Figure 2: Applications of EOP (enzyme-on-peptide)-mode pep-ELISA. In this experiment, the fluorescence intensity can be regarded to be proportional to the amount of the enzyme CatE since it generates the fluorescent substrate by cleaving the fluorogenic substrate. a) Measurement of CatE levels of different specimens (rat spleen and gastric mucosa extracted from cathepsin E-gene knockout and wild rats). Since the peptide used (pp2) has a binding (and inhibitory) activity at pH 4.5, these specimens in physiological conditions (at a neutral pH) were pre-treated to adjust the pH to pH 4.5. The aggregates generated in this process were removed (supernatant) or not removed (crude) during the enzyme activity assay. b) The effect of the change in the peptide aptamer composing EOP on the CatE-binding. The peptides used are (b1) non-CatE-binding octapeptide (the sequence of PCSELINT), (b2) pp2 (CatEinhibitor at pH4.5), and (b3) pp1015 (CatE-activator at pH4.5). The specimens used were plasma extracted from wild type (WT) and knockout (KO) rats. c) CatE measurement by the EOP-mode pep-ELISA made of a peptide aptamer (pa4043) selected at a neutral pH (pH7.4). In this case, the trapping of CatE was performed at a neutral pH, where the peptide pa4043is active, while the enzyme activity was monitored at pH 4.5using the supernatant of centrifugationtreated plasma solution(the same origin as in b)which had been subjected topH-alteration from a physiological pH to pH4.5(see Materials and Methods). Note that CatE does not need to be bound to the peptide when its activity is monitored at pH4.5 (where the peptide selected at pH7.4 will probably lose its binding affinity to CatE). Throughout the above experiments, the CatE activity at pH 4.5 was compared, making the comparison of activity easy. Statistics were taken from three independent experiments except b1 (n=2).