Abstract
Background
SARS-CoV-2 Spike protein Receptor Binding Domain neutralizing antibodies (NAbs-RBD) inhibit the viral binding to angiotensin-converting enzyme 2 (ACE2) receptors. We compared an ELISA and a fluorescence immunochromatography (FIC) method in NAbs-RBD detection after COVID-19 immunization.
Method
Serum samples from healthcare workers (HCWs) vaccinated with BNT162b2 were collected one and four months after the second dose. NAbs-RBD (%) detection was performed using ELISA cPass™ (FDA approved) and FIC n-AbCOVID-19® assays.
Results
Samples from 200 HCWs [median age (IQR): 45(35−53)] were tested with both assays. There was a good qualitative agreement between the two methods [AUC: 0.92(95%C.I.: 0.89–0.94, P-value:0.007)]. NAbs-RBD (%), one and four months after immunization, were significantly lower with FIC compared to ELISA for all age groups (P-value<0.0001). The quantitative comparison between FIC and ELISA detected slight agreement one month after the second dose [(Lin’s Concordance Correlation Coefficient (CCC): 0.21(95%CI: 0.15–0.27)] which improved four months after the second dose [CCC: 0.6(95%CI: 0.54–0.66)].
Conclusion
FIC had good qualitative agreement with ELISA in the detection of positive NAbs-RBD (%) and could be an alternative for rapid NAbs-RBD (%) testing.
Keywords: SARS-CoV-2, COVID-19, Neutralizing antibodies, Enzyme-linked immunosorbent assay, Immunofluorescence, Vaccine
1. Introduction
Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic with more than 600 million confirmed cases worldwide (Center for Systems Science and Engineering, 2021).
The SARS-CoV-2 spike protein is responsible for viral attachment and fusion with the membrane of a host cell. SARS-CoV-2 can successfully enter human cells through the receptor for angiotensin converting enzyme 2 (ACE2) as a result of increased affinity (Xu et al., 2020). The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a major target for specific antibodies that block viral entry and replication in host cells (Dogan et al., 2021).
Total and neutralizing antibodies against the Receptor Binding Domain of SARS-CoV-2 Spike protein (TAbs-RBD and NAbs-RBD, respectively) are commonly used to evaluate humoral immune response after SARS-CoV-2 vaccination. NAbs-RBD are considered of great importance for the protection of breakthrough SARS-CoV-2 infection, since they directly inhibit the binding of the SARS-CoV-2 spike protein through the RBD region to ACE2 receptors (Dogan et al., 2021).
Several SARS-CoV-2 serological immunoassays based on different methodology principles have been developed for the detection of NAbs-RBD. The enzyme-linked immunosorbent assay (ELISA) is considered the gold standard method for evaluating neutralizing activity after COVID-19 immunization (EUA Authorized Serology Test Performance, FDA, 2022). New, rapid, easy-to-use, and cost-effective point-of-care tests, usually based on immunofluorescence, are required to be standardized for NAbs-RBD determination. However, the agreement between the gold standard and point of care immunoassays for NAbs-RBD detection is still unknown.
The purpose of this study was to compare the performance of two NAbs-RBD (%) detection immunoassays, the gold standard ELISA, and the rapid fluorescence immunochromatography (FIC) methods, in healthcare workers (HCWs) who received two doses of Pfizer/BioNTech BNT162b2 mRNA COVID-19 vaccine. The results were evaluated against epidemiological and clinical parameters.
2. Materials and methods
2.1. Study design and participants
This is a cohort study involving HCWs of “Aghia Sophia” Children’s Hospital in Athens, which is the largest tertiary pediatric hospital in Greece. The cohort of the study included healthcare professionals (medical doctors, nurses, technicians) who received the first two doses of the Pfizer/BioNTech BNT162b2 mRNA COVID-19 vaccine in January 2021.
Neutralizing antibodies have the highest values one month and significantly decline approximately four months after the second dose of BNT162b2 vaccine (Levin et al., 2021). Thus, evaluating neutralizing antibodies at these time points allow us to investigate the agreement of the two methods at both high and lower levels.
A demographic and clinical data collection form was completed by each participant. HCWs with a history of laboratory-confirmed natural SARS-CoV-2 infection or immunocompromised conditions during the study period were excluded from the study.
Serum samples were collected in two time points for all participants: one month and four months after the second dose of the BNT162b2 mRNA COVID-19 vaccine. Blood samples were collected in SST tubes (Becton Dickinson, New Jersey, USA) and centrifuged for 10 min at 3400 rpm. Serum samples were tested by the two methods in parallel. ELISA was performed prospectively, while immunofluorescence was performed retrospectively with the same tube. Serum samples were thawed once.
The study protocol was approved by the Scientific and Bioethics Committee of the ‘Aghia Sophia’ Children’s Hospital (No. 2794) and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.
2.2. Anti-RBD neutralization assays
The neutralizing activity of antibodies against the receptor binding domain (NAbs-RBD; %) were measured using two different assays. ELISA was performed prospectively, while FIC was performed retrospectively. All the reagents used in both assays had the same lots.
The first assay was the SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript Biotech Corporation, Piscataway, New Jersey, USA). This method is a blocking ELISA using the horseradish peroxidase conjugated recombinant SARS-CoV-2 RBD fragment and the human ACE2 receptor protein. The optical density (OD) was measured at 450 nm in the Labtech LT-4500 microtiter plate reader. The percentage of RBD-specific neutralization antibodies is calculated by the following type: Percentage signal inhibition (%) = (1-OD value of sample/OD value of negative control)* 100. Percentages of ≥ 30% are considered as positive. This method has been approved by FDA due to its high sensitivity and specificity rates (100%) (EUA Authorized Serology Test Performance, FDA, 2022) and was regarded as the gold-standard method for the purposes of this study.
The second assay was rapid fluorescence immunochromatography (FIC) testing, which was performed on the Axceed-P200 analyzer (Bioscience Diagnostic Technology Co., Ltd., Tianjin, China) using the n-AbCOVID-19® reagent (DyonMed S.A., Glyka Nera, Greece). When NAbs-RBD are present in the serum, they bind to the fluorescent RBD protein in the detection area of membrane (Test zone) during a 15-minute incubation in room temperature. Then, the fluorescence signal is measured by the analyzer. The interpretation of the results is based on the calculation of the degree of inhibition [(negative control sample to be tested)/negative control* 100%)]. Percentages ≥ 20% are considered positive. According to manufacturer, sensitivity and specificity of FIC are 95% and 99%, respectively.
2.3. Statistical analysis
Data are presented with median values and interquartile range (IQR) due to skewed distribution. Normality was tested with Anderson-Darling and Shapiro-Wilk tests. The comparison of continuous data was tested with the independent non-parametric test Kruskal-Wallis for more than two categories. Multiple comparisons were checked with the pairwise Wilcoxon rank sum test with Bonferroni correction. The comparison of dependent continuous data was tested with the Wilcoxon Signed Rank test. The Receiver operating characteristic (ROC) curve was calculated to detect the positive percentage of agreement between the cut-off points of these two methods. The area under the curve (AUC) designates the goodness of the test. AUC values < 0.5 indicate that the test is not useful, 0.5–0.6 bad, 0.6–0.7 sufficient, 0.7–0.8 good, 0.8–0.9 very good and 0.9–1.0 an excellent diagnostic precision, respectively (Šimundić, 2009). The P-values of AUCs (Mann-Whitney) and the comparisons (Venkatraman test) were computed with pROC package. The 95% CI were calculated with 2000 stratified bootstrap replicates. Positive percentage agreement (PPA), negative percentage agreement (NPA), overall percentage agreement (OPA), and Cohen’s Kappa coefficient were calculated to detect the concordance between the results of ELISA and FIC. Kappa values ≤0.2 indicate slight agreement, 0.2–0.4 fair agreement, 0.4–0.6 moderate agreement, 0.6–0.8 substantial agreement and ≥0.8 almost perfect agreement (Wongpakaran et al., 2013). The ideal agreement between two methods could be a linear relationship with zero intercept and a slope equal to one. An intercept that is not statistically different from zero indicates that there is no deviation between the two methods. When the gold standard method is zero, then the method of interest will be zero. A slope that is not statistically different from one indicates that there is no overestimation or underestimation between these two methods. An increment in gold standard method (ELISA) by 1 unit corresponds to an increase in the method of interest (FIC) by 5 units. Deming regression is an improved method for regression analysis that is free from the usual simplifying assumptions and is generally applicable to linearly related method-comparison data. Deming regression produces statistically unbiased estimates of systematic bias and reliable confidence intervals of bias for all cases (Martin, 2000). Lin’s concordance correlation coefficient (CCC) and Bland-Altman plots reflect the agreement between ELISA and FIC concerning the reliability of their values. CCC and 95% CI were evaluated using epiR package at both time points using bootstrap methods. Accuracy index is the observed proportional agreement between ELISA and FIC and was calculated as the sum of the diagonal values divisible by the sum of all the values of the table.
3. Results
3.1. Study population
A total of 200 HCWs who received the first two doses of the BNT162b2 vaccine 21 days apart were enrolled in the study. The median age (IQR) of the participants was 45 (35−53) years old. HCWs were divided into 3 age groups; 20–35 y: 53 (26.5%), 35–50 y: 80 (40%) and 50–65 y: 67 (33.5%). The median value of BMI (IQR) was 24.2 kg/m2 (21.6–27.1 kg/m2). Among the 200 HCWs, 147 (73.5%) were females, 62 (43.06%) had O blood type, 52 (26%) had allergies, 44 (22%) had a history of autoimmune disease, 42 (21%) were smokers and 16 (8%) had hyperlipidemia.
3.2. Qualitative comparison of results from ELISA and FIC Neutralizing activity
ROC analysis of ELISA and FIC showed that FIC could accurately predict the number of HCWs with positive and negative neutralizing activity with AUC= 0.92 (95% C.I.: 0.89–0.94, P-value: 0.007) for both time points of the study ( Fig. 1). The estimated AUC from the ROC analysis one and four months after the second dose did not vary significantly (P-value: 0.722). One month after the second dose, AUC was estimated at 0.98 (95% C.I.: 0.49–1, P-value: 0.051), while four months after the second dose, AUC was estimated at 0.86 (95% C.I.: 0.6–1, P-value: 0.04).
Fig. 1.
Receiver operating characteristic (ROC) analysis of ELISA and fluorescence immunochromatography for the whole study period, as well as one and four months after the second dose of the COVID-19 BNT162b2 mRNA vaccine respectively. Cut-off values of ELISA and fluorescence immunochromatography are 30% and 20%, respectively according to the manufacturer’s instructions. The estimated AUCs from the ROC analysis one and four months after the second dose were estimated at 0.975 (95% CI: 0.489–0.999, P-value: 0.051) and 0.861 (95% C.I.: 0.597–0.999, P-value: 0.04), respectively.
For ELISA and FIC, PPA, NPA and OPA were estimated at 98.2% (95% confidence interval (CI): 98.4–100), 25% [20.8–29.2] and 97.5% (96−99) for the whole study period, respectively. Cohen’s kappa coefficient was estimated at 0.17 (95% CI: 0.11–0.24) for the whole study period.
3.3. Quantitative comparison of results from ELISA and FIC
The quantitative comparison of ELISA and FIC on the neutralizing activity between one and four months after the second dose are presented in Table 1, Table 2 and Fig. 2.
Table 1.
Quantitative comparison of the median NAbs-RBD values (%) between ELISA and FIC per age group. Statistical analysis was performed using the Wilcoxon signed rank test(a) or Kruskal-Wallis test (b). Statistically significant differences (P-value<0.05) are marked in italics. Values represent median [IQR] (%).
| Time after mRNA immunization | 1 Month |
4 Months |
||||
|---|---|---|---|---|---|---|
| Age (Years) | ELISA (%) | FIC (%) | P-value | ELISA (%) | FIC (%) | P-value |
| 20–35 n = 53 (26.5%) |
96.1 [94.5–96.8] |
78.3 [76.1–80.3] |
< 0.001a | 94.3 [87.7–96.5] |
77.6 [72.2–80] |
< 0.001a |
| 35–50 n = 80 (40%) |
95.1 [93–96.5] |
76.5 [72.4–79.2] |
< 0.001a | 86.9 [70.9–94.2] |
71.3 [64.9–77.1] |
< 0.001a |
| 50–65 n = 67 (33.5%) |
94.5 [89.6–96.4] |
75.4 [71.4–79.9] |
< 0.001a | 78.5 [55.9–91.4] |
67.2 [54.4–76.4] |
< 0.001a |
| P-Value | 0.0189b | 0.008b | < 0.001b | < 0.001b | ||
Table 2.
Contingency table between the categorized rates of neutralizing antibody detection methods of ELISA and fluorescence immunochromatography (FIC). Cut-off values of ELISA and fluorescence immunochromatography are 30% and 20%, respectively. The accuracy index is 36.8% showing fair agreement.
| FIC (%) |
||||
|---|---|---|---|---|
| ELISA (%) | [0–20) | [20–50) | [50–80) | [80–100] |
| [0–30) | 1 | 4 | 2 | 1 |
| [30–50) | 0 | 4 | 8 | 0 |
| [50–80) | 2 | 6 | 59 | 0 |
| [80–100] | 0 | 1 | 229 | 83 |
Fig. 2.
Deming Regression, Lin’s Concordance Correlation Coefficient and Bland-Altman plots for ELISA and fluorescence immunochromatography one (Panel A) and four (Panel B) months after the second dose of the COVID-19 BNT162b2 mRNA vaccine. In Deming Regression plots, the black cut line represents the real agreement, and the blue line represents the ideal agreement between the two methods. Pearson’s r was estimated at 0.513 and 0.801 one and four months after the second dose, respectively. In the Blant-Altman plots, the mean proportion bias and 95% confidence intervals are represented with black and red lines, respectively.
One month after the second dose, the median values (IQR) of NAbs-RBD (%) detected with ELISA and FIC were 95.3% (92.8–96.6%) (range: 11.8–98%) and 76.9% (72.7–80%) (range: 10–88.1%), respectively (P-value<0.0001). Four months after the second dose, the median values (IQR) of NAbs-RBD (%) detected with ELISA and FIC were 87.9% (71.5–94.9) (range: 14.3–98%) and 72.2% (63–78.2%) (range: 10–88.8%), respectively (P-value<0.0001).
Ιn both study time points, the study, median values of NAbs-RBD (%) were significantly lower with FIC compared to ELISA (P-value<0.0001) for all age groups (Table 1). The majority of the participants had significantly higher NAbs-RBD (%) values with ELISA compared to FIC in both time-points (P -value<0.0001) (Supplementary Figure 1). The highest median difference was detected in the age group 50–65 years one month after the second dose (ELISA: 94.5% vs FIC: 75.4%, P-value<0.0001), while the lowest median difference was observed in the age group 50–65 years four months after the second dose (78.1% vs 67.3%, P-value<0.0001) (Table 1). Quantitative comparison of the median NAbs-RBD values (%) between ELISA and FIC per age group after increasing the NAbs-RBD (%) values of FIC method by 10% is presented in Supplementary Table 1. This analysis showed no significant differences between the two methods only for the age group 50–65 years four months after the second dose (P-Value: 0.485) (Supplementary Table 1).
We classified NAbs-RBD (%) values measured by ELISA and FIC in four different cut-points (0 to cut-off, cut-off to 50, 50–80, 80–100) for each method to investigate any possible declinations between the two methods regardless of age and are presented in Table 2. The accuracy index between ELISA and FIC, calculated as the sum of the diagonal values divisible by the sum in all values of the table, was estimated at 36.75% (147/400). NAbs-RBD (%) values measured by ELISA and FIC in 4 different cut-points after correcting for plus 10% units in FIC are presented in Supplementary Table 2 and the accuracy index was estimated at 83.8% (335/400). One month after the second dose, there were no participants negative with both methods, while 2/200 (1%) participants were negative in ELISA and positive in FIC and 1/200 (0.5%) was negative in FIC and positive in ELISA. Four months after the second dose, 5/200 (2.5%) participants were negative in ELISA and positive in FIC, 2/200 (1%) were negative in FIC but positive in ELISA and 1/200 (0.5%) was negative for both methods (Table 2).
There is a high correlation (Pearson’s r = 0.801 > 0.8) between the two methods four months after the second dose but the agreement is moderate (CCC= 0.6 (95% C.I.: 0.5–0.7). Deming Regression estimates (Fig. 2), after taking into account the error ratios, are 17.7 (95% C.I.: 7.8–25.8) and 0.6 (95% C.I.: 0.6–0.8) for intercept and slope respectively, without contain 0 or 1 respectively. For both study time points, the study, Lin’s CCC was estimated at 0.37 (95% CI: 0.31–0.42). CCC increased significantly from one to four months after the second dose (P-value<0.001). CCC was estimated at 0.21 (95% CI: 0.15–0.27) and 0.6 (95% CI: 0.54–0.66) one and four months after the second dose, respectively (Supplementary Figure 2).
In Fig. 2 , the Bland-Altman plots estimate the quantitative agreement between the two assays. Between the one- and four-months period after the second dose, FIC and ELISA showed less bias from − 16.7% (−36.9% to 3.4%) to − 11.2% (−34.3% to 11.8%). The proportionate biases were estimated at − 0.3% (95% CI: −0.5%, −0.2%) and − 0.4% (95% CI: −0.5%, −0.3%) one and four months after the second dose, respectively. This indicates that the difference between the two methods is not constant across the range of measurement (P-value at one month <0.001, P-value at four months <0.001). The systematic biases were estimated at − 16.7% (95% CI: −18.1%, −15.3%) and − 11.2% (95% CI: −12.9%, −9.6%) one and four months after the second dose, respectively. This indicates that the ELISA measurements showed higher NAbs-RBD (%) values than FIC (P-value at one month <0.001, P-value at four months <0.001).
4. Discussion
Evaluation of SARS-CoV-2 neutralizing antibody activity is important for assessing protection after natural infection or COVID-19 immunization. Several immunoassays have been developed for the detection of neutralizing antibodies, but their utility for the evaluation of immunological response is still uncertain and requires further investigation. Data on comparison between ELISA and FIC for measuring SARS-CoV-2 neutralizing antibody activity are limited because most commercially available fluorescence immunoassays, approved by the FDA, are designed for the detection of total antibodies against SARS-CoV-2 antigens (EUA Authorized Serology Test Performance, FDA, 2022). FIC is a less expensive, faster, and easier-to-use method compared to ELISA.
In the present study, we compared the performance of an FIC method with an FDA approved ELISA to test for neutralizing antibody activity after SARS-CoV-2 vaccination in 200 HCWs, who received two doses of BNT162b2 vaccine. We selected two time points (one and four months) as there is evidence supporting the waning humoral immunity following SARS-CoV-2 vaccination at least one month after the second vaccine dose (Levin et al., 2021).
Comparing the two assays, high qualitative agreement one and four months after the second mRNA vaccine dose was detected. However, differences in qualitative neutralizing antibody results were found, which were lower four months after the second dose. This could be attributed to the different cut-off values of the two assays. Despite moderate quantitative agreement between the two immunoassays when neutralizing antibody values are high (one month after the second dose), the deviation of the two methods decreased as time after vaccination and the age of the vaccinees increased.
It is important to highlight that the cut-off values of the ELISA and FIC (30% and 20% respectively) for the NAbs-RBD (%) are not well established regarding their clinical relevance as a surrogate marker of protection from symptomatic infection.
NAbs-RBD (%) are considered the primary immune predictors of protection against symptomatic SARS-CoV-2 infection and their levels are positively associated with vaccine efficacy. (Dogan et al., 2021) A study from Israel in HCWs vaccinated with BNT162b2 highlighted that NAbs-RBD titers were negatively associated with the risk of breakthrough SARS-CoV-2 infection and infectivity rates (Bergwerk et al., 2021). However, the levels of neutralizing antibodies that prevent infection from advancing have not yet been determined.
Several studies show fair agreement between the cPass ELISA method and chemiluminescent immunoassays (Nandakumar et al., 2021), (Nam et al., 2021), (Dou et al., 2022) (Valdivia et al., 2021), plaque reduction neutralization tests (Taylor et al., 2021) or other competitive ELISA methods (Krüttgen et al., 2022). Duan et al. compared the cPass ELISA method with a dual-detection fluorescence immunochromatographic assay in 266 serum samples, and found fair agreement (Kappa: 0.82, OPA: 91%) (Duan et al., 2022). Another study by Jung et al. - using a different immunochromatographic assay - showed fair agreement (Kappa: 0.91) between cPass ELISA and BZ-nAb. However, the methods were performed 0–2 months after SARS-CoV-2 natural infection (Jung et al., 2021). In contrast, a study by Saker et al. showed sensitivity between 49.2% and 66.7% and specificity between 68.4% and 77.3% of a qualitative immunochromatographic method (Dynamiker Biotechnology) compared to quantitative ELISA (TECO) (Saker et al., 2022).
A limitation of this study was the retrospective evaluation of NAbs-RBD (%) with FIC, compared to the prospective measurement with ELISA, which may have affected the quality of the samples. However, the study included two time points for the same participants and detected a high qualitative agreement of ELISA and immunofluorescence in the detection of negative and positive results, which could support immunofluorescence as an alternative to ELISA with point-of-care NAbs-RBD testing procedures.
5. Conclusions
FIC had good qualitative agreement with ELISA for the detection of neutralizing SARS-CoV-2 antibodies and could be an alternative for neutralizing antibodies testing. However, there was low quantitative agreement possibly due to different cut-off values of the two methods, which need further investigation. Further studies are required to determine the levels and kinetics of neutralizing antibodies that are a possible surrogate of protection to guide public health decisions for booster immunization.
CRediT authorship contribution statement
Filippos Filippatos: Data curation, Investigation, Methodology, Validation, Roles/Writing – original draft, Writing – review & editing. Elizabeth-Barbara Tatsi: Data curation, Investigation, Methodology, Project administration, Validation, Writing – review & editing. Christos Papagiannopoulos: Formal analysis, Methodology, Software, Validation, Visualization. Vasiliki Syriopoulou: Conceptualization, Methodology, Project administration, Resources, Validation, Writing – review & editing. Athanasios Michos: Conceptualization, Methodology, Project administration, Resources, Supervision, Validation, Roles/Writing – original draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The study was partially funded by DyonMed S.A., Glyka Nera, Greece, providing only the reagents for the FIC assay.
Footnotes
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jviromet.2023.114728.
Appendix A. Supplementary material
Supplementary material
.
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