A-385 A Point-of-Care Test to Assess Vaccine Response and Recent Infection by Quantitative Measurement of Neutralizing Antibody, Anti-Nucleocapsid, and Anti-Spike Levels
Authors: Perebikovsky A , Lim J Y , Munoz H , Caparas M C , Fiore A , Srinivasan A , Okrongly D , Liu Y , Janwari H , Murray G , Martinez B , Le B , Rashidi K , Kido H , Roberts A C , Lu H , Pollack B , Shamloo E , Miller B D , Wright D , Davis R E 
Published: September 2023

Abstract

Background
Following vaccination against SARS-CoV-2 (SC2), the human immune system produces neutralizing antibodies (NAb) that block the virus from entering host cells. These NAb levels are highly predictive of protection against SC2 infection and progression to severe disease [1]. Unfortunately, individual humoral responses vary and NAb titers decay over time, making measurement of NAb important in timing vaccine boosters, especially for at-risk individuals.
Microneutralization assays (MNA) can be used to measure vaccine effectiveness by quantifying virus-specific NAb titers. However, these assays have long turnarounds, require a BSL3 lab, and have high inter-lab variability [2].
The Q-NAb™ test was developed to quantitatively measure NAb levels and correlate with MNA [3]. Q-NAb is available as both an ELISA for central laboratories, and a multiplexed POC centrifugal disc assay that measures NAb, anti-Nucleocapsid protein (NP), and anti-Spike antibodies.

Methods
The heart of the Q-NAb test is a recombinant fusion protein (FP) that blocks anti-RBD non-neutralizing antibodies (NNAb). FP consists of human ACE2 fused to the Wuhan-RBD of the SC2 Spike. Designed to conceal the the NAb binding epitopes of RBD, FP serves as a specific depleting agent for NNAb. The pretreated sample is then incubated with immobilized Wuhan-RBD to obtain a NAb readout.
Both Q-NAb formats were calibrated to WHO international standard 20/136 using a set of 7 calibrators and correlated to MNA results. NAb levels were determined using &gt;200 clinical samples collected 2–12 weeks post monovalent or bivalent vaccine booster and compared across age groups (&lt;60, 60–70, 70–80, 80+ years).

Results
Both Q-NAb Disc and ELISA show correlation to each other (n = 167, WLS, R2 = 0.85) and are traceable to international standards. Q-NAb also correlated with MN50: ELISA (n = 137, Spearman's ρ = 0.88) and Disc (n = 208, Spearman's ρ = 0.91). After receiving a bivalent booster, no difference in median NAb levels across age groups was observed (Kruskal-Wallis, P = 0.3). While 99.4% of samples had a positive anti-Spike, using a NAb cutoff of 1000 IU/mL derived from the literature [2, 4–5], we found the percentage of individuals below that cutoff increased with age (9.8% for &lt;60 years, 20.4% for 60–70 years, 19.5% for 70–80 years, and 34.1% for 80+ years). In addition, 32% of samples had an anti-NP level indicating recent infection.

Conclusion
The Q-NAb test, available in both central lab and POC formats, measures NAb levels, correlates to MNA, and is traceable to international standards. Q-NAb was used to assess humoral responses following vaccine boosters in a clinical sample set. While virtually all subjects had anti-Spike levels, the percentage of individuals falling below a NAb cutoff of 1000 IU/mL ranged from 9.8% to 34.1%, depending on age. Additionally, about one-third of patients had anti-NP activity, indicating NAb levels resulted from a combination of vaccination and infection. This data shows Q-NAb is an easily accessible and more accurate tool to identify a subset of at-risk patients that anti-Spike antibody tests alone do not discriminate.