Reportable range of quantitative assays for SARS-CoV-2 antibodies determination: An overlooked issue?

Dear Editor,

There is some evidence showing that anti-SARS-CoV-2 antibody concentrations differ according to the severity of the disease. ¹ , ² Even in the context of assessing the strength and duration of the antibody response, both after illness and after vaccination, the quantification of antibody concentrations may have a significant role. However, it was possible to propose a real quantification only with the introduction of methods with appropriate calibration curves. One way to identify the quantification capability of an immunoassay method is to evaluate the linearity of the reportable range of measurement with respect to the calibration curve. ³ This evaluation was carried out for anti-SARS-CoV-2 QuantiVac ELISA IgG (Euroimmun, Lubeck, Germany) and the recently released SARS-CoV-2 IgG anti-RBD (SNIBE, Shenzen, China).

Both assays were performed according to the manufacturer’s instructions. In the ELISA Euroimmun method, patient samples were prediluted 1:100 with sample buffer, and the concentrations were determined through the interpolation with a six-point calibration curve (from 1 to 120 RU/mL). The Maglumi method was carried out on the Maglumi 800 analyzer (SNIBE; Shenzen, China), and the samples concentrations were determined through the interpolation with a nine-point master curve (from 0.5 to 100 AU/mL) periodically adjusted by a two-point calibration.

The claimed reportable ranges are 6–120 RU/mL for the ELISA method (positivity threshold 11 RU/mL) and 0.18–100 AU/mL for the Maglumi method (positivity threshold 1 AU/mL).

Twenty-nine serum specimens with different degrees of positivity were evaluated, both from vaccinated subjects and from patients who recovered from COVID-19. Nine of these specimens were used for the dilution tests with the Maglumi method, while 20 specimens were used with the ELISA method. The dilutions were carried out using the respective diluents supplied by the companies. The linearity of the dilution was evaluated according to the method suggested by Emerson et al., ⁴ using ±3 zeta-score as the limit of acceptability. The results are shown in Figure 1(a). For the ELISA method, the data resulted within three zeta-scores and the recovery resulted within ± 20% of the initial value in all samples with initial values <100 RU/mL. Among the six cases with initial values between 100 and 120 RU/mL, four showed a non-linear trend. The subsequent dilutions (when multiplied by the corresponding dilution factor) yield constant results, but higher than the undiluted value (Table 1 in Supplemental file). For the Maglumi method, an underestimation of values higher than 50 AU/mL was detected, while for concentrations lower than 50 AU/mL, the linearity seems confirmed (Figure 1(b)). Since the Maglumi method does not provide the predilution of the samples, two samples were also diluted using a negative plasma sample, to maintain the same matrix over the dilution curve. The results were similar to those found using the diluent of the kit (Table 2 in Supplemental file).

Figure 1.

Dilution test. In abscissa were reported the measured concentration of each dilution, in ordinate the final concentration of each dilution (multiplied by the respective dilution factor). The dotted lines connect the concentrations significantly different. The horizontal line identifies the level below which the test resulted linear. (a) Dilution test with ELISA method and (b) Dilution test with Maglumi method.

Both evaluated assays use a wide range standard curve, which provides an accurate quantification over the measurement range. However, it is not guaranteed that high concentrations of detectable antibodies do not saturate the analytical system before reaching the response corresponding to the highest calibration point.

An evaluation of dilution curves showed that the QuantiVac-ELISA from Euroimmun and the IgG anti-RBD from SNIBE could satisfactorily quantify anti-SARS-CoV-2 antibodies, provided that the highest detectable value is fixed to 50 AU/mL for Maglumi and, possibly, 100 RU/mL for ELISA. Beyond these concentrations, the sample should be diluted.

Supplemental Material

sj-pdf-1-acb-10.1177_00045632211020047 - Supplemental material for Reportable range of quantitative assays for SARS-CoV-2 antibodies determination: An overlooked issue?

Supplemental material, sj-pdf-1-acb-10.1177_00045632211020047 for Reportable range of quantitative assays for SARS-CoV-2 antibodies determination: An overlooked issue? by Ruggero Dittadi, Isabella Bertoli and Paolo Carraro in Annals of Clinical Biochemistry