Reproducibility problem in an ELISA assay method - fibroblast growth factor 23 (FGF-23) as an example
Henrik Alfthan1,*, Helene Markkanen1, Esa Hämäläinen1
1HUSLAB, Helsingfors, Finland
Introduction: Fibroblast growth factor-23 ( FGF -23) is produced by bone and its main function is to maintain phosphate hoemostasis in man. FGF-23 decreases serum phosphate levels by increasing phosphate excretion in urine and by reducing adsorption of phosphate in the intestine. Increased FGF-23 activity has been demonstrated to cause hypophosphatemia in several diseases.
Objectives: Kainos Laboratories FGF-23 ELISA assay was used by manual pipetting. During preliminary testing a remarkably large variation between replicates was observed, which made the method unacceptable for routine use. In order to eliminate this we tested side by side two different washing techniques.
Methods: Serum samples (n = 169) were routine patient samples ordered for FGF-23 and volunteer control samples from the laboratory personel. The assay was performed according to the manufacturer's instructions. Assays were carried out in parallel using two different washing techniques. The microtiter wells were either washed with an automatic washer or manually.
Results: By using an automatic washer, the median CV% for duplicates was 25.9% (range 0.0 - 120.1%). The corresponding figures for manual wash was 4.8% (range 0.0 - 30.7%). Correlation coefficient between the duplicates was R2 = 0.0061 for automatic wash and R2 = 0.8349 for manual wash.
Conclusion: The reasons for the extremely large variation in duplicates when usin automatic wash has not been studied in detail but a similar phenomenon has been observed by others. A return to manual washing was unevitable and a powerful way to get around the problem.