Correlation of the Commercial Anti-SARS-CoV-2 Receptor Binding Domain Antibody Test with the Chemiluminescent Reduction Neutralizing Test and Possible Detection of Antibodies to Emerging Variants

Abstract

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22-0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

Keywords: convalescent; high throughput; neutralizing antibodies; receptor-binding domain; seroconversion.

FIG 1

Neutralization and anti-RBD antibody levels. (A) Neutralization of pseudotyped viruses measured by CRNT (serum dilution, ×100). (B) Anti-RBD antibody levels measured by commercial test. (C) ROC curves to classify the symptomatic confirmed COVID-19 patients in the convalescent phase and the unscreened individuals. (D) Relationship between test results and time from symptom onset or close contact to blood sampling in COVID-19 patients. Symptomatic and asymptomatic individuals are presented in red and blue (blue arrowhead for overlapping cases), respectively. ***, P < 0.001 by unpaired Kruskal-Wallis test and Dunn’s multiple comparison using the unscreened group as control. Ac, acute phase; Co, convalescent phase; S, symptomatic; AS, asymptomatic; Unscr., unscreened; PCR negative, SARS-CoV-2 PCR negative; AUC, area under the curve; COV, cut-off value; Sn, sensitivity; Sp, specificity.

FIG 2

Relationship between CRNT and anti-RBD antibody test. (A) Comparison of neutralization levels and anti-RBD antibody results. Concordant samples are red (positive for both tests) or white (negative for both tests). Discordant samples are green (positive for anti-RBD antibody) or yellow (positive for CRNT). Dotted line for CRNT indicates 50% infectivity (IC₅₀). (B) Anti-RBD antibody test as a function of neutralizing activity. Sera positive for CRNT (diluted 1:100) were serially diluted up to 1:6,400 and the dilution yielding >IC₅₀ was defined as neutralizing titer. Boxes indicate median and interquartile. Error bars indicate minima and maxima.

FIG 3

Neutralizing activities against SARS-CoV-2 variants in Wuhan-CRNT-positive sera. (A) Neutralizing sensitivity of SARS-CoV-2 pseudotyped variants. Neutralization by wild-type (WT) spike protein (Wuhan) CRNT-positive sera (serum dilution, ×100) was assessed against pseudotyped viruses displaying the mutant spike proteins (B.1.1.7 and B.1.351-derived variants). Box indicate median and interquartile. Error bars indicate minimum to maximum. ***, P < 0.001; ns, not significance. (B) Relationship between anti-RBD-antibody test results and neutralization of B.1.1.7 and B.1.351 variants (serum dilution 1:100).