Accurate Determination of Soluble Axl by Enzyme-Linked Immunosorbent Assay.
Levels of soluble Axl (sAxl) are routinely assessed in human sera by sandwich enzyme-linked immunosorbent assay (ELISA). Although sAxl values are suggested to diagnose different types of disorders, no uniform ELISA method is available, allowing the reliable interassay comparison between results. Furthermore, little is known about the stability of sAxl under storage conditions, which is a relevant parameter for biomedical trials. The evaluation of sAxl stability under various stress conditions and the determination of proper conditions to use the sAxl ELISA for routine clinical applications are of great interest. In this study, serum samples were subjected to freeze-thaw cycles and incubation at different temperatures to analyze the stability of sAxl by ELISA. Dilution and spike-in experiments were carried out to examine the impact of serum and diluent components on the ELISA performance. Various diluents and media were employed to resolve masking effects of the serum. The assay components were further optimized for long-term usability by treatment with stabilizers and validation under temperature stress. Indeed, sAxl showed long-term stability in serum during freeze-thaw cycles and incubation under temperature stress conditions. The dilution experiments revealed that unknown components in the serum caused masking effects that can be reduced by proper dilutions. The assay performance was further increased by using a standardized buffer system to dilute serum samples. Stabilization of coated plates and of streptavidin-horseradish peroxidase allowed long-term storage for up to 6 months. In sum, our data demonstrate proper ELISA conditions, allowing the accurate analysis of sAxl levels in human serum.
1 Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna , Vienna, Austria .
Mirko
M
Dengler
1 Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna , Vienna, Austria .
Heidemarie
H
Huber
2 Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna , Vienna, Austria .
Christian J
CJ
Müller
3 Candor Bioscience GmbH , Wangen im Allgäu, Germany .
Angela
A
Zellmer
3 Candor Bioscience GmbH , Wangen im Allgäu, Germany .
Peter
P
Rauch
1 Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna , Vienna, Austria .
Wolfgang
W
Mikulits
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D002417:Cattle; D002626:Chemistry, Pharmaceutical / Q000379:methods / Q000592:standards*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood*; D011518:Proto-Oncogene Proteins / Q000097:blood*; D020794:Receptor Protein-Tyrosine Kinases / Q000097:blood*; D012710:Serum Albumin, Bovine / Q000032:analysis; D000094963:Axl Receptor Tyrosine Kinase
D011518:Proto-Oncogene Proteins; D012710:Serum Albumin, Bovine; D020794:Receptor Protein-Tyrosine Kinases; D000094963:Axl Receptor Tyrosine Kinase
ELISA; biomarker; matrix effect; soluble Axl; stability