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HCoV-NL63 Antibody Profiling: Spike RBD Antigen

This workflow and its products described here are for research use only and is not to be used for any other purposes, including, but not limited to, in vitro diagnostics, clinical diagnostics, or use in humans. The document and its content are proprietary to Fluidic Analytics and is intended for use only in connection with the products described herein and for no other purposes.

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1. Intended use

This user guide describes microfluidic affinity antibody profiling (MAAP) against the HCoV-NL63 Spike RBD protein (RBD) directly in serum samples by use of the Fluidity One-W Serum instrument.

2. Background

COVID-19 is a respiratory disease caused by the coronavirus SARS-CoV-2 that produces mild symptoms in most individuals. However, dependent on underlying health issues and other factors such as age, gender, or genetic makeup, symptoms can vary dramatically in severity. A complete and accurate immune response assessment of COVID-19 patients or vaccinated individuals based on the two key determining factors, namely antibody concentration and affinity, is key to:

  • Assessing the strength and duration of acquired immunity
  • Evaluating the mechanisms and immunological effects of virus-neutralizing antibodies
  • Characterizing cross-reactivity with other, less harmful, coronaviruses

This user guide serves to evaluate background immunity and cross-reactivity with the receptor binding domain of the spike protein of HCoV-NL63. HCoV-NL63 is a common coronavirus that binds to the same host-cell receptor (ACE2) as SARS-CoV-2 and causes respiratory infections in humans.

3. Microfluidic affinity antibody profiling – assay principle

This user guide describes all steps required to characterize the immune response against the HCoV-NL63 RBD directly in minimally diluted serum samples. After RBD labeling, three independent serum titration curves are measured to enable the simultaneous determination of antibody affinity and concentration.

Flow_diagram - FA0004-01

4. Before you start

Good laboratory practice when working with serum samples

For use of this user guide, we recommend working with serum rather than plasma. Serum collected from COVID-19 patients (whether newly infected or recovered), and uninfected people who could still harbour infectious agents must be handled using a high level of precaution and at the required biosafety level for your country’s regulations. In addition, all serum samples should be handled as if capable of transmitting infectious agents.

 For the full protocol download the user guide below                            Download User Guide

7.2. Results

After data submission, background subtraction will be performed for each measurement and the resulting data for all three titration series subjected to a global, non-linear fit to determine the following parameters:

Parameter Definition Constraints
Rh, free Hydrodynamic radius of the unbound
Alexa Fluor™ 647–RBD (nm)
Shared, must be greater than zero
Rh, complex Hydrodynamic radius of the antibody-bound Alexa Fluor™ 647–RBD (nm) Shared, must be greater than zero
A Antibody concentration in the sample
serum (nM)
Shared, must be greater than zero
n Stoichiometry factor Fixed to 1.0
P Concentration of Alexa Fluor™ 647–RBD in each experiment (nM) Fixed to 10, 40 and 100 nM respectively
KD Dissociation constant (nM) Shared, must be greater than zero

Please note: The shape of the binding curves will vary between patient samples. If neither of the three curves reaches a plateau at the highest serum concentrations, samples might contain low amounts of antibodies. Please contact
technical support for more information.

 
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