Monitoring the activity of specific antibodies to pathogens based on biobank materials

Aim. To study the effect of storage temperatures (-20 and -70 оC) and exposure duration (6 months and 6 years) on the preservation and stability of specific anti-hepatitis B virus HBs antigen IgG antibodies (anti-­HBs IgG) in serum samples stored in a biobank.

Material and methods. An experimental study was conducted on 180 serum samples. Anti-­HBs IgG levels were determined by enzyme-­linked immunosorbent assay (ELISA) using Vector-­Best test systems (Russia) in native samples and after storage according to two following scenarios: 1) storage at -20 and -70 оC for 6 months; 2) storage at -70 оC for 6 months and 6 years. Statistical analysis was performed using nonparametric methods (Wilcoxon test, median, interquartile range, Spearman correlation coefficient).

Results. The study using the first scenario found that storage at -20 оC for 6 months led to an almost 2-fold increase in the median anti-­HBs IgG level (from 35,5 (7,8-76,8) to 65,3 (26,0-105,0) IU/ml) and 9 false-­positive results. In the second scenario study, storage at -70 оC for 6 months was accompanied by a slight decrease in antibody levels (from 72,5 (27,9-132,0) to 49,3 (4,0-98,4) IU/ml) without false positive results. After 6 years of storage at -70 оC, a further decrease to 33,3 (6,15-88,4) IU/ml was noted, with 2 samples becoming false negative. A strong correlation was found between antibody levels in native samples and after storage (р=0,97-0,99; p<0,05).

Conclusion. Long-term (up to 6 years) storage of serum samples at -70 оC is reliable and does not critically distort the results of population serological studies of anti-­HBs IgG prevalence. Storage at -20 оC is only acceptable for short-term storage, as after just 6 months it leads to false-­positive results and an overestimation of the average antibody level, making it unsuitable for biobanking.

1. Semenenko TA, Akimkin VG. Seroepidemiology in the surveillance of vaccine-­preventable diseases. Journal of microbiology, epi­de­miology and immunobiology. 2018;95(2):87-94. (In Russ.) doi:10.36233/0372-9311-2018-2-87-94.

2. Hörber S, Klein R, Peter A. Effects of Long-­Term Storage on Se­rum Free Light Chain Stability. Clin Lab. 2019;65(5). doi:10.7754/Clin.Lab.2018.181107.

3. Kozlova VA, Metelskaya VA, Pokrovskaya MS, et al. Stability of serum biochemical markers during standard long-term storage and with a single thawing. Cardiovascular Therapy and Pre­ven­tion. 2020;19(6):2736. (In Russ.) doi:10.15829/1728-8800-2020-2736.

4. Hendriks J, Stals C, Versteilen A, et al. Stability studies of bin­ding and functional anti-vaccine antibodies. Bioanalysis. 2014; 6(10):1385-93. doi:10.4155/bio.14.96.

5. Olmeda P, Díaz-­Regañón D, Villaescusa A, et al. Evaluating the stability of antibody titres against Leishmania infantum deter­mined by IFAT in long-term stored canine frozen samples. Parasit Vectors. 2025;18(1):327. doi:10.1186/s13071-025-06982-x.

6. Engelmaier A, Butterweck HA, Weber A. Stability assessment of anti-bacterial antibodies in immunoglobulin G-depleted serum with validated immunoassays. Immunotherapy. 2023;15(17): 1459-76. doi:10.2217/imt-2023-0127.

7. Laner-­Plamberger S, Siller A, Lauth W, et al. Stable SARS-CoV-2 antibody levels and functionality in serum and COVID-19 con­valescent plasma after long-term storage. Vox Sang. 2025; 120(8):784-92. doi:10.1111/vox.70059.

8. Shurrab FM, Al-­Sadeq DW, Amanullah F, et al. Effect of multiple freeze-thaw cycles on the detection of anti-­SARS-CoV-2 IgG an­ti­bo­dies. J Med Microbiol. 2021;70(8):001402. doi:10.1099/jmm.0.001402.

9. Nozdracheva AV, Semenenko TA. Assessment of the reliability of serological monitoring results based on biobank materials. Cardiovascular Therapy and Prevention. 2023;22(11):3709. (In Russ.) doi:10.15829/1728-8800-2023-3709.

10. Nozdracheva AV, Semenenko ТA. Influence of long-term sto­ra­ge of blood serum samples in a biobank for population-­ba­sed se­ro­epi­demiologic studies. Cardiovascular Therapy and Pre­ven­tion. 2022;21(11):3407. (In Russ.). doi:10.15829/1728-8800-2022-3407.

11. Akimkin VG, Semenenko TA, Nikitina Gyu, et al. Epidemiology of hepatitis B and C in health care institutions. Moscow. 2013. p. 216. (In Russ.) ISBN: 978-5-9903962-1-0.

12. Tu T, Douglas MW. Hepatitis B Virus Infection: From Diagnostics to Treatments. Viruses. 2020;12(12):1366. doi:10.3390/v12121366.

13. Lin CL, Kao JH. Development of hepatocellular carcinoma in treated and untreated patients with chronic hepatitis B virus infection. Clin Mol Hepatol. 2023;29(3):605-22. doi:10.3350/cmh.2022.0342.

14. Qin Y, Liao P. Hepatitis B virus vaccine breakthrough infec­tion: surveillance of S gene mutants of HBV. Acta Virol. 2018; 62(2):115-21. doi:10.4149/av_2018_210.

15. Bazhenov AI, Elgort DA, Feldsherova AA, et al. Detection of anti­bodies to mutant forms of HBsAg in persons immunized against hepatitis B with vaccines of different subtypes. Epidemiology and Vaccinal Prevention. 2011;5(60):49-53. (In Russ.)

16. Conners EE, Panagiotakopoulos L, Hofmeister MG, et al. Screening and Testing for Hepatitis B Virus Infection: CDC Re­com­mendations, 2023. MMWR Recomm Rep. 2023;72(1):1-25. doi:10.15585/mmwr.rr7201a1.

17. Kochetova EO, Shamsheva OV, Polesko IV, et al. Features of the for­mation of specific immunity after vaccination against viral hepatitis B in children and young people. Lechaschi Vrach. 2023;26(6):7-15. (In Russ.) doi:10.51793/OS.2023.26.6.001.

18. Semenenko TA. Immune response after vaccination against hepa­titis B in patients with immunodeficiency. Epidemiology and Vaccinal Prevention. 2011;1(56):51-8. (In Russ.)

19. Marjani A, Alavian SM, Nassiri Toosi M, et al. Hepatitis B virus in­fec­tion after immunization: How serious it is? An updated review. Clin Exp Med. 2025;25(1):113. doi:10.1007/s10238-025-01645-8.

20. Soloviev DV, Korabelnikova MS, Kudryavtseva EN, et al. Sero­lo­gical monitoring as an indicator of population immu­nity against he­patitis B in the population of the Russian Federation in 2017–2022. Epidemiology and Vaccinal Prevention. 2024;23(5):24-32. (In Russ.) doi:10.31631/2073-3046-2024-23-5-24-32.

21. Cuhadar S, Koseoglu M, AtayA, Dirican A. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochem Med. 2013;23(1):70-7. doi:10.11613/bm.2013.009.

22. Anisimov SV, Granstrem OK, Meshkov AN, et al. National as­sociation of biobanks and biobanking specialists: new community for promoting biobanking ideas and projects in Russia. Biopreserv Biobank. 2021;19(1):73-82. doi:10.1089/bio.2020.0049.

23. Aladawy AI, Elnakib M, Fattah MA, et al. Impact of -20 оC cryo­pre­servation on serum factors from schistosomiasis patients at different storage durations: insights into serum bio-banking. J Pa­rasit Dis. 2025;49(1):162-72. doi:10.1007/s12639-024-01734-7.

24. Castro AR, Jost HA. Effect of multiple freeze and thaw cycles on the sensitivity of IgG and IgM immunoglobulins in the sera of patients with syphilis. Sex Transm Dis. 2013;40(11):8701. doi:10.1097/OLQ.0000000000000036.