Detection of Anti-SARS-CoV-2 IgG, IgM and IgA Using Advanced Flow Cytometry
Advance your COVID-19 research by obtaining a comprehensive profile of antibodies against SARS-CoV-2 using a multiplex and high throughput serological assay.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to persist due to constant mutation and evasion. Because development of a humoral immune response against this virus is critical for protection, there is an ongoing need to accurately assess the quantity and duration of antibody responses against SARS-CoV-2, particularly antibodies targeting the SARS-CoV-2 Spike protein receptor binding domain (RBD). The SARS-CoV-2 Spike RBD mediates viral entry into human cells by binding to the angiotensin-converting enzyme 2 (ACE2) and is the primary target of many COVID-19 vaccines currently in use. Antibodies against the SARS-CoV-2 Spike RBD generally have potent viral neutralizing activity and correlate with protective immunity against the virus. Serological testing to quantitate the three major isotypes (IgG, IgM and IgA) of antibodies specific for SARS-CoV-2 Spike RBD can be used to facilitate characterization and monitor the duration of antibody responses in both COVID-19 patients and vaccinated individuals, detect asymptomatic infections and survey past infection prevalence in a population.
There are several methods for detecting anti-SARS-CoV-2 Spike RBD IgG, IgM or IgA antibodies, including a traditional enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay and flow cytometry. However, these methods have limitations:
- Provide only qualitative results
- Low sensitivity and specificity
- Can be labor-intensive and may not support high-throughput applications
- Do not allow multiplexing analysis of IgG, IgM and IgA antibodies
- Require large amounts of reagents
The iQue® SARS-CoV-2 (IgG, IgM and IgA) kit provides a rapid, robust and sensitive serological assay for the measurement of IgG, IgM and IgA anti-SARS-CoV-2 Spike RBD antibodies in a single well of a 96 or 384-well plate with antibody detection levels in the pg/mL range. Combined detection of all three antibody isotypes saves time and precious patient samples and provides a dynamic, comprehensive profile of humoral immune responses over time following natural infection with SARS-CoV-2 or immunization with COVID-19 vaccines. Accelerate research with the iQue® SARS-CoV-2 (IgG, IgM and IgA) kit!
The iQue® SARS-CoV-2 (IgG, IgM and IgA) Kit is a bead-based immunoassay designed for simultaneous detection of human IgG, IgM and IgA antibodies specific for SARS-CoV-2 Spike protein RBD produced by the immune system in response to viral infection or vaccination.
The iQue® SARS-CoV-2 (IgG, IgM and IgA) Kit has been optimized for robust, multiplexed analysis on the iQue® platform with a VBR configuration. Qualitative and quantitative analysis of anti-SARS-CoV-2 Spike RBD antibodies from human blood samples (sera and plasma) is accomplished with a streamlined workflow and pre-set templates for ease of use.
- Add pre-diluted patient samples to 96 or 384-well plate
- Add SARS-CoV-2 Spike RBD-coupled Beads to capture anti-SARS-CoV-2 Spike RBD antibodies
- Add SARS-CoV-2 Antibody Detection Cocktail containing fluorescently labeled anti-human IgG, IgM and IgA antibodies
- Analyze the plate on iQue® platform (VBR configuration).
Obtain accurate assessment of immune response - Sensitive and simultaneous measurement of anti-SARS-CoV-2 Spike RBD IgG, IgM and IgA antibodies induced in response to natural infection or vaccination.
Maximize your productivity - Multiplexed assay enables high-throughput analysis of the three major isotypes (IgG, IgM and IgA) of antibodies specific for SARS-CoV-2 Spike RBD in a single well for comprehensive analysis of the humoral immune response.
Save precious samples - Streamlined workflow conserves precious samples - requires only a single 10 µL sample diluted at least 100-fold.
Obtain accurate assessment of immune response
Sensitive and simultaneous measurement of anti-SARS-CoV-2 Spike RBD IgG, IgM and IgA antibodies induced in response to natural infection or vaccination.
Figure 1. Analysis of anti-SARS-CoV-2 Spike RBD IgG, IgM, and IgA antibodies in (A) serum from recovered COVID-19 patients (P1-4). Serum collected from two individuals prior to the pandemic were included as negative controls (Neg. 1-2). (B) Analysis of serum collected from two individuals (VA & VB) prior to immunization (VA-0, VB-0), and again after the first (VA-1, VB-1) and second injection (VA-2, VB-2) with the BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine. Serum samples were diluted 1:100 in Assay Diluent and then serially diluted 1:5 in triplicate to generate dilution curves.
Maximize your productivity
Multiplexed assay enables high throughput analysis of the three major isotypes (IgG, IgM and IgA) of antibodies specific for SARS-CoV-2 Spike RBD in a single well for comprehensive analysis of the humoral immune response with real-time data analysis and novel visualization tools.
Figure 2. Analysis of anti-SARS-CoV-2 Spike RBD IgG, IgM, and IgA antibodies in serum from recovered COVID-19 patients or individuals vaccinated with the BNT162b2 (Pfizer-BioNTech) vaccine. Serum samples were diluted 1:100 in Assay Diluent and then serially diluted 1:3 in triplicate. Serum collected from two COVID-19 negative individuals were included as negative controls (Neg. Ctl). Each color represents triplicate dilution curves which were repeated in different quadrants of plate. (A.) Plate layout (B) Heat Map of anti-SARS-CoV-2 Spike RBD IgG concentrations per well (ng/mL).
Frequently Asked Questions
What care should I take in disinfecting the flow cytometer when using samples contaminated with SARS-CoV-2 or blood pathogens?
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 oC 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® instrument automatically cleans itself by default after each run and at shutdown. As with any flow 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® Advanced Flow 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 high-throughput iQue® Advanced Flow 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® 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.