Stability of important antibodies for kidney disease: pre-analytic methodological considerations

PeerJ. 2018 Jul 9:6:e5178. doi: 10.7717/peerj.5178. eCollection 2018.


Background: The importance of circulating antibodies as biomarkers of kidney disease has recently been recognized. However, no study has systematically described the methodology of sample preparation and storage regarding antibodies as biomarkers of kidney disease. It remains unknown whether repetitive freeze-thaw cycles, physical disturbances, storage at different temperatures or for different periods of time, or haemolytic or turbid serum samples affect antibody measurements. The aim of this study was to investigate the stabilities of antibodies associated with kidney disease in serum samples under various relevant clinical and research conditions.

Methods: We stored serum samples in the following different conditions: repetitive freeze-thaw cycles (1, 6 or 12 times), long-term storage (7 or 12 months at -80 °C), physical disturbance (1 or 8 h), and storage at 4 °C (1, 3 or 6 weeks) and room temperature (1 or 7 days). The stabilities of the anti-phospholipase A2 receptor (anti-PLA2R), anti-glomerular basement membrane, anti-myeloperoxidase and anti-proteinase 3 antibodies were evaluated with enzyme-linked immunosorbent assays (ELISA).

Results: We found that repetitive freeze-thaw cycles did not have a significant effect on the stabilities of the abovementioned antibodies in clear serum samples. The ELISA readings of haemolytic and turbid serum samples tended to increase and decrease, respectively. Neither long-term storage at -80 °C nor physical disturbance had a significant effect on anti-PLA2R antibody stability in sealed serum samples. The concentrations of most of these antibodies increased in unsealed serum samples that were stored at 4 °C for more than 6 weeks or at room temperature for more than 7 days.

Discussion: Our findings revealed that the abovementioned circulating antibodies that are used as biomarkers for kidney disease had stable physicochemical properties, structures and immunoreactivities such that they were not influenced by repetitive freeze-thaw cycles, physical disturbances or long-term storage at -80 °C. However, the ELISA readings tended to change for haemolytic, turbid and unsealed serum samples.

Keywords: Stability; Storage; anti-GBM antibodies; anti-MPO antibodies; anti-PLA2R antibodies; anti-PR3 antibodies.

Grants and funding

This work was supported by the National Key R&D Program of China (2016YFC1305500 and 2016YFC1305404), the National Natural Science Foundation of China (Nos. 61471399 and 61671479), National Clinical Research Center for Kidney Disease (No. 2013BAI09B05), the Key Program of Joint Funds of the National Natural Science Foundation of China (No. U1604284) and the Natural Science Foundation of Henan province (No. 162300410282). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.