Introduction

Measures of SARS-CoV-2 antibody levels, such as those reported in multiple publications [1–3], are used to determine whether or not a person has been exposed to an antigen, characterize the extent of an individual’s immune response, and to infer rates of both infection and vaccination in populations [4–6]. The prozone effect occurs in immunoassays when excessively high antibody concentration oversaturates and disrupts the antigen-antibody bridging between immobile phase and chemiluminescent signal, resulting in a spuriously low reported result. This phenomenon is well documented in serological testing of other infectious diseases, including syphilis, HIV, and malaria [7,8]. Here, we report a prozone or ‘hook’ effect adversely influencing measurement of SARS-CoV-2 spike protein antibody levels using the Roche Elecsys® Anti-SARS-CoV-2 S immunoassay from participants in the Texas Coronavirus Antibody Research survey (Texas CARES), an ongoing prospective, longitudinal antibody surveillance program.

Materials and methods

Implementation of standardized serial dilution protocol

Beginning October 12, 2022, all study samples with initial neat results of 0.8–250 U/mL were subjected to a dilution algorithm with initial retest at 1:10 and reviewed for possible prozone effect (Fig 1). Initial (neat) results greater than or equal to 0.8 U/mL and less than 250 U/mL underwent 1:10 dilution. The dilution-adjusted values were compared to the initial neat values, and if the two values were within 20% of each other, the initial neat result was reported. If the dilution-adjusted value exhibited a greater than 20% difference from the initial neat value, the sample was classified as a prozone case. Among these prozone cases, if the dilution-adjusted value was greater than or equal to 2500 U/mL, a value of >2500 U/mL was reported to participants; if the dilution-adjusted value was less than 2500 U/mL, an additional 1:50 dilution was performed to rule out persistent prozone effect.

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Fig 1. Dilution protocol for anti-SARS-CoV-2 spike protein immunoassay, implemented October 12, 2022.

https://doi.org/10.1371/journal.pone.0301232.g001

Results and discussion

The results of the exploratory dilution studies are presented in Table 1. Participants A, B, and C represent the three samples first retested under the 1:50 pre-dilution procedure: final endpoint results of these ranged from 8,600 to 66,100 U/mL. Of the eleven participants asked to submit additional serum samples, seven provided new samples. These seven participants’ (D-J in Table 1) samples demonstrated prozone effect using 1:50 predilution with final antibody levels ranging from 3,845 to 20,450 U/mL as given in Table 1.

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Table 1. Prozone effect in anti-SARS-CoV-2 spike protein immunoassay testing, August 2022.

https://doi.org/10.1371/journal.pone.0301232.t001

Following implementation of the standardized serial dilution protocol in October 2022, additional prozone cases were identified. Between October 12, 2022, and February 28, 2023, there were 74 additional instances of prozone effect detected out of 1,720 participants’ samples. Median initial (neat) result for these 74 samples was 177 U/mL, interquartile range: 122 U/mL to 210 U/mL (S1 Table). Following 1:10 predilution and retesting, all 74 samples had final (reported) results of >2,500 U/mL, in accordance with the updated reporting protocol.

Discussion

The results of this report add to the growing body of evidence suggesting that the prozone effect may artificially decrease anti-SARS-CoV-2 spike antibody titer results in certain immunoassays [13–15]. To the best of our knowledge, the prozone effect resulted in spuriously low positive results but did not produce a negative antibody result using the Roche Elecsys S antibody assay in the Texas CARES program. The consistency of our findings with those of independent contemporaneous investigations such as those published by Abedon, et al. [13] are indicative of the scale and clinical relevance of this phenomenon. As COVID-19 becomes endemic globally and immunization protocols evolve, people experiencing numerous successive natural and/or artificial SARS-CoV-2 exposures, may undergo more extreme immune responses. In response to the rapid increase in reported levels of anti-SARS-CoV-2 antibody titers, some laboratory protocols now report results up to 250,000 U/mL [16]. While a clinical antibody-based threshold of protection against COVID-19 infection has not yet been defined, serological results remain important in clinical decision-making, such as vaccination protocols for immunosuppressed patients.

Conclusion

The probability of a prozone effect obscuring high antibody concentrations will certainly only increase as populations become repeatedly exposed to SARS-CoV-2 spike protein through multiple immunizations or serial infections, making awareness and mitigation of this issue paramount. To address the likelihood of prozone effect obscuring very high antibody titer results, laboratories should be aware of this phenomenon to consider implementing defined and standardized dilution protocols as appropriate for clinical, research, and public health applications.

Supporting information

Acknowledgments

Author contributions. MNS, JAR, MAS, SJP and EB designed and coordinated the study. MNS performed the analysis and, together with MDS, performed initial data interpretation. SPM and MAS performed the experiments. Every author contributed to the initial draft of the manuscript and agreed on submission for publication. All authors reviewed the manuscript and approved the final version.

This analysis would not have been possible without the partnership of many. We would like to acknowledge The University of Texas Health Science Center at Houston, School of Public Health’s Texas CARES investigative team for their contribution to participant recruitment, data collection, statistical analysis, and data visualization including Sarah E Messiah, PhD, MPH; Melissa Valerio-Shewmaker, PhD, MPH; Steven Kelder, PhD, MPH; Harold W Kohl, PhD, MSPH; Kimberly Aguillard, PhD, MPH; Stacia DeSantis, PhD; Ashraf Yaseen, PhD; Luis León-Novelo, PhD; Lindsay Padilla, MPH; Ximena Flandes Amavisca, MPH; Amy Casebier, BA; Yashar Talebi, MS; Frances Brito, MS; Michael Gonzalez, MS; Leqing Wu, PhD; Rhiana Abbas, MS; Onyinye OmegaNjemnobi, MBBS, MPH; Shiming Zhang, MS; Joy Yoo, BS; Tianyao Hao, MS; Cesar Pinzon Gomez, MD, MPH; Michelle Obiesie, BS; Elizabeth Grunder, BS; Breanna Ellis, BS; Hazel Flores, MPH; Kourtney Lopez, MPH; Henal Alepsh Gandhi, MDS; Anuja Rajendra Godbole, MBBS; Han Haoting, BS; Rachit Sabharwahl, MS; David Lakey, MD; Jennifer Shuford, MD, MPH. The Texas CARES investigation team would like to thank Clinical Pathology Laboratories, Federally Qualified Health Care Centers statewide, and the Texas Association for Community Health Centers for assisting with sharing information with families about this survey.