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Article

Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation

by
Gulnur Nakhanova
1,
Olga Chervyakova
1,
Kamshat Shorayeva
1,
Aigerim Zhakypbek
1,
Sabina Moldagulova
1,*,
Aknur Ulankyzy
1,
Alisher Omurtay
1,
Yeraly Shayakhmetov
1,
Temirlan Baiseit
1,
Zharkinay Absatova
1,
Aisha Issabek
1,
Gaukhar Shynybekova
1,
Sandugash Sadikaliyeva
1,
Aziz Nakhanov
1,
Kuanysh Jekebekov
1,
Ainar Kossylganova
2,
Karlygash Zhaparkulova
2,
Assem Kalykova
2,
Tolkyn Bekezhanova
2,
Albina Atakanova
2,
Zakir Yershebulov
3 and
Kuandyk Zhugunissov
1add Show full author list remove Hide full author list
1
Research Institute for Biological Safety Problems, National Holding QazBioPharm, Gvardeiskiy 080409, Kazakhstan
2
School of Pharmacy, Asfendiyarov Kazakh National Medical University, Almaty 050000, Kazakhstan
3
Limited Liability Partnership “OtarBioPharm”, QazBioPharm National Holding JSC, Gvardeiskiy 080409, Kazakhstan
*
Author to whom correspondence should be addressed.
Methods Protoc. 2026, 9(4), 113; https://doi.org/10.3390/mps9040113
Submission received: 2 June 2026 / Revised: 15 July 2026 / Accepted: 17 July 2026 / Published: 28 July 2026
(This article belongs to the Section Molecular and Cellular Biology)
Versions Notes

Abstract

Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D2) and antigen concentration (R2 = 0.956–0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625–10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations.
Keywords: brucellosis; recombinant proteins; single radial immunodiffusion; method validation; vaccine quality control brucellosis; recombinant proteins; single radial immunodiffusion; method validation; vaccine quality control

Share and Cite

MDPI and ACS Style

Nakhanova, G.; Chervyakova, O.; Shorayeva, K.; Zhakypbek, A.; Moldagulova, S.; Ulankyzy, A.; Omurtay, A.; Shayakhmetov, Y.; Baiseit, T.; Absatova, Z.; et al. Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation. Methods Protoc. 2026, 9, 113. https://doi.org/10.3390/mps9040113

AMA Style

Nakhanova G, Chervyakova O, Shorayeva K, Zhakypbek A, Moldagulova S, Ulankyzy A, Omurtay A, Shayakhmetov Y, Baiseit T, Absatova Z, et al. Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation. Methods and Protocols. 2026; 9(4):113. https://doi.org/10.3390/mps9040113

Chicago/Turabian Style

Nakhanova, Gulnur, Olga Chervyakova, Kamshat Shorayeva, Aigerim Zhakypbek, Sabina Moldagulova, Aknur Ulankyzy, Alisher Omurtay, Yeraly Shayakhmetov, Temirlan Baiseit, Zharkinay Absatova, and et al. 2026. "Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation" Methods and Protocols 9, no. 4: 113. https://doi.org/10.3390/mps9040113

APA Style

Nakhanova, G., Chervyakova, O., Shorayeva, K., Zhakypbek, A., Moldagulova, S., Ulankyzy, A., Omurtay, A., Shayakhmetov, Y., Baiseit, T., Absatova, Z., Issabek, A., Shynybekova, G., Sadikaliyeva, S., Nakhanov, A., Jekebekov, K., Kossylganova, A., Zhaparkulova, K., Kalykova, A., Bekezhanova, T., ... Zhugunissov, K. (2026). Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation. Methods and Protocols, 9(4), 113. https://doi.org/10.3390/mps9040113

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