key: cord-0961709-nb1vfi1h
authors: Taylor, Sean C.; Hurst, Beth; Martiszus, Ian; Hausman, Marvin S.; Sarwat, Samar; Schapiro, Jeffrey M.; Rowell, Sarah; Lituev, Alexander
title: Semi-quantitative, high throughput analysis of SARS-CoV-2 neutralizing antibodies: Measuring the level and duration of immune response antibodies post infection/vaccination
date: 2021-08-06
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.07.098
sha: d1283adbaafb1206f2696f6482f11aa0aa25c7ee
doc_id: 961709
cord_uid: nb1vfi1h

The question associated with efficacy and longevity of SARS-CoV-2 protection post-vaccination is paramount. The cPass surrogate virus neutralization test (sVNT) has gained popularity globally as a dual application assay for: 1. Accurate SARS-CoV-2 population surveillance (seroprevalence) analysis and 2. Revealing the presence of antibodies that block and effectively neutralize the interaction between the SARS-CoV-2 receptor binding domain and the host cell ACE2 receptor in recovered or vaccinated individuals. This study describes an approach for accurate quantification of neutralizing antibodies using the cPass sVNT with an automated workflow on the Tecan EVO and Dynex Agility platforms that is applicable to other liquid handling systems. This methodology was used to assess the stability of SARS-CoV-2 neutralizing antibodies between freeze/thaw and refrigerated sample storage conditions. Furthermore, a subset of twenty-five samples from SARS-CoV-2 infected/recovered individuals revealed a 600-fold difference in the neutralizing antibody response where low titers were represented in about half of the samples. Finally, pre- and post-vaccination samples were tested for neutralizing antibodies using the qualitative and semi-quantitative cPass sVNT protocols revealing undetectable or relatively low levels after the first vaccine dose and a decline in levels longitudinally over the months following the second dose. This wide range in neutralizing (blocking) antibodies from both natural infection and vaccination supports a differential immune response that may be attributed to several physiological and genetic factors underlining the potential for measuring SARS-CoV-2 neutralizing antibody titer levels post-vaccination to help ensure robust and prolonged immunity.

low levels after the first vaccine dose and a decline in levels longitudinally over the months following the second dose. This wide range in neutralizing (blocking) antibodies from both natural infection and vaccination supports a differential immune response that may be attributed to several physiological and genetic factors underlining the potential for measuring SARS-CoV-2 neutralizing antibody titer levels post-vaccination to help ensure robust and prolonged immunity.

The cPass sVNT has proven to serve as a highly accurate serology assay that detects and measures the functional response of circulating antibodies that "block or neutralize" the interaction of the SARS-CoV-2 receptor binding domain (RBD) to the host cell ACE2 receptor [1] [2] [3] [4] [5] . These dual detection/screening and functional properties uniquely positions the test for: 1. population surveillance (seroprevalence) [6, 7] ; 2. vaccine development, associated clinical trials and post-vaccination follow-up testing [8, 9] ; 3. convalescent donor plasma and drug screening [10, 11] and 4. longitudinal testing to track neutralizing antibody levels post-vaccination. Considering the broad applications and potential large-scale need for cPass sVNT, migration to an automated liquid handling platform is critical. However, differences between the cPass sVNT competition/inhibition test methodology and those of more traditional antigen-coated, ELISA-based SARS-CoV-2 assays create unique challenges [4, [12] [13] [14] .

The current gold standard virus neutralization tests require live cells and virus in a BSL3 containment lab.

These complex, manual assays span two to four days, require specialized equipment and highly trained technicians [15] [16] [17] . With the advent of SARS-CoV-2 global vaccination programs with vaccines of varying efficacies [18] [19] [20] , it is important to correlate post-vaccination immune responses with the duration of protection against reinfection. Understanding this temporal component may be important in preventing future SARS-CoV-2 pandemics and outbreaks.. Recent studies from SARS-CoV-2 infected individuals show antibody titers decline after recovery, distinct immunotypes between infected individuals, and weakened immune responses in older adults [21] [22] [23] [24] [25] [26] . This may warrant regular measurement of neutralizing antibody titers post-vaccination. Thus, a technology that permits the direct comparison of neutralizing antibody levels between samples while circumventing time-consuming and cost-prohibitive live cell neutralizing antibody assays like the plaque reducing neutralizing antibody test (PRNT) could be beneficial [17, 27, 28] . The cPass sVNT has been shown to give comparable data to live cell tests without the extensive processing and complexity in a 96-well plate-based assay that requires approximately 1.5 hours to qualitatively interrogate up to 92 samples [1] [2] [3] [4] [29] [30] [31] . While the improved workflow with the sVNT is beneficial, further improvements can be made by transforming the assay to a fully automated and semi-quantitative test. This would facilitate at-scale, continued longitudinal assessment of neutralizing antibody titers in post-vaccination populations.

Accurate SARS-CoV-2 antibody testing also requires knowledge of neutralizing antibody stability in serum/plasma samples at 4 o C and -80 o C with multiple freeze/thaw cycles. Although the data are sparse, some work has been performed in the past with dengue, measles, mumps, and rubella as well as anticardiolipin immune response antibodies that give varying degrees of stability [32] [33] [34] . Considering the real-world variability in sample acquisition [35, 36] , processing and storage [37] , a better understanding concerning how those conditions impact assay results could improve downstream data accuracy and the conclusions drawn from this semi-quantitative test.

A methodology for automation and production of semi-quantitative data from the cPass sVNT is described. A set of SARS-CoV-2 positive and negative samples were qualitatively screened and delineated with this platform with selected samples processed using a novel, semi-quantitative protocol.

Longitudinally collected samples from individuals both pre-and post-vaccination were tested using the qualitative and semi-quantitative cPass sVNT protocols. Finally, a subset of samples was assessed for stability under a number of freeze/thaw and refrigerated conditions to reveal the effect of storage on the measured antibody titers. Taken together the results pave a practical path forward to achieve accurate, high-throughput and semi-quantitative SARS-CoV-2 immune response testing in a post-COVID vaccination world

Seventy-four COVID-19 presumed positive samples collected for clinical diagnostic purposes were subsequently de-identified and released to the Kaiser Permanente (KP) Research Bank for secondary use. Fifty-seven of the presumed positive samples were used to compare the performance of cPass sVNT with two commercial IgG binding assays (see subsection "SARS-CoV-2 ELISA Tests" below). The remaining 17 samples were allocated to refrigerated and freeze/thaw stability studies. Twenty-nine serum samples collected prior to August 2019 (pre-pandemic) were randomly selected from the KP Research Bank Biorepository and subsequently deidentified. The SARS-CoV-2 positive serum specimens were collected from 3 to 15 weeks post PCR testing, processed in BD Vacutainer Serum Separation Transport Tubes (SST) according to the standard clinical protocol in CAP/CLIA certified Kaiser Permanente laboratories. These samples were stored for 3-15 days refrigerated prior to shipment to the KP Research Bank laboratory. All specimens except those used for stability testing, were introduced to one freeze/thaw cycle. Pre pandemic specimens were stored at -80 o C prior to testing.

The data in Figure 5A Pathologists (CAP) accredited (CAP ID # 9511943) BSL2 facility. The Biorepository adheres to industry best practices for processing, long-term storage, retrieval and distribution of specimens. The data generated from vaccinated individuals was derived from Cayman Chemical Company and Cure-Hub LLC.

The SARS-CoV2 cPass Surrogate Virus Neutralizing Test (sVNT) (GenScript sVNT (Piscataway, NJ) #L00847) utilizes the recombinant receptor binding domain (RBD) of the SARS-CoV-2 spike protein to detect antibodies that block the RBD from binding to hACE2 receptor [4] . A neutralizing antibody standard curve was designed to validate the kit and semi-quantitatively assess neutralizing antibody titers in diluted samples. Monoclonal Neutralizing Antibody (MAB) (GenScript -#A02051) was diluted in negative control matrix (SARS-CoV-2 negative serum diluted 1:10 in kit-specific dilution buffer) to a concentration of 300 ng/mL. This stock solution was then serially diluted 1:2 in negative matrix control (ie: a pool of SARS-CoV-2 negative serum diluted 1:10 in the kit-provided sample dilution buffer) for standards 2 through 7. Since each dilution is mixed with an equivalent volume of RBD conjugated horseradish peroxidase (RBD-HRP), the final, starting concentration for the MAB standard curve was 150 ng/ml ( Figure 3A ). Diluted negative matrix control alone was used for background wells as well as the kit 

The samples were tested using Tecan EVO200 liquid handler with 8 Liquid Handling Arm (LiHa), 96 channels Multichannel arm (MCA) and robotic manipulator arm (ROMA), integrated 1D/2D Ziath scanner and Tecan M200 Infinite multimode detector. Two Tecan EVO scripts have been developed for each respective method: 1) Batch testing for qualitative analysis and 2) Sample of interest serial dilutions for semi-quantitative testing. Serial dilutions were prepared using 8 channels arm without changing the tips from high to low dilution. 96 MCA was used to decrease processing time and ensure simultaneous introduction of all test samples, controls and dilution series to various reagents or reaction components. The kit components and test sample have various liquid properties and pipetting volumes.

Customized liquid classes have been developed/tested to accommodate various liquid properties, minimize droplet formation, ensure proper mixing, decrease dead volumes and minimize sample losses due to the tip retention. Integrated Tecan M200 Infinite multimode detector was used for on-deck dark ambient incubation and for absorbance detection. During all steps of the sample testing, sample dilution and test plates were moved using the Robotic Manipulator inside the instrument enclosure. To support completely unsupervised testing, photosensitive reagents (TMB) should be kept in a light protective trough or pipet the reagent directly from the container covered by perforated foil. Instead of manual test plate patting after wash steps, maximizing the removal of residual buffer is recommended by adjusting the MCA aspiration high (Z max close to the plate wells bottom) without touching the well surface.

The Dynex Agility was programmed to run the cPass sVNT with scripts produced for both qualitative (delineation between positive and negative) and quantitative (determination of neutralization antibody titers) sample analysis. Since the Dynex systems use a single channel pipette for sample transfer and mixing solutions and an eight-channel wash station, scripting to ensure consistent well-to-well incubation times was the major challenge. Minimizing incubation lag times and accounting for any extended lag times through software programming ensured high quality data.

For both the Tecan and Dynex systems, the cPass sVNT kit instructions for use were modified to a fully room temperature protocol to: a) eliminate pipetting lag times and intraplate data drift and b) produce a more streamlined protocol that is more adaptable to automation platforms that may not be equipped with temperature-controlled incubation. The incubation times were modified as follows:

1. Neutralization Reaction: changed from 30 minutes at 37 degrees to 45 minutes at room temperature.

2. Incubation of neutralization reaction mixture in the ACE2-coated assay plate: altered from 15 minutes at 37 degrees to between 20 to 25 minutes at room temperature (depends on lab temperature).

3. TMB substrate reaction: altered from 15 minutes at 25 degrees to between 18 to 25 minutes at room temperature (depends on lab temperature).

Also, to accommodate the dilution effect on TMB from residual wash buffer giving a reduced OD450 signal, the volume of TMB was adjusted to from 100µL to 200µL for the Tecan and 125µL for the Dynex systems.

GraphPad Prism software was used for all graphical representations of the data as well as statistical analysis.

The product insert for the cPass sVNT calls for 37 degree and ambient temperature incubations at specific steps in the protocol. Using the Tecan automation platform, an optimized ambient temperature procedure was contrasted with the standard protocol giving comparable results (ie: within 20% CV for the second and third dilutions within the linear range) for both OD450 (Figures 1A1 and 1A3 ) and % neutralization ( Figures 1A2 and 1A4) . However, the ambient, automated protocol requires a manual manipulation of the plate to remove excess wash buffer by patting the inverted plate against paper towel. The residual wash buffer remaining in the wells after washing was about 20µL which significantly diluted the 100µL of TMB required for the standard protocol leading to a decreased OD450. The effect of 20µL residual wash buffer was tested in the presence of 100uL, 150uL and 200uL TMB against a control without residual wash buffer revealing a significant difference on OD450 ( Figure 1B2 ) but virtually none on % neutralization ( Figure 1B1 ). Similar data were obtained using the Dynex Agility system which had a smaller volume of residual wash buffer (about 7µL) thus requiring 125µL of TMB.

Key considerations to achieve high quality data for semi-quantitative analysis of SARS-CoV-2 immune response with automated liquid handling platforms

To avoid system induced data drift, it is of critical importance for cPass sVNT (and most competitionbased ELISA tests) that incubation times are strictly adhered and care is taken to minimize well-to-well differences to avoid introducing system induced drift in the data. Thus, especially for single but also for multichannel pipetting robotic manifolds, pre-diluting the samples, standards and controls into a "sample/standards plate" will minimize lag times. Pre-diluted samples/standards can then be mixed with the RBD-HRP substrate (see materials and methods) in a separate "neutralization reaction plate". Furthermore, when transferring the neutralizing reactions to the ACE2-coated assay plate, the well-towell transfer lag time should be factored into the plate wash protocol. The following key points should be considered for programming the pipetting and plate washing with an automated liquid handling platform cPass sVNT:

1. Pre-dilute samples, standards and controls into a 96-well "Sample Plate".

2. Pre-Dilute the RBD-HRP into a pipetting trough or 96-well "RBD-HRP Plate".

Reaction Plate" accounting for lag times between wells to ensure all wells are incubating for the same time (45min at room temperature or 30 minutes at 37 o C) prior to transfer to the ACE2coated assay plate. 4 . Wash the ACE2-coated assay plate such that the lag time in transferring the neutralization reaction mixtures to the ACE2-coated assay plate is factored to ensure each well has incubated for a specific time in the 20 to 25 minute range at room temperature or 15 minutes at 37 o C before washing. 5 . Add TMB to the washed ACE2 coated assay plate such that all wells have been incubating for a specific time in the 18 to 25 minute range at room temperature or 15 minutes at 25 o C in the dark before adding Stop Solution.

6. Add Stop Solution accounting for pipetting lag times between wells to ensure all wells were incubating for the same time with TMB (see step 5 above).

Room temperature scripts were written and successfully tested for the Tecan EVO and Dynex Agility systems enabling both qualitative and semi-quantitative sample analysis using cPass sVNT in a complete hands-off, walkaway, automated solution. The cPass sVNT, MAB dilution series (see Materials and

Methods subsection "SARS-CoV-2 ELISA Tests") can be applied to each column over a full plate to validate intra-plate precision at each dilution.

A set of 57 positive and 29 pre-pandemic negative samples were assessed with cPass sVNT, Platelia SARS-CoV-2 Total Ab and Lumit™ Dx SARS-CoV-2 Immunoassay tests (Figure 2 -red dots were false negative samples shared between the tests). The associated data were tabulated to summarize specificity, sensitivity and accuracy (Table 1 ). In order to migrate the test from the qualitative (delineation between SARS-CoV-2 positive and negative samples using a 30% cutoff) format to a semiquantitative measurement of neutralization antibody titers, each sample may require dilution that is dependent on the individual immune response to infection or vaccination. This dilution step reveals OD450 values that fall within the linear quantitative dynamic range for interpolation against a standard curve ( Figure 3) . The lower limit of quantification (LLOQ) of the standard curve ( Figure 3D) is defined by the OD450 that equates to the 30% neutralization cutoff derived by plotting the OD450 vs % inhibition ( Figure 3B ). The upper limit of quantification (ULOQ) ( Figure 3D ) for a 4PL fitted standard curve is commonly defined by the 95% confidence interval in the lower plateau [38] . Alternatively, the ULOQ can be set at a signal to noise of three [39] . For this test, the noise is the lowest measured OD from either the most concentrated standard curve dilution or the positive control and the LLOQ would be the OD450 of the noise multiplied by three.

A subset of the qualitatively assessed low ( Figure 4A and C) and high ( Figure 4B and D) positive samples were serially diluted and interrogated for antibody response with cPass sVNT. The summarized results from 25 samples revealed large neutralizing antibody titer differences ranging up to about 600-fold (Table 2 -"Fold Difference to Sample 1 By Titer"). However, the differences between the samples were much smaller or non-existent when using the % neutralization data obtained through qualitative analysis (Table 2 -"Fold Difference to Sample 1 By % Neutralization"). Figure 5A and 5B) or semi-quantitatively (ng/ml titers) ( Figure   5C and 5D). Although the Moderna and Pfizer vaccines gave a high % neutralization after the second dose, several samples were below the 30% cut-off and therefore negative for neutralizing antibodies after receiving the first dose.

Of the three people who received the Johnson & Johnson vaccine, one was previously infected and recovered from COVID-19 who had detectable levels of neutralizing antibodies prior to vaccination and very high levels 14 days post-vaccination ( Figure 5A ). Whereas the remaining two people were negative up to 2 weeks after vaccination and exhibited neutralizing antibody levels just above the 30% cut-off 39 days post-vaccination ( Figure 5B ).

Four subjects receiving the Pfizer vaccine were tested semi-quantitatively with cPass sVNT giving low or undetectable levels of neutralizing antibodies up until the second dose where levels rose rapidly and peaked within days ( Figure 5C ). At peak concentration, an approximate 3-fold difference in titer was observed between individuals (compare samples 154 and 156) and all samples exhibited a sharp initial decline in neutralizing antibodies that began to tail off approximately 30 days after the second dose. For sample 152, the % neutralization was qualitatively assessed using the standard 1:20 sample dilution at each time point to reveal a marked difference between the neutralizing antibody levels measured qualitatively versus quantitatively ( Figure 5D ).

With the combined semi-quantitative format and automated platform for cPass sVNT, stability testing was straightforward and fast. A set of 17 samples were semi-quantitatively tested according to the protocol described in Figure 3 for various refrigerated (9 samples) and freeze/thaw (8 samples) storage conditions to assess stability of measured antibodies. The data reveal a high degree of stability for circulating neutralizing antibodies with excellent tolerance to up to ten freeze/thaw cycles ( Figure 6A1 and A2) and refrigerated storage up to one week with a two-fold decrease after two weeks ( Figure 6B1 and B2).

An ambient temperature protocol with compensated incubation times was optimized for cPass sVNT and found to be comparable to the standard methodology ( Figure 1A ) which is useful for the many single channel liquid pipetting platforms that cannot support the temperature-controlled incubations.

Furthermore, since plate inversion and patting to remove residual wash buffer prior to addition of TMB is challenging on robotics liquid handlers, the addition of 100µL additional TMB for a total of 200µL was found to rescue the dilution effect on OD450 from residual wash buffer with the Tecan system ( Figure   1B2 ). For the Dynex Agility, 125µL was required (data not shown). However, the inhibitory effect of wash buffer on the % neutralization was minimal at all antibody dilutions ( Figure 1B1 ). The scripts for qualitative population surveillance (seroprevalence) and semi-quantitative analysis of immune response neutralizing antibody titers using cPass sVNT were produced for both the Tecan EVO and Dynex Agility.

SARS-CoV-2 positive and pre-pandemic negative samples were qualitatively screened to delineate positive samples ranging from low (31%) to high (96%) neutralization in addition to a pre-pandemic negative group (Figure 2 ). These data substantiate the high sensitivity, specificity and accuracy of the cPass sVNT test (Table 1 ) versus other commercial serology assays with previous studies [2-4, 29-31, 40] .

The two cPass sVNT-derived false negative samples were shared with the Lumit test and one was shared with the Platelia assay ( Figure 2 -red dots). Both samples (red dots) were clinically characterized as asymptomatic and likely true negatives for SARS-CoV-2 infection.

In simple terms, vaccination should illicit a robust and general immune response that stimulates T-cell, memory B-cell and frontline neutralization antibodies that together ensure long term protection from the disease for several months or years [18, 19, 41] . However, for SARS-CoV-2, recent studies have shown that the immune response from infected/recovered individuals may be more complicated with declining immune response antibodies and differential T and B-cell levels post-infection particularly in the aging population [21-26, 42, 43] . Furthermore, a recent study has shown that neutralizing antibodies are highly predictive of immune protection from SARS-CoV-2 infection [44] . Thus, periodic assessment of neutralizing antibody titers post-vaccination may be warranted to determine cutoff titer levels triggering booster vaccinations for ongoing protection.

Since individual neutralizing antibody response to infection and vaccination can vary widely [8, 9, [45] [46] [47] , the dilution of samples is required to ensure the data fall within the linear quantitative dynamic range of the assay (Figure 3 ). However, it is important to note that some variability in the data will be inherent to the effect of matrix dilution on the antibody activity [48, 49] . This was exemplified by examining the neutralizing antibody titers derived from multiple dilutions within the linear range of the same samples revealing up to a 40% difference between the dilution-factor corrected concentrations (samples 8029054639 and 8029054652 in Figure 4B (data not shown)). However, the larger % differences between the dilution-factor corrected concentrations that exceeded 20% were more associated with comparing sample dilutions that were near the ULOQ and LLOQ where variability increases. This underlines the "semi" quantitative nature of serology assays requiring different sample dilutions to achieve data points within the linear quantitative range of the standard curve whereby variability can arise from both matrix dilution and standard curve interpolation.

A subset of 25 positive samples qualitatively delineated between 31% and 96% neutralization ( Table 2 "% Neutralization using 1:10 Dilution Factor (Qualitative Analysis)") were serially diluted 1:3 from an Table 2 "% Neutralization using 1:10 Dilution Factor (Qualitative Analysis)"). The data support a diverse response to SARS-CoV-2 infection with a broad range of neutralization titers ranging up to 600-fold (Table 2 -"Fold Difference to Sample 1 By Titer") that is likely dependent on multiple factors including age, general health and environment [45] .

For vaccination, the neutralizing antibody response was generally low to negative after the first dose of both the Pfizer and Moderna vaccines ( Figure 5A Figure 5B ) or within two weeks after the second dose (Pfizer and Moderna). However, there remains the question concerning how long immunity persists after vaccination and although the sample set was limited to four subjects, there was a consistent and rapid initial decline in neutralizing antibody titers after the second dose of Pfizer vaccine ( Figure 5C ). Furthermore, the qualitative (% Neutralization) data did not reveal the downward quantitative trend in neutralizing antibody titers (ng/ml) as shown with sample 152 ( Figure 5D ) which is a direct consequence to the sample dilutions required semi-quantitative titer analysis.

Given that cPass sVNT, gives highly comparable results to the gold standard live cell neutralization tests [1, 3, 4, 29] , these data support the use of the semi-quantitative protocol for cPass sVNT in comparing neutralizing antibody titers between SARS-CoV-2 vaccinated or infected/recovered individuals longitudinally. At the very least, samples can initially be measured two-weeks post-vaccination using the qualitative cPass sVNT protocol at a single 1:20 dilution factor to ensure the presence of neutralizing antibodies. As immunity wanes over time, subjects can potentially submit samples several months postvaccination for semi-quantitative analysis (Figure 3 ) to determine and more closely follow the titer levels.

The stability of circulating antibodies from viral infections has remained a topical and important subject for sample testing globally [33, 34] . Selected samples quantified using cPass sVNT underwent successive freeze/thaw cycles ( Figure 6A ) and refrigerated storage up to two weeks ( Figure 6B ). Both refrigerated storage and freeze/thaw cycles showed little difference (well within a log change) in antibody stability.

However, between one and two weeks at 4 degrees did give an approximate two-fold decrease that was statistically significant ( Figure 6B2 ). Since the collection and storage of samples at various locations does not permit storage under optimal laboratory conditions, these data provide evidence that even suboptimal storage conditions do not greatly affect antibody stability such that the samples may remain useful for downstream analysis.

The cPass sVNT provides a highly accurate dual functional test for population surveillance and semiquantitative analysis of neutralizing antibody titers post-infection and vaccination. The assay protocol was modified to permit ambient temperature incubations ( Figure 1A ) with accommodation of residual wash buffer ( Figure 1B) permitting straightforward migration to automated liquid handing systems.

Using automation supports accurate population screening for seroprevalence and contact tracing ( Figure 2 and Table 1 ). The application of cPass sVNT for semi-quantitative analysis of immune response antibodies was achieved by broadly diluting a subset of samples (Figure 3) to reveal a wide range (600 fold in our sample set) in neutralizing antibody titers between recovered individuals (Table 2 -"Fold Difference to Sample 1 By Titer"). Furthermore, the qualitative protocol permitted the longitudinal delineation of neutralizing antibody positive and negative samples both pre-and post-vaccination and the quantitative protocol revealed a decline in neutralizing antibody titers post-vaccination ( Figure 5 ).

Finally, using assay automation and semi-quantitative analysis permitted stability testing of positive samples to elucidate the effect of various sample storage conditions ( Figure 6 ).

The cPass SARS-CoV-2 Neutralization Antibody Test is CE Marked for diagnostic use in European Union Figure 3C ) to achieve semi-quantitative titers (red, blue and green).

The LLOQ is defined in Figure 3B and the upper limit of quantification (ULOQ) is determined from either the 95% confidence interval within the standard curve lower plateau [38] or by using a signal to noise of three (ie: 3 multiplied by the lowest OD450 between the most concentrated point on the standard curve or the positive control) [39] . E. Calculate the final Neutralizing Titer. Calculate the product of the interpolated titers from the standard curve ( Figure 3D ) and the sample dilution factors required to achieve the linear range ( Figure 3C ) as shown for samples with high (red) medium (blue) and low (green) neutralization titers. All steps were performed with the Tecan EVO automation system. freeze/thaw cycles were assessed for samples with high and low neutralization antibody titers (A1) with no statistically significant difference between them as measured with a one way ANOVA (multiple comparisons) test on the combined, normalized sample data (A2). B. Refrigerated storage. Samples stored for one and two weeks at 4 degrees were tested (B1). Although, there was a significant difference between the baseline/1 week and the 2 week refrigerated cycles the decrease in titers were only about two-fold (B2). For both refrigerated and frozen stability, all samples were tested semi-quantitatively as described in Figure 3 . Figure 3D ). The titers are then multiplied by the associated sample dilution factor (described in Figure 3E ) used for standard curve interpolation to give the "Final Neutralizing Titer" from which the "Fold Difference to Sample 1 By Titer" can be calculated. Also represented are the "% Neutralization Using 1:20 Dilution Factor" values used for qualitative analysis (first column) and the associated "Fold Difference to Sample 1 By % Neutralization" (last column).

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We thank Lisa Wilson and FourthWall Testing LLC as well as and Cure-Hub LLC for their generous contribution in longitudinal data from vaccinated individuals. This work was supported by the Kaiser Permanente Research Bank (KPRB) and the Kaiser Permanente NCAL TPMG Regional Laboratory, Medical Director Dr. Jeffrey Schapiro, Technical Director, Microbiology LaRonda S. Frazier and Lab Clinical R&D Scientist Ivy Yeung. We also wanted to acknowledge contribution of KPRB Lab Support Specialists, Shiyun Liang, Joe Nunoo and Shigeshi Yamamoto. We are grateful to Dynex for conducting the feasibility study of cPass sVNT on their Agility automated liquid handling systems.