DEVELOPMENT OF NEUTRALIZING ANTIBODY ASSAYS: PITFALLS AND CHALLENGES

Sharique Ahmad; Farhana; Yoshita Agnihotri; Pushpendra D Pratap; Subuhi Anwar; Tuba Saud

doi:10.29121/granthaalayah.v13.i3.2025.5988

Authors

Sharique Ahmad Professor, Dept. Of Pathology & Blood Bank, Era's Lucknow Medical College & Hospital, Era University, Lucknow, Uttar Pradesh, India-22600
Dr. Farhana Associate Professor Department of Pathology F.H. Medical College and Hospital, Agra, Uttar Pradesh, India- 283204
Dr. Yoshita Agnihotri Junior Resident, Department of Pathology, Era’s Lucknow Medical College Hospital, Era University, Lucknow, Uttar Pradesh, India 226003
Dr. Pushpendra D Pratap Research Analyst, Department of Biochemistry, Era’s Lucknow Medical College & Hospital, Era University, Lucknow, Uttar Pradesh, India.
Subuhi Anwar Research Assistant, Department of Pathology, Era's Lucknow Medical College and Hospital, Era University, Lucknow, Uttar Pradesh, India-226003
Tuba Saud MBBS Student, Era’s Lucknow Medical College Hospital, Era University, Lucknow, Uttar Pradesh India 226003

DOI:

https://doi.org/10.29121/granthaalayah.v13.i3.2025.5988

Keywords:

Antibody, Pathogen, COVID-19, Immunology, Infectious Disease

Abstract [English]

Neutralizing antibody assays are vital in evaluating immune responses to infectious agents and vaccines while assessing the capacity of antibodies to inhibit pathogen infection or replication. These represent a cornerstone for determining vaccine efficacy and therapeutic antibody potential. Yet, a multitude of challenges accompanies the development of reliable and accurate nAb assays. The review deals with nAb testing, uncovering the most frequent pitfalls and challenges in their development, which are classified into the selection of appropriate assay formats, assay protocol standardization, biological variability management, and interpretation of results. Each of the four major assay formats-namely, plaque reduction neutralization tests, microneutralization assays, pseudovirus-based assays, and cell-based assays-offers specific benefits and limitations. Ensuring protocol standardization across laboratories is mandatory to obtain results that are comparable and reproducible. Assay outcomes are substantially affected by biological variability stemming from a host of factors: differing pathogen strains; timing of sample collection. Given the possible options, interpretation of results from nAb tests becomes exceedingly complex due to defining appropriate neutralization thresholds and connecting these to correlates of protection. Addressing some of the aforementioned challenges shall lead to better reliability and reproducibility of nAb assays, propelling the advancement of immunology and infectious disease research. Examples of cases will also be discussed to bolster the argument with warm bodies, such as the fast-track development of nAb assays during the COVID-19 pandemic, while future directions in nAb assays will be outlined, underlining the need for HIV collaborators to outpace demand.

Downloads

Download data is not yet available.

References

Amanat, F., et al. (2020). Rapid Development of nAb Assays During COVID-19. Cell, 182(4), 1013-1022.e18.

Bachmann, M. F., & McKee, T. (2021). Multiplex Assays for Neutralizing Antibodies. Nature Reviews Immunology, 21(12), 673-685.

Baum, A., et al. (2020). Pseudovirus-Based Neutralization Assays. Nature Protocols, 15(1), 14-30.

Earle, K. A., et al. (2021). Quantifying the Relationship Between SARS-CoV-2 Neutralizing Antibody Titers and Protective Immunity. Nature Medicine, 27(7), 1205-1211.

Gaebler, C., et al. (2021). Host Variability in Neutralizing Antibody Responses. Cell Reports, 34(6), 108621.

Geers, D., et al. (2021). Multiplex Assays for SARS-CoV-2 Neutralizing Antibodies. Nature Immunology, 22(5), 638-644.

Gilbert, P. B., et al. (2020). Establishing Correlates of Protection. The Lancet Infectious Diseases, 20(12), 1350-1360. https://doi.org/10.1016/S1473-3099(20)30699-X DOI: https://doi.org/10.1016/S1473-3099(20)30699-X

Goh VS, Ang CC, Low SL, Lee PX, Setoh YX, Wong JC. (2024). Evaluation of Three Alternative Methods to the Plaque Reduction Neutralizing Assay for Measuring Neutralizing Antibodies to Dengue Virus Serotype 2. Virology Journal, 21(1), 208. https://doi.org/10.1186/s12985-024-02459-y DOI: https://doi.org/10.1186/s12985-024-02459-y

Goo, L., et al. (2018). Cell-Based Assays for Measuring Viral Entry Inhibition. Journal of Immunological Methods, 457, 24-34.

Harvey, W. T., et al. (2021). Next-Generation Sequencing for nAb Epitope Mapping. Nature Reviews Genetics, 22(6), 321-332.

Hensley, S. E., et al. (2019). Challenges in Influenza Neutralization Assays. Current Opinion in Virology, 34, 97-105.

Hogan, C. A., et al. (2021). Data Management Systems in Neutralizing Antibody Assays. Clinical Chemistry, 67(3), 499-508.

John Pirro (2024). "Best Practices for Optimizing NAB Assay Performance with Suggestions for Improved Accuracy & Efficiency.". https://swordbio.com/blog/best-practices-for-optimizing-nab-assay-performance-with suggestions-for-improved-accuracy-efficiency/

Katherine Berman, Greta Van Slyke, et al. (2024). "Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots." ASM J. Microbiology Spectrum. 12 (12). https://doi.org/10.1128/spectrum.00846-24 DOI: https://doi.org/10.1128/spectrum.00846-24

Khoury, D. S., et al. (2021). Quantitative and Qualitative Data Balance in nAb Assays. Nature Communications, 12(1), 3135.

Knezevic, I., et al. (2017). Standard Operating Procedures for nAb Assays. Vaccine, 35(3), 466-471.

Krammer, F., et al. (2020). Antibody Correlates of Protection from SARS-CoV-2 infection. Nature Reviews Immunology, 20(6), 383-392. https://doi.org/10.1038/s41577-020-0359-5 DOI: https://doi.org/10.1038/s41577-020-0359-5

Liu KT, Han YJ, Wu GH, Huang KY, Huang PN. (2022). Overview of Neutralization Assays and International Standard for Detecting SARS-CoV-2 Neutralizing Antibody. Viruses, 14(7), 1560. https://doi.org/10.3390/v14071560 DOI: https://doi.org/10.3390/v14071560

Liu, Y., et al. (2021). Advanced Analytical Tools for Neutralizing Antibody Assays. Analytical Chemistry, 93(8), 3962-3970.

Long, T. F., et al. (2021). Automation and High-Throughput in Neutralizing Antibody Assays. Methods in Enzymology, 635, 169-195.

Lu, L., et al. (2021). Advances in High-Throughput Neutralizing Antibody Assays. Viruses, 13(2), 190.

McGuire, A. T., & Gray, M. D. (2020). The Plaque Reduction Neutralization Test. Journal of Virology Methods, 276, 113768.

Meyer, B., et al. (2020). Inter-laboratory Comparison of Neutralization Assays. Journal of Clinical Virology, 129, 104455. https://doi.org/10.1016/j.jcv.2020.104455 DOI: https://doi.org/10.1016/j.jcv.2020.104455

Mouquet, H., & Nussenzweig, M. C. (2011). Antibodies that Fight Viruses: Cross-Reactivity and Neutralization. Nature Reviews Microbiology, 9(7), 392-402.

Nachbagauer, R., et al. (2019). Challenges in Influenza Virus Neutralization Assays. Frontiers in Immunology, 10, 1236.

Pazos, M., et al. (2020). High-Throughput Screening and Miniaturization. Journal of Clinical Microbiology, 58(9), e01248-20.

Planas, D., et al. (2021). Considerations for the Use of Neutralizing Antibody Assays. Nature Reviews Immunology, 21(12), 732-747.

Plotkin, S. A. (2020). Correlates of Vaccine-Induced Protection. Vaccine, 38(33), 5080-5088. https://doi.org/10.1016/j.vaccine.2019.10.046 DOI: https://doi.org/10.1016/j.vaccine.2019.10.046

Plotkin, S. A., et al. (2018). Correlates of Protection Against Viral Infections. Clinical Infectious Diseases, 67(2), 312-319.

Robinson, M. L., et al. (2021). Standardization of Neutralizing Antibody Assays: Challenges and strategies. Viral Therapy, 26(4), 242-256.

Sanders, R. W., et al. (2020). Temporal Aspects of Neutralizing Antibody Responses. Annual Review of Virology, 7, 343-363.

Scheid, J. F., et al. (2021). Host and Pathogen Variability in nAb Assays. Cell Host & Microbe, 29(4), 529-542.e6.

Shu, B., et al. (2020). Implications of SARS-CoV-2 Variants for Vaccine Development. The Lancet Infectious Diseases, 20(12), 1346-1348.

Starr, T. N., et al. (2020). Pseudovirus-Based Neutralization Assays for SARS-CoV-2. Nature Reviews Microbiology, 18(12), 762-777.

Sullivan, N. J., et al. (2020). Genetic Diversity and Neutralization Sensitivity. Nature Reviews Immunology, 20(5), 321-335. https://doi.org/10.1038/s41577-019-0269-6 DOI: https://doi.org/10.1038/s41577-019-0269-6

Suthar, M. S., et al. (2020). Rapid Neutralizing Antibody Development During COVID-19. Cell Reports Medicine, 1(3), 100040. DOI: https://doi.org/10.1016/j.xcrm.2020.100040

WHO Expert Committee. (2013). Biological Standardization: Report on the International Conference. WHO Technical Report Series, 987.

Wang, P., et al. (2021). Temporal Variability in Neutralizing Antibody Titers. Nature Medicine, 27(4), 635-640.

Weissenhorn, W., et al. (2019). Microneutralization Assays: A High-Throughput Alternative. Methods in Molecular Biology, 2048, 121-134.

Wu, F., et al. (2021). Next-generation Sequencing Insights into nAb Responses. Nature Communications, 12(1), 228.

Young, B. E., et al. (2021). Balancing Throughput with Accuracy in Neutralizing Antibody Assays. Journal of Clinical Microbiology, 59(2), e02403-20.

Yu S, Zhao Q, Zhang C, Fu D, Zhu X, Zhou J, Ma W, Dong Z, Zhai X, Jiang L, Han X. (2024). Methodological Validation and Inter-Laboratory Comparison of Microneutralization Assay for Detecting Anti-AAV9 Neutralizing Antibody in Human. Viruses. 16(10), 1512. https://doi.org/10.3390/v16101512 DOI: https://doi.org/10.3390/v16101512

Yuan, M., et al. (2021). Structural Basis of a Shared Antibody Response to SARS-CoV-2. Science, 373(6554), 84-88.

Zhu L, Mao N, Yi C, Simayi A, Feng J, Feng Y, He M, Ding S, Wang Y, Wang Y, Wei M. (2023). Impact of Vaccination on Kinetics of Neutralizing Antibodies Against SARS-CoV-2 by Serum Live Neutralization Test Based on a Prospective cohort. Emerging Microbes & Infections. 12(1), 2146535. https://doi.org/10.1080/22221751.2022.2146535 DOI: https://doi.org/10.1080/22221751.2022.2146535

Zost, S. J., et al. (2020). Multiplex Assays for nAb Activity. Cell Host & Microbe, 28(3), 387-398.e6.