Validation of a Novel Fluorescent Lateral Flow Assay for Rapid Qualitative and Quantitative Assessment of Total Anti-SARS-CoV-2 S-RBD Binding Antibody Units (BAU) from Plasma or Fingerstick Whole-Blood of COVID-19 Vaccinees

Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL.


Introduction
Serological immunoassays are rapidly emerging with varying degrees of sensitivity and specificity [1,2]. These include enzyme-linked immunosorbent assays (ELISAs), chemiluminescence assays (CLIA), electrofluorescent assays (ELFA), and lateral flow assays (LFAs). Most serological assays measure the binding antibodies (Abs) response against specific SARS-CoV-2 antigens. Measuring binding Abs is insufficient for assessing protective immunity since these Abs do not play significant roles in virus neutralization [3,4]. Neutralizing antibodies (nAbs) are more indicative of protective immunity due to their ability not only to bind S-RBD, but also to block viral entry to the host cells [3,[5][6][7][8]. Therefore, neutralization assays remain the gold standard for measuring the nAbs titer against SARS-CoV-2 [9]. Nevertheless, nAb assays require high-level biosafety laboratories, highly trained personnel, and often take several days to complete [9]. To avoid such limitations, GenScript Biotech Corporation developed a reliable surrogate virus-neutralizing assay (known as sVNT or cPass) to measure the nAb. The cPass GenScript assay is now recommended by the World Health Organization (WHO) and has also received full USA FDA approval to be used as a reference assay for quantitative measurement of the nAb [10].
One of the major challenges for the commercially available serology assays is that their binding Ab results are provided in arbitrary units per milliliters (ARU/mL). Thus, the results between assays are highly variable, although they target the same SARS-CoV-2 antigen, usually the S-protein. To minimize the discrepancies between serological assays, the WHO introduced an international standard to harmonize the Abs immune response assessment and recommended reporting the assay results for binding activity in binding antibody unit per milliliter (BAU/mL) instead of arbitrary ARU/mL [11]. One of the best ways to identify a reliable assay that can be used as a surrogate assay to assess the quantity of neutralizing Abs post-vaccination is by performing correlation studies between these assays and a reference neutralization assay.
LFAs are attractive for small or non-laboratory settings and population surveillance. They are affordable and rely on easily accessible specimens such as fingerstick whole blood and provide the result within minutes. According to the literature, none of the few commercially available fluorescent LFAs that provide quantitative measurement of SARS-CoV-2 (in BAU/mL) to the S-RBD protein were validated. The FinecareTM2019-nCoV S-RBD Ab Test is a fluorescence immunoassay used along FinecareTM FIA Meters (reader) (Model No.: FS-113) for quantitative detection of S-RBD BAU in human fingerstick whole blood, venipuncture whole blood, and serum or plasma specimens [12]. For more information about the assay principle, see Figure S1. In this study, we evaluated the performances of FinecareTM and its fluorescent reader (FinecareTM-FIA Meter) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); (ii) three highly performing commercially available automated anti-SARS-CoV-2 immunoassays; Mindray CL-900i ® SARS-CoV-2 S-RBD IgG, BioMérieux VIDAS ® 3 [13,14], and Ortho VITROS ® [13,14].

Samples Collection and Ethical Approval
Participants who received two BNT162b2 or mRNA-1273 vaccine doses were eligible for inclusion. A total of 488 EDTA whole-blood samples (5.0 mL) were collected between April and October 2021 from staff and students at Qatar University, the largest national university in Qatar. Plasma was separated from whole venous blood and stored at −80 • C until performing the test. Demographic information and information on the previous infection with SARS-CoV-2 were collected through a self-administered questionnaire. Out of the total samples, 156 participants in the vaccinated group performed the FinecareTM test with fingerstick whole blood. The study was reviewed and approved by the Institutional Review Board at Qatar University (QU-IRB 1537-FBA/21). All analyses were conducted according to the ethical standards of the Declaration of Helsinki of the World Medical Association (WMA).

Fingerstick Samples
Briefly, the area of the fingertip was lanced with an alcohol pad. Then, the skin was punctured using a sterile lancet, and 20 µL of fingerstick blood was collected with the capillary sampler. The specimen was squeezed out into the detection buffer tube. After that, 75 µL of sample mixture was loaded into the sample well and was inserted into the test cartridge holder of FinecareTM FIA Meters. The reaction time was 15 min. The results unit was displayed as a relative fluorescence unit (RFU, AU/mL). To obtain the results in BAU/mL, the AU/mL values were multiplied by the WHO standard (20) that is provided by the manufacturer.

Plasma Samples
According to the standard phlebotomy procedure, venipuncture whole blood specimens were collected from each participant in EDTA tubes. Then, plasma was separated from blood immediately, and the test was performed as described above in Section 2.2.1.

cPass GenScript sVNT
A SARS-CoV-2 surrogate virus neutralization test (sVNT) (Cat. No. L00847, GenScript Biotech, Piscataway, NJ, USA) was utilized as a reference test for detecting nAbs against SARS-CoV-2. The test was conducted following the manufacturer's specifications. Briefly, samples and controls (provided within the kit) were diluted 1:10 with the sample dilution buffer provided with the kit. Then, 125 µL of sample/control was mixed in a 1:1 ratio with HRP-RBD solution and incubated at 37 • C for 30 min. After that, 100 µL of each sample/control was added to the hACE2-coated plate in duplicates. The plate was sealed and incubated for 15 min at 37 • C. Wells were then washed 4x with 200 µL of the wash solution provided. Then, 100 µL of the TMB solution was added per well and the plate was incubated in the dark for 15 min at room temperature. Finally, 50 µL of stop solution was added to each well to quench the reaction, and absorbance was read at 450 nm. % inhibition of ≥30% was considered positive and percent inhibition of <30% was considered negative. The conversion tool for cPass % inhibition to IU/mL of the WHO International Standard (NIBSC code 20/136) was calculated as established in [10].

Statistical Method
Correlation and linear regression analyses between FinecareTM and each commercial serological immunoassay and the sVNT were performed. Pearson correlation coefficient (r) was calculated. For absolute values of Pearson's r, 0-0.19 was denoted as very weak, 0.2-0.39 as weak, 0.40-0.59 as moderate, 0.6-0.79 as strong, and 0.8-1 as very strong correlation [19].
Concordance analysis between FinecareTM and the three automated assays was conducted, which includes the overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA), accuracy/efficiency as well as Cohen's Kappa statistic. Cohen's Kappa measure is a robust metric that estimates the level of agreement between two diagnostic tests. A Kappa value <0.40 denotes poor agreement, 0.40-0.59 denotes fair agreement, 0.60-0.74 denotes good agreement, and ≥0.75 denotes excellent agreement [20]. The significance level was indicated at 5%, and a 95% confidence interval (CI) was reported for each metric. All statistical analysis was performed using GraphPad Prism software (Version 8.2.1. San Diego, CA, USA).

FinecareTM Is a Very Specific and Sensitive Assay
To assess the specificity of FinecareTM, we tested 100 pre-pandemic plasma samples using FinecareTM as well as sVNT as a reference method. None of the tested samples were positive by any of the two assays, indicating 100% specificity of FinecareTM. Regarding the sensitivity, 156 plasma samples were tested by sVNT and FinecareTM. In total, 155 samples were positive by sVNT and FinecareTM. However, only one sample tested negative with sVNT but positive with FinecareTM, indicating 99.8% sensitivity of FinecareTM. Interestingly this sample tested negative when fingerstick whole blood samples were tested. The discrepancy between the results of the plasma and fingerstick could be due to a technical error between the two different technicians who performed the test.

FinecareTM Fingerstick and Plasma Results Are Comparable
We assessed the performance of FinecareTM using fingerstick whole blood samples. A total of 156 participants provided informed consent to receive a fingerstick and had venous blood drawn to generate matched plasma samples. These samples were tested using FinecareTM and the results were compared as shown in Figure 1. A very strong correlation was observed (r = 0.9, p < 0.0001) between fingerstick and venous plasma samples. Similarly, linear regression analysis showed that the constructed model could strongly predict the dependent variable (R 2 = 0.8, p < 0.0001). Most importantly, the interpretation concordance was 100%, demonstrating equivalency between fingerstick and venous plasma.

Moderate Correlation between FinecareTM and sVNT Using Fingerstick Whole Blood Samples
We evaluated the performance of FinecareTM in comparison to sVNT using whole blood samples (n = 156). Moderate significant correlation between FinecareTM and sVNT with r-value of 0.5 (p < 0.001) is shown in Figure 2. Most importantly, the interpretation concordance was 99.4%, as only one sample tested negative with sVNT but positive with Finecare TM . using FinecareTM and the results were compared as shown in Figure 1. A very strong correlation was observed (r = 0.9, p < 0.0001) between fingerstick and venous plasma samples. Similarly, linear regression analysis showed that the constructed model could strongly predict the dependent variable (R 2 = 0.8, p < 0.0001). Most importantly, the interpretation concordance was 100%, demonstrating equivalency between fingerstick and venous plasma.

Moderate Correlation between FinecareTM and sVNT Using Fingerstick Whole Blood Samples
We evaluated the performance of FinecareTM in comparison to sVNT using whole blood samples (n = 156). Moderate significant correlation between FinecareTM and sVNT with r-value of 0.5 (p < 0.001) is shown in Figure 2. Most importantly, the interpretation concordance was 99.4%, as only one sample tested negative with sVNT but positive with FinecareTM.

Moderate to Strong Correlation between FinecareTM and Three Commercially Available Automated Anti-SARS-CoV-2 Immunoassays Using Plasma Samples
The correlation and linear regression analysis between the readings obtained from FinecareTM and each automated immunoassay are illustrated in Figure 3. Pearson's correlation coefficients (r) showed a statistically significant positive correlation for

Moderate Correlation between FinecareTM and sVNT Using Fingerstick Whole Blood Samples
We evaluated the performance of FinecareTM in comparison to sVNT using whole blood samples (n = 156). Moderate significant correlation between FinecareTM and sVNT with r-value of 0.5 (p < 0.001) is shown in Figure 2. Most importantly, the interpretation concordance was 99.4%, as only one sample tested negative with sVNT but positive with FinecareTM.

Moderate to Strong Correlation between FinecareTM and Three Commercially Available Automated Anti-SARS-CoV-2 Immunoassays Using Plasma Samples
The correlation and linear regression analysis between the readings obtained from FinecareTM and each automated immunoassay are illustrated in Figure 3. Pearson's cor relation coefficients (r) showed a statistically significant positive correlation fo

Moderate to Strong Correlation between FinecareTM and Three Commercially Available Automated Anti-SARS-CoV-2 Immunoassays Using Plasma Samples
The correlation and linear regression analysis between the readings obtained from FinecareTM and each automated immunoassay are illustrated in Figure 3. Pearson's correlation coefficients (r) showed a statistically significant positive correlation for FinecareTM with all three automated assays (p < 0.001). The strongest correlation coefficient was shown with VIDAS ® 3 (r = 0.6, Figure 3A), followed by VITROS ® (r = 0.5, Figure 3C), and CL-900i ® (r = 0.4, Figure 3B). Linear regression analysis showed that all constructed models could statistically significantly predict the dependent variable (Fine care (BAU/mL)). The best regression model fitting the data was shown by VIDAS ® 3 (R 2 = 0.3), followed by VITROS ® (R 2 = 0.3) and CL-900i ® (R 2 = 0.2).

Excellent Agreement between FinecareTM and the Three Automated Anti-SARS-CoV-2 Immunoassays
The tests' agreements were studied in a pairwise fashion by applying inter-rater agreement statistics (Cohen's Kappa statistic, k) ( Table 2). The OPA, PPA, and NPA were 100% between FinecareTM and the three automated immunoassays. In addition, Cohen's Kappa statistic denoted excellent agreement between FinecareTM and the three automated anti-SARS-CoV-2 immunoassays (κ = 1.00). Most importantly, the interpretation concordance was 100%, demonstrating equivalency between FinecareTM and the three automated anti-SARS-CoV-2 immunoassays.

Discussion
Despite this rapid progress in vaccines development, it is unlikely that COVID-19 will be eradicated in the near future. At best, COVID-19 infections can be brought under control to the point where life can return to "pre-COVID" normality [21]. A key part of this approach will be the deployment of high-throughput testing to determine who is immune and the duration of immune response [21]. The rapid LFA-POC Ab tests are simple, cheap, and fast. They do not require qualified personnel for interpretation and could be conducted in non-laboratory settings. The usefulness of rapid tests for the detection of SARS-CoV-2 Abs has been questioned regarding their inferiority compared to other serology assays. This is because none of the few commercially available LFAs provide quantitative measurement of SARS-CoV-2 (in BAU/mL) to the S-RBD. The FinecareTM S-RBD Ab Test used along FinecareTM reader is the first quantitative LFA for the detection of S-

Discussion
Despite this rapid progress in vaccines development, it is unlikely that COVID-19 will be eradicated in the near future. At best, COVID-19 infections can be brought under control to the point where life can return to "pre-COVID" normality [21]. A key part of this approach will be the deployment of high-throughput testing to determine who is immune and the duration of immune response [21]. The rapid LFA-POC Ab tests are simple, cheap, and fast. They do not require qualified personnel for interpretation and could be conducted in non-laboratory settings. The usefulness of rapid tests for the detection of SARS-CoV-2 Abs has been questioned regarding their inferiority compared to other serology assays. This is because none of the few commercially available LFAs provide quantitative measurement of SARS-CoV-2 (in BAU/mL) to the S-RBD. The FinecareTM S-RBD Ab Test used along FinecareTM reader is the first quantitative LFA for the detection of S-RBD BAU in human fingerstick whole blood, venipuncture whole blood, and serum or plasma specimens [12]. WHO International Standard (BAU/mL) is essential for facilitating the standardization of SARS-CoV-2 serological methods and allowing for the comparison and harmonization of datasets across laboratories. This is critical for determining the Ab levels that are needed for efficacious vaccines and protection from emerging variants such as Omicron [22]. Recent studies have shown that a two-dose COVID-19 vaccine regimen does not induce enough neutralizing Abs against the Omicron variant [22].
One of the major advantages of FinecareTM is using fingerstick whole blood samples and obtaining quantitative results within 15 min. Although Abs are more stable in serum/plasma samples, the whole blood samples are more convenient to use. Therefore, we assessed the performance of FinecareTM using fingerstick whole blood samples compared to venous plasma samples. A very strong correlation was observed (r = 0.9, p < 0.0001) between fingerstick and venous plasma samples. In addition, the interpretation concordance was 100%, demonstrating equivalency between fingerstick whole blood and venous plasma samples. Our results confirmed the feasibility of using fingerstick whole blood for SARS-CoV-2 IgG detection and showed excellent concordance with plasma values using FinecareTM assay. Collection of fingerstick whole blood samples in Microtainer tubes is quick and easy. It does not require a phlebotomist, making it an attractive alternative to venepuncture for use in SARS-CoV-2 drive-through testing sites. The simplicity of performing the LFA with whole blood also eliminates the need for centrifugation and plasma separation steps, reducing the cost and complexity of obtaining a result.
The correlation and linear regression analyses between the readings obtained from FinecareTM and each automated anti-SARS-CoV-2 immunoassay were evaluated. Each assay was selected for previously determined performance [13,14], ease-of-use characteristics (automated serology), and availability. Pearson's correlation coefficients (r) showed a statistically significant positive correlation between FinecareTM and all three automated assays (p < 0.001). The strongest correlation was shown with VIDAS ® 3 (r = 0.6), followed by VITROS ® (r = 0.5) and CL-900i ® (r = 0.4). The best correlation with VIDAS ® 3 was expected, as both assays are based on fluorescence immunoassay technology and solely target Abs against S-RBD. Cohen's Kappa statistic denoted excellent agreement between FinecareTM and the three automated anti-SARS-CoV-2 immunoassays. Interestingly, the interpretation concordance was 100%, demonstrating equivalency between FinecareTM and the three automated immunoassays. The excellent concordance between the POC FinecareTM and the automated serological assay makes it an attractive alternative for serological assays that do not require laboratory settings. The ability to rapidly, accurately, and affordably determine seroprevalence in a population will be an important tool in the growing arsenal of SARS-CoV-2 diagnostic testing and assessment of Ab response post-vaccination, particularly in resource-limited areas.
The major drawback of FinecareTM is the detection limit. Similar to other quantitative assays, false negative results might occur if the Abs concentration against the SARS-CoV-2 S-RBD is below the detection limit. If the concentration is above the detection limit, serial dilution of the samples would be necessary. On the other hand, false positive results might occur due to cross-reactivity from pre-existing Abs to other homologous and common antigens.
In conclusion, SARS-CoV-2 continues to spread and threatens to disrupt human activity for years to come. Fortunately, we already have vaccines that effectively reduce disease and are now being rolled out in many countries, but countering that, many countries have low vaccine coverage and ongoing high rates of infection, raising the potential for immune-mediated selection escape variants. FinecareTM should contribute to offering more widespread access to immunity testing for many populations, including those in resourcepoor areas with limited access to laboratories and equipment for more conventional virus neutralization tests or ELISA-based inhibition assays.