Effect of multiple freeze-thaw cycles on detection of anti-SARS-CoV-2 IgG antibodies

Several studies have investigated the effect of repeated freeze-thaw (F/T) cycles on RNA detection for SARS-CoV-2. However, no data is available regarding the effect of repeated F/T cycles on SARS-CoV-2 antibody detection in serum. We investigated the effect of multiple F/T cycles on anti-SARS-CoV-2 IgG detection using an ELISA test targeting the nucleocapsid antibodies. Ten positive and one negative SARS-CoV-2 IgG sera from 11 participants, in replicates of five were subjected to a total of 16 F/T cycles and stored at 4 degrees celsius until tested by ELISA. Statistical analysis was done to test for F/T cycle effect. Non-of the 10 positive sera turned into negative after 16 F/T cycles. There was no significant difference in the OD average reading between the first and last F/T cycles, except for one serum with a minimal decline in the OD. The random-effect linear regression of log (OD) on the number of cycles showed no significant trend with a slope consistent with zero (B=-0.0001; 95% CI -0.0008; 0.0006; p-value=0.781). These results suggest that multiple F/T cycles had no effect on the ability of the ELISA assay to detect the SARS-CoV-2 IgG antibodies.

The random-effect linear regression of log (OD) on number of cycles showed no significant 1 0 2 trend with a slope consistent with zero (B=-0.0001; 95% CI -0.0008; 0.0006; P-value=0.781), 1 0 3 indicating that the multiple F/T cycles had no effect on the SARS-CoV-2 IgG antibody titers in 1 0 4 the serum. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. each serum plotted against the number of freeze-thaw cycles.   Figure 2 Antibody levels for SARS-CoV-2 for 438 specimens against the number of freeze-thaw cycles. The blue and red solid horizontal lines delineate the cut-off for negative at optical density 0.2655 and the cut-off for positive at 0.3245. 6 for of ve . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 19, 2021. 7 4. Discussion: Frozen serum banks are an important source of scientific and clinical information and are 1 1 1 essential to infectious disease and vaccine research. It is commonly believed that antibodies keep 1 1 2 their stability if serum is stored below -20C o (9). Nevertheless, evidence suggests that just one 1 1 3 F/T cycle can significantly reduce IgG antibody levels (9). To date, studies investigating the 1 1 4 effects of F/T cycles on the detection of SARS-CoV-2 antibodies have yielded inconsistent 1 1 5 results, and the data availability on the effects of multiple F/T cycles, sample size, and storage  The present study investigated the effect of 16 repeated F/T cycles on SARS-CoV-2 IgG in sample reactivity between the first and last F/T cycle, and none of the reactive sera became  In agreement with our findings, other studies have tested the effect of repeated F/T cycles 1 2 6 on measles, mumps, rubella, syphilis, anti-nuclear antibodies (ANA), and anti-neutrophil that there was no clinically or statistically significant difference in the antibody levels after 1 3 0 several F/T cycles. Interestingly, another study has shown that 174 F/T cycles on anti-treponemal 1 3 1 sera did not affect the stability, the reactivity of antibodies, nor the samples' quality when tested 1 3 2 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.04.13.21255379 doi: medRxiv preprint 8 by a chemiluminescence assay (1). The data generated from our study and the previous studies 1 3 3 provide concrete knowledge regarding antibody stability in serum, which allows the maximum 1 3 4 potential use of serum, especially for those that have undergone multiple F/T cycles.

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Additionally, our generated results can be beneficial to the design of serum banks, where it is 1 3 6 important to monitor the integrity of sample components.

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We conclude that 16 F/T cycles did not interfere with the detection of SARS-CoV-2 IgG 1 3 8 antibodies, and with no effect on assay sensitivity. However, in this study, we tested the effect of   Acknowledgements: CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ;

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We would like to thank Dr. Nahla Afifi, Director of Qatar Biobank (QBB), Ms. Tasneem Al-     . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 0 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)