Establishment of a Sensitive Indirect ELISA for Fish Parvalbumin quantification; Method Optimization and Preliminary Plasma Analysis

Abstract

Parvalbumin is a small Ca2+-binding protein found in various organisms, including fish. Previous research has shown that fish-specific parvalbumin, f-PVB, has been able to be measured in plasma samples. The aim of this thesis was to develop and optimize an indirect ELISA for fish parvalbumin in plasma samples. Two primary antibodies, 3E1 and PARV19, were compared in signal behavior, specificity, and suitability for plasma analysis. A total of eight ELISA experiments were performed using different detection methods, calcium concentrations and sample dilutions. The results showed that the antibody PARV19 gave lower signal in meat controls compared to the antibody 3E1, which indicates that PARV19 has higher specificity for fish parvalbumin compared to 3E1. In addition, there was no clear distinction between vegan and fish diet plasma signals, using the antibody 3E1. These results indicated that PARV19 should be the go to antibody. Addition of Calcium chloride (CaCl2) during the steps of the ELISA improved the ELISA performance by increasing signal intensity and improving antibody binding, likely because calcium stabilizes the Parvalbumin structure. A CaCl2 concentration of 1mM resulted in higher signal intensity and improved reproducibility compared to 0 and 5 mM CaCl2. Furthermore, we tested two different detection methods, a chemiluminescent (SuperSignal, Bio-Rad) and a colorimetric method (TMB, ThermoFisher). Chemiluminescence detection with SuperSignal provided higher sensitivity and more consistent standard curves than using TMB. In the end it was deemed that the antibody PARV19 diluted in TBS-tween 20 with 1 mM CaCl2 using chemiluminescent detection was the best option. In this ELISA, The PVB signal from fish eater plasma was higher than the PVB signal from the vegan diet plasma (214 %). Unfortunately, this result could not be reproduced in further assays, indicating that further studies are needed. Overall, this study highlights methodological challenges associated with parvalbumin detection in tissue extract samples and plasma samples and demonstrates the importance of antibody specificity and assay optimization.