Importance of sample dilution in the evaluation of the antibody response after SARS-CoV-2 vaccination
Authors: Julien Favresse, Jonathan Douxfils
Journal: Journal of Infection (2021)

•All participants had high antibody titers after 3 months with the mRNA-BNT162b2 vaccine.•Nevertheless, a highly significative antibody decrease was observed at 3 months, only when a dilution factor was applied.•Non saturated quantitative signals are needed to study the real kinetics of antibodies. We read with great interest the recently published article of Tré-Hardy et al. reporting on the time-related changes in the serological response of healthcare workers having received the mRNA-1273 vaccine.1Tré-Hardy M. Cupaiolo R. Wilmet A. Beukinga I. Blairon L. Waning antibodies in SARS-CoV-2 naïve vaccinees: results of a three-month interim analysis of ongoing immunogenicity and efficacy surveillance of the mRNA-1273 vaccine in healthcare workers.J Infect. 2021; (2021-06-01)Abstract Full Text Full Text PDF Scopus (25) Google Scholar Among 205 individuals, 161 (78.5%) were initially seronegative at baseline while 44 (21.5%) already developed antibodies directed against SARS-CoV-2. The antibody response was assessed 2 weeks after the first vaccine injection (T1), 2 weeks after the second vaccine injection (T2) and 3 months after the first injection (T3). The quantitative analysis of the anti-SARS-CoV-2 IgG antibodies directed against the subunits (S1) and (S2) of the virus spike protein was carried out using the LIAISON® SARS-CoV-2 IgG kit (DiaSorin®, Saluggia, Italy). Almost all samples at T1 and all samples at T2 and T3 in the seropositive cohort were above the maximum quantification value of the assay kit, i.e. >400 AU/mL on neat samples. In the discussion, the authors reported that, in previously seropositive subjects (n = 44), no drop in antibody between T2 and T3 was observed. In order to share our experience on that important topic, we would like to present the results we obtained 3 months post-vaccination in the CRO-VAX HCP study (EudraCT registration number: 2020–006,149–21), an ongoing multicenter study in healthcare workers having received BNT162b2, another mRNA vaccine (Pfizer-BioNTech, Mainz, Germany).2Favresse J. Bayart J.L. Mullier F. Dogne J.M. Closset M. Douxfils J. Early antibody response in health-care professionals after two doses of SARS-CoV-2 mRNA vaccine (BNT162b2).Clin Microbiol Infect. 2021 May 8; (PubMed PMID:33975007. Pubmed Central PMCID: PMC8106520. Epub 2021/05/12)Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Among the 200 individuals who were followed up to 3 months, 58 (29%) were seropositive and 142 (71%) were seronegative at baseline.3Favresse J., Bayart J.-.L., Mullier F., Elsen M., Eucher C., Eechhoudt S.V., et al. Antibody titers decline 3-month post-vaccination with BNT612b2. 202. Emerg Microbes Infect. 2021 Jul 7;1-8. doi:10.1080/22221751.2021.1953403. Online ahead of print.Google Scholar Antibodies against the SARS-CoV-2 receptor binding domain of the S1 subunit of the spike protein (anti-S; Elecsys® anti-SARS-CoV-2 spike quantitative ECLIA, Cobas 801, Roche Diagnostics®, Machelen, Belgium) were measured. As the Roche system permits samples dilution to increase the range of measurement, we diluted our samples 10 or 100 times when signal was out of range according to the manufacturer recommendations. Similar timepoints as Tré-Hardy et al., i.e. baseline, 14 days, 42 days and 3 months, were collected in our cohort and analyzed. Using neat or 10-fold diluted samples, we did not observe an antibody drop in seropositive individuals between T1, T2 and T3 (p >0.05, Fig. 1), a finding which is similar to that of Tré Hardy et al.1Tré-Hardy M. Cupaiolo R. Wilmet A. Beukinga I. Blairon L. Waning antibodies in SARS-CoV-2 naïve vaccinees: results of a three-month interim analysis of ongoing immunogenicity and efficacy surveillance of the mRNA-1273 vaccine in healthcare workers.J Infect. 2021; (2021-06-01)Abstract Full Text Full Text PDF Scopus (25) Google Scholar However, a highly significant drop in antibody titers was observed at 3 months if a 100-fold dilution was performed (p <0.001; from 16,935 U/mL to 8919 U/mL; a decrease of 47.3%) (Fig. 1). Such dilution factor permits to increase the range of measurement until 25,000 U/mL on the Roche assay. Considering seronegative individuals, a highly significant antibody drop was also shown when a 10- or 100-fold dilution was applied (p <0.001, Table 1). The application of a 10 or 100-fold dilution (depending on the sample) with our kit permits to show an important difference between the previously seronegative and seropositive subjects (1863 U/mL versus 15,856 U/mL at day 42 and 1262 U/mL versus 8919 U/mL at 3 months), a difference which is not observed when neat samples are used (Table 1). Analytical kits that do not allow a wide range of measurement may thus hide a difference of serological response between previously seronegative and seropositive subjects and does not permit to appreciate the drop in antibody titers in both groups (Table 1).Table 1Evolution of SARS-CoV-2 spike antibodies (U/mL) in previously seronegative and seropositive subjects according to the dilution factor. Means (95% confidence intervals) are shown.SeronegativeSeropositivep valueNo dilution(up to 250 U/mL)Before first dose0.40 (0.39–0.41)102.2 (80.3–124)<0.000114 days36.6 (27.4–45.7)246 (238–254)<0.000142 days247 (243–251)250 (250–250)0.982690 days246 (243–245)250 (250–250)0.9753Dilution 1/10(up to 2500 U/mL)Before first dose0.40 (0.39–0.41)131.8 (86.1–178)<0.000114 days38.2 (27.7–48.6)2457 (2371–2543)<0.000142 days1503 (1380–1625)2400 (2303–2496)<0.000190 days1173 (1057–1288)2477 (2431–2523)<0.0001Dilution 1/100(up to 25,000 U/mL)Before first dose0.40 (0.39–0.41)132 (86.1–178)<0.000114 days38.2 (27.7–48.6)15,540 (13,606–17,473)<0.000142 days1863 (1613–2113)15,856 (13,968–17,824)<0.000190 days1262 (1104–1420)8919 (7201–10,637)<0.0001 Open table in a new tab Currently, data about the long-term kinetics of antibodies in vaccinees are scarce. Two studies found an time-dependent antibody decline with the mRNA-1273 vaccine in only 33 and 34 participants while Tré-Hardy et al. followed more than 200 subjects.4Doria-Rose N. Suthar M.S. Makowski M. O'Connell S. McDermott A.B. Flach B. et al.Antibody persistence through 6 months after the second dose of mRNA-1273 vaccine for Covid-19.N Engl J Med. 2021 Apr 6; (PubMed PMID:33822494. Epub 2021/04/07)Crossref Scopus (488) Google Scholar,5Widge A.T. Rouphael N.G. Jackson L.A. Anderson E.J. Roberts P.C. Makhene M. et al.Durability of responses after SARS-CoV-2 mRNA-1273 vaccination.N Engl J Med. 2021 Jan 7; 384 (PubMed PMID:33270381. Pubmed Central PMCID: PMC7727324. Epub 2020/12/04): 80-82Crossref PubMed Scopus (527) Google Scholar Nevertheless, in previous investigations, sample dilutions were applied to allow a better discrimination between previously seronegative and seropositive subjects.6Manisty C. Otter A.D. Treibel T.A. McKnight A. Altmann D.M. Brooks T. et al.Antibody response to first BNT162b2 dose in previously SARS-CoV-2-infected individuals.Lancet. 2021 Mar 20; 397 (PubMed PMID:33640038. Pubmed Central PMCID: PMC7972310. Epub 2021/03/01): 1057-1058Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 7Salvagno G.L. Henry B.M. di Piazza G. Pighi L. De Nitto S. Bragantini D. et al.Anti-SARS-CoV-2 receptor-binding domain total antibodies response in seropositive and seronegative healthcare workers undergoing COVID-19 mRNA BNT162b2 vaccination.Diagnostics. 2021; 11 (PubMed PMID: doi): 832https://doi.org/10.3390/diagnostics11050832Crossref PubMed Scopus (70) Google Scholar, 8Prendecki M. Clarke C. Brown J. Cox A. Gleeson S. Guckian M. et al.Effect of previous SARS-CoV-2 infection on humoral and T-cell responses to single-dose BNT162b2 vaccine.Lancet. 2021 Mar 27; 397 (PubMed PMID:33640037. Pubmed Central PMCID: PMC7993933. Epub 2021/03/01): 1178-1181Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 9Capetti A.F. Stangalini C.A. Borgonovo F. Mileto D. Oreni L. Dedivitiis G. et al.Impressive boosting of anti-S1/S2 IgG production in COVID-19-experienced patients after the first shot of the BNT162b2 mRNA COVID-19 Vaccine.Clin Infect Dis. 2021 Mar 6; (PubMed PMID:33693610. Pubmed Central PMCID: PMC7989538. Epub 2021/03/12)Crossref PubMed Scopus (5) Google Scholar Compared to the antibody response observed in past-COVID-19 patients, where none or few samples needed to be diluted,2Favresse J. Bayart J.L. Mullier F. Dogne J.M. Closset M. Douxfils J. Early antibody response in health-care professionals after two doses of SARS-CoV-2 mRNA vaccine (BNT162b2).Clin Microbiol Infect. 2021 May 8; (PubMed PMID:33975007. Pubmed Central PMCID: PMC8106520. Epub 2021/05/12)Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,9Capetti A.F. Stangalini C.A. Borgonovo F. Mileto D. Oreni L. Dedivitiis G. et al.Impressive boosting of anti-S1/S2 IgG production in COVID-19-experienced patients after the first shot of the BNT162b2 mRNA COVID-19 Vaccine.Clin Infect Dis. 2021 Mar 6; (PubMed PMID:33693610. Pubmed Central PMCID: PMC7989538. Epub 2021/03/12)Crossref PubMed Scopus (5) Google Scholar,10Tre-Hardy M. Wilmet A. Beukinga I. Dogne J.M. Douxfils J. Blairon L. Validation of a chemiluminescent assay for specific SARS-CoV-2 antibody.Clin Chem Lab Med: CCLM /FESCC. 2020 Jul 28; 58 (PubMed PMID:32447328. Epub 2020/05/25): 1357-1364Crossref PubMed Scopus (68) Google Scholar the antibody response in vaccinees is significantly higher and will certainly require dilutions to obtain the real quantitative value with some assays (i.e. not rounded to the upper limit of measurement). In conclusion, we agree with Tré-Hardy et al. that a persistent antibody response was observe following the administration of the mRNA vaccine, as observed elsewhere using various assays.1Tré-Hardy M. Cupaiolo R. Wilmet A. Beukinga I. Blairon L. Waning antibodies in SARS-CoV-2 naïve vaccinees: results of a three-month interim analysis of ongoing immunogenicity and efficacy surveillance of the mRNA-1273 vaccine in healthcare workers.J Infect. 2021; (2021-06-01)Abstract Full Text Full Text PDF Scopus (25) Google Scholar, 2Favresse J. Bayart J.L. Mullier F. Dogne J.M. Closset M. Douxfils J. Early antibody response in health-care professionals after two doses of SARS-CoV-2 mRNA vaccine (BNT162b2).Clin Microbiol Infect. 2021 May 8; (PubMed PMID:33975007. Pubmed Central PMCID: PMC8106520. Epub 2021/05/12)Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Favresse J., Bayart J.-.L., Mullier F., Elsen M., Eucher C., Eechhoudt S.V., et al. Antibody titers decline 3-month post-vaccination with BNT612b2. 202. Emerg Microbes Infect. 2021 Jul 7;1-8. doi:10.1080/22221751.2021.1953403. Online ahead of print.Google Scholar, 4Doria-Rose N. Suthar M.S. Makowski M. O'Connell S. McDermott A.B. Flach B. et al.Antibody persistence through 6 months after the second dose of mRNA-1273 vaccine for Covid-19.N Engl J Med. 2021 Apr 6; (PubMed PMID:33822494. Epub 2021/04/07)Crossref Scopus (488) Google Scholar,7Salvagno G.L. Henry B.M. di Piazza G. Pighi L. De Nitto S. Bragantini D. et al.Anti-SARS-CoV-2 receptor-binding domain total antibodies response in seropositive and seronegative healthcare workers undergoing COVID-19 mRNA BNT162b2 vaccination.Diagnostics. 2021; 11 (PubMed PMID: doi): 832https://doi.org/10.3390/diagnostics11050832Crossref PubMed Scopus (70) Google Scholar However, the absence of "antibody drop" between T1, T2 and T3 observed in their cohort of previously seropositive could depend on the analytical kit used and the application of a dilution factor in case of signal saturation if such procedure is permitted and documented by the manufacturer. The results of the CRO-VAX HCP study showed that the use of undiluted or diluted samples led to different conclusions regarding the antibody kinetics. The fact that the signal is not saturated with the Roche assay also permit to derived more precise pharmacokinetic models which could latter better predict the probable persistence of antibodies.3Favresse J., Bayart J.-.L., Mullier F., Elsen M., Eucher C., Eechhoudt S.V., et al. Antibody titers decline 3-month post-vaccination with BNT612b2. 202. Emerg Microbes Infect. 2021 Jul 7;1-8. doi:10.1080/22221751.2021.1953403. Online ahead of print.Google Scholar Such data are important, especially since the question about a third dose has been raised.