Quantification of CSF Protein Biomarkers

The following commercially available ELISAs were used to determine CSF protein levels of AGT, ENPP-2 and TRX-1: [AGT (JP27412; TECAN, IBL International GmbH, Europe), ENPP-2 (DENP20; R&D Systems, Europe), TRX-1 (RAB1756; Sigma-Aldrich, Merck KGaA, Germany)]. All ELISAs were performed according to the manufacturers’ protocols with samples diluted 1:200 for AGT, 1:40 for ENPP-2 and 1:2 for TRX-1. NG and NFL were determined by ELISA, as previously described.^{5 (link),26 (link)} TRX-80 was quantified by an in-house ELISA, as previously published.^{10 (link),27 (link)} Samples were tested in duplicate, and the average was considered for the statistical analyses. The calculated intra- and inter-assay coefficients of variation (CV%) were <6 and <15%, respectively, for the overall ELISAs. Samples with duplicate CV above 15% were remeasured or excluded from the analyses. Absorbance was measured with a microplate reader (Tecan Life Sciences, Männedorf, Switzerland). Concentrations were calculated by interpolation from the standard curves using GraphPad Prism 9 software, through a 4PL curve fit.

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Daniilidou M., Eroli F., Alanko V., Goikolea J., Latorre-Leal M., Rodriguez-Rodriguez P., Griffiths W.J., Wang Y., Pacciarini M., Brinkmalm A., Zetterberg H., Blennow K., Rosenberg A., Bogdanovic N., Winblad B., Kivipelto M., Ibghi D., Cedazo-Minguez A., Maioli S, & Matton A. (2023). Alzheimer’s disease biomarker profiling in a memory clinic cohort without common comorbidities. Brain Communications, 5(5), fcad228.

Publication 2023

JP27412 DENP20 RAB1756 microplate reader Prism 9

Assay Elisas Enpp Prism Protein

Corresponding Organization : Imperial College London

Other organizations : Swansea University, University of Gothenburg, Sahlgrenska University Hospital, University College London, UK Dementia Research Institute, University of Eastern Finland, Karolinska University Hospital, Sanofi (France)

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Variable analysis

independent variables

Dilution factor for AGT (1:200)
Dilution factor for ENPP-2 (1:40)
Dilution factor for TRX-1 (1:2)

dependent variables

CSF protein levels of AGT
CSF protein levels of ENPP-2
CSF protein levels of TRX-1
CSF protein levels of NG
CSF protein levels of NFL
CSF protein levels of TRX-80

control variables

Manufacturers' protocols for all ELISAs
Intra-assay coefficient of variation (CV%) < 6%
Inter-assay coefficient of variation (CV%) < 15%
Samples with duplicate CV above 15% were remeasured or excluded from the analyses
Absorbance measured with a microplate reader (Tecan Life Sciences, Männedorf, Switzerland)
Concentrations calculated by interpolation from the standard curves using GraphPad Prism 9 software, through a 4PL curve fit

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