Sars cov 2 surrogate virus neutralization test kit

Detection and semiquantitation of neutralizing antibodies were determined by using SARS-CoV-2 Surrogate Virus Neutralization Test Kit (Genscript, https://www.genscript.com) according to the manufacturer’s instructions. All samples from 7–29 dpi were assessed, including archived negative serum samples and kit-supplied negative controls. We considered values above the manufacturer’s recommended cutoff of 20% positive for neutralization.

To detect viral neutralizing antibodies the SARS-CoV-2 Surrogate Virus Neutralization Test kit was utilized (Genscript, #L00847) according to the standard protocol. Samples were run in duplicate with blocking values averaged. This kit detects antibodies that can block the interaction between the receptor-binding domain of the viral spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) cell surface receptor and has been approved by the FDA for emergency use. Plasma samples along with positive (anti-RBD antibody) and negative (buffer only) were incubated with a Horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD fragment. The mixture was then added to a capture plate that was coated with the human ACE2 protein. The unbound HRP-RBD will bind to the plate. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to develop the HRP signal and was read at 450 nm in a microtiter plate reader. The absorbance of the sample is inversely dependent on the titer of the anti-SARS-CoV-2 neutralizing antibodies. Inhibition was calculated by (1 − OD value of sample/OD value of negative control) × 100 which gives percent inhibition. A cutoff of ≥ 30% is considered positive for SARS-CoV-2 neutralizing antibody. Plasma samples were diluted 1:10 for all samples and 1:100 for a more dilute assay of seropositive samples at week3 and week 7 timepoints.

Blocking of the RBD-ACE2 interaction by the mouse sera was assessed using a SARS-CoV-2 surrogate virus neutralization test kit (GenScript) (37 (link)) according to the manufacturer’s instructions. Briefly, sera from bleed 3 were diluted in PBS, before further dilution in the provided sample buffer at a 1:9 ratio. Samples and controls were then mixed with HRP-RBD, incubated in 37°C for 30 min, and added to the ACE2-coated wells. The plate was incubated at 37°C for 15 min and washed four times. TMB solution was added to the reactions, followed by a 15-min incubation in the dark at room temperature. The absorbance at 450 nm was read immediately following quenching with the provided stop solution (Synergy H1 hybrid multi-mode reader; BioTek). The IC₅₀ was estimated using a three-parameter log-logistic regression fit in GraphPad Prism. The absorbance at 450 nm was modeled in response to serum dilution.