Detection of Anti-SARS-CoV-2 IgG, IgM and IgA Using High-Throughput Screening (HTS) by Cytometry

Unless working in a BSL-3 laboratory, COVID-19 patient samples should be treated to inactivate any potential viral contamination prior to working with them in a BSL-2 lab.  There are several validated virus inactivation methods reported in the literature, including heating at 56 °C for an hour, or treatment with certain chemical detergents.  Effective virus inactivation should be verified for whatever method is utilized.  The samples used in our assay were inactive with 4% Triton X-100 at RT for 5-10 min.  After sample collection, the iQue^® High-Throughput Screening (HTS) Cytometer automatically cleans itself by default after each run and at shutdown.  As with any cytometry system, the waste generated should be treated with a proper decontamination solution before disposal, and waste disposal should be done according to local regulations.

The kit is available in several formats = enough for 1 or 5x96 well plates, or for 1 or 5x 384 well plates.  The actual number of samples depends on how many dilutions and multiples of samples are included in your experiment.

The assay was developed for use with the iQue^® High-Throughput Screening (HTS) Cytometry Platform – including the compensation adjustments, and pre-defined analysis templates that autogenerate concentration response curves.  The low sample volume needed is also unique to the iQue^® High-Throughput Screening (HTS) Cytometry Platform.  In particular, this assay requires only 10 µL of pre-diluted serum or plasma - and then after the final wash, only 10 µL of buffer is added before acquisition - thanks to the low volume of sample required by the iQue^® HTS Platform.  Other systems may require substantially more of your sample.

The RBD is from the original / WT strain of SARS-CoV-2 – which is the same as what's utilized in the current vaccines.  

This kit allows users to run the assay qualitatively or quantitatively and generally can detect a range from low picogram to the high nanogram concentrations.  For example, qualitative analysis may be useful for initial screening studies on a large number of samples.  The qualitative assay detection levels are as low as 30-60 pg/mL – which is actually far below the negative threshold cut-off values based on COVID-19 negative plasma samples.  For quantitative analysis, the lower level of quantification (LLOQ) is higher, and varies slightly between Ab isotypes.  For example, the linear range for accurate quantification of IgG Abs is between approximately 50 ng/mL down to 300 pg/mL when using the Ab standard included in the kit and preparing a 1:3 serial dilution curve across a row of a 96 well plate or a column of a 384 well plate.

Samples can be run in as many replicates as desired when adding to 96 or 384 well plates.  Samples can also be split across multiple plates, and then all test samples can then be interpolated using the same standard antibody curve.