Measurements of SARS-CoV-2 antibody dissociation rate constant by chaotrope-free biolayer interferometry in serum of COVID-19 convalescent patients

Abstract

Kinetics measurements of antigen-antibody binding interactions are critical to understanding the functional efficiency of SARS-CoV-2 antibodies. Previously reported chaotrope-based avidity assays that rely on artificial disruption of binding do not reflect the natural binding kinetics. This study developed a chaotrope- and label-free biolayer interferometry (BLI) assay for the real-time monitoring of receptor binding domain (RBD) binding kinetics with SARS-CoV-2 spike protein in convalescent COVID-19 patients. An improved conjugation biosensor probe coated with streptavidin-polysaccharide (SA-PS) led to a six-fold increase of signal intensities and two-fold reduction of non-specific binding (NSB) compared to streptavidin only probe. Furthermore, by utilizing a separate reference probe and biotin-human serum albumin (B-HSA) blocking process to subtracted NSB signal in serum, this BLI biosensor can measure a wide range of the dissociation rate constant (k_off), which can be measured without knowledge of the specific antibody concentrations. The clinical utility of this improved BLI kinetics assay was demonstrated by analyzing the k_off values in sera of 24 pediatric (≤18 years old) and 63 adult (>18 years old) COVID-19 convalescent patients. Lower k_off values for SARS-CoV-2 serum antibodies binding to RBD were measured in samples from children. This rapid, easy to operate and chaotrope-free BLI assay is suitable for clinical use and can be readily adapted to characterize SARS-CoV-2 antibodies developed by COVID-19 patients and vaccines.

Keywords: Avidity; Biolayer interferometry; COVID-19; Dissociation rate constant; Kinetics; SARS-CoV-2.

Fig. 1

Schematic illustration and polyclonal antibody validation of the SARS-CoV-2 antibody kinetics assay. A) BLI kinetics measurement in SARS-CoV-2 antibody positive serum samples. The SA-PS probe is loaded with Biotin-RBD and then saturated with Biotin-HSA as blocker. The probe is subsequently incubated into the sample well and then dissociation buffer to capture RBD specific antibodies and measure the dissociation rate (k_off) respectively. another probe with unloaded RBD is used as reference probe for NSB measurement. B) Data processing steps to calculate the specific binding signal and the k_off values.

Fig. 2

Improved performance of streptavidin polysaccharide (SA-PS) coated probe. A) NSB signal of pooled negative samples measurements by SA and SA-PS probe. B) Specific binding signal of PAb antibody sample measurements by SA and SA-PS probe. Grey area represents standard deviation (SD). Error bars represent as mean ± SD.

Fig. 3

Assay validation in patient serum samples. A) Three representative SARS-CoV-2 antibody positive samples (PS) measurements of total binding (TB) and non-specific binding (NSB). B). Three representative SARS-CoV-2 antibody negative samples (NS) measurements of total binding (TB) and non-specific binding (NSB). C) Specific binding signal calculated by subtracting the NSB from the total binding.

Fig. 4

The k_off measurement is SARS-CoV-2 antibody concentration independent. A) Specific binding signal measurement in original, 2-fold and 4-fold diluted samples. B) specific binding signal and k_off measurement of the original and diluted samples. C) Comparing specific binding signal of patient samples between original and 2-fold diluted samples (paired, two-tail t-test). D) Comparing k_off values between original and 2-fold diluted patient samples (paired, two-tail t-test).

Fig. 5

Dissociation kinetics assessment in SARS-CoV-2 antibody positive sera of convalescent children and adults. A) k_off vs specific binding signal of convalescent COVID-19 pediatric and adult serum samples (r, P values were test by Spearman correlation coefficient). B) specific binding signal and C) k_off of patient samples comparison in pediatric and adult serum. Error bars represent as median with IQR.