Abstract   Tripeptidyl-peptidase II (TPPII), is present in most eukaryotic cells. It cuts tripeptides from the N-terminus of peptides and is especially important for degrading peptides longer than 15 amino acids. TPPII also tailors long peptides into suitable substrates for the enzymes which transport and produce the peptides that MHC I present. Increased levels of TPPII have also been found in certain cancer cells, thus it is of interest to determine if TPPII could be used as a tumour marker. The aim of this study was to optimise and evaluate 3 different methods for quantifying TPPII. Western blot, enzyme-linked immunosorbent assay (ELISA) and fluorophore-linked immunosorbent assay (FLISA) protocols were optimised regarding incubation times and antibody dilutions. Sensitivity and linearity were the most important parameters when evaluating the results. The coefficient of determination of western blot was R2=0.98-1 within the range of 1.29-250ng TPPII/well and ELISA had a coefficient of determination of R2=0.96 within the range of 0.03-250ng TPPII/well. Presently western blot is the only one of these methods to yield reliable results with impure samples, but ELISA is superior regarding sensitivity and throughput. Thus further optimisation of ELISA is interesting to pursue.

Tripeptidyl peptidse II (TPPII) is an enzyme forming a complex of 4,5 MDa. It is located in the cytosol and participates in protein turnover where it degrades oligopeptides to smaller peptides and tripeptides. Moreover, some of its products might take part in antigen presentation through MHC-class 1. High expression of the enzyme is believed to correlate with tumor progression. A decreased concentration seems to contribute to increased susceptibility to infection. The aim of this project was to develop an enzyme linked immusorbent assay (ELISA) specific for TPPII and compare it to two commercial kits. TPPII was detected through western blot and activity assay to ensure which samples were containing the enzyme. Samples with purified enzyme from different species, erythrocyte lysate and plasma were used to develop an indirect ELISA as well as compare all the assays and evaluate which one performed the best. The commercial kits were based on sandwich and competitive techniques. It was observed that the developed ELISA was able to detect the purified enzyme of different concentrations whereas the commercial kits could not. On the other hand, the commercial kits were able to generate their own standard curve which leads to suspicion that these might be non-specific to TPPII. Though, all assays did detect the enzyme in erythrocyte lysate. This study presents that it is possible to develop an ELISA specific for TPPII using an indirect technique which also performed better than commercial kits.