SnoopTagged VHHs were added. When required, complexes were separated from uncoupled VHHs by Strep-Tactin based affinity chromatography according the manufacturer’s instructions (IBA).