Method for the determination of hydrolytic activity

A method for determining hydrolase activity in a sample, wherein the hydrolase is an esterase capable of hydrolysis of carboxylic acid esters producing an alcohol and a carboxylic acid, the method comprising the following steps:

• a) incubating the sample or an aliquot thereof and a buffered solution to form an incubated sample,
• wherein the buffered solution comprises a buffer substance, an artificial hydrolase substrate, a salt and bovine serum albumin,
• wherein the sample comprises a therapeutic polypeptide,
• wherein the artificial hydrolase substrate comprises a single ester bond covalently linking a carboxylic acid residue, which is conjugated to a first label, and an alcohol residue, which is conjugated to a nucleic acid tag and a second label, which is different from the first label, wherein the artificial hydrolase substrate is represented by the following formula:
• wherein, n and m each independently of each other is zero or a positive integer value, R1 comprises the first label, and R2, R3 comprises the second label and the nucleic acid tag,
• wherein the ester bond in the artificial hydrolase substrate is cleaved into a free alcohol residue and a free carboxylic acid residue if hydrolase activity and/or a hydrolase is present in the sample,
• b) removing the free carboxylic acid residue and artificial hydrolase substrate from the incubated sample to obtain a depleted sample, wherein the removing is carried out by contacting the incubated sample with a first solid phase, which specifically binds to the first label, and separating the free carboxylic acid and artificial hydrolase substrate-depleted incubation mixture from the first solid phase thereafter,
• c) determining hydrolase activity and/or the presence of a hydrolase in the sample by determining the second label in the depleted sample,
• wherein the determining is carried out by capturing the free alcohol residue on a second solid phase via the nucleic acid tag followed by detecting the solid-phase captured free alcohol residue with an antibody specifically binding to the second label, which antibody is conjugated to one or more detectable labels.