A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

Abstract

BackgroundKallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.MethodsThe coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.ResultsAnti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.ConclusionsThe ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-017-9140-6) contains supplementary material, which is available to authorized users.

Beyond liquid biopsy: Toward non-invasive assays for distanced cancer diagnostics in pandemics

“… Type of cancer Type of biomarker Method of detection/Targeted Biomarkers/Ref. Ovarian Amino acids Immunocapture/parallel reaction monitoring mass spectrometry - mat-KLK9 ( Filippou et al, 2017 ) Lung Metabolites Qualitative tandem liquid chromatography quadrupole time of flight mass spectrometry - Suberic acid, tetrahexose, trihexose, nonanedioic acid & monoglyceride ( Calderón-Santiago et al, 2015 ) Lung, Gastric, Prostate, Head, Kidney, pancreas Chiral alcohol Gas chromatography with mass spectrometry - 2-ethyl-1-hexanol, hexanal, octanal & 2,6 dimethyl-7-octen-2-ol ( Monedeiro et al, 2020a ) Head and Neck Metabolites Headspace solid-phase micro extraction (SPME) gas chromatography (GC) mass spectrometry (MS) – 81 VOMs as potential tumor markers ( Opitz and Herbarth, 2018 ) Prostate Amino acids Mass spectrometry – KLK4 ( Karakosta et al, 2016 ) Various Nucleic acids Real-time RT-PCR - DCD ( Sakurada et al, 2010 ) …”

“…The authors found that in all cases, KLK9 concentration in sweat was stable after 24 h. At RT, on day seven, the concentration decreased by about 40%. KLK9 spiked in serum, instead, decreased by about 60% after 24 h incubation and this was attributed to the formation of heterocomplexes with endogenous serine protease inhibitors ( Filippou et al, 2017 ).…”

Kallikrein-related peptidases protein expression in lymphoid tissues suggests potential implications in immune response

“…Tissue cytosolic extracts were prepared from snap-frozen tissues as previously described [24]. Briefly, fresh frozen human tissues were pulverized to a fine powder in 0.2 g/ml of extraction buffer.…”

Optical Fingerprints of Proteases and Their Inhibited Complexes Provided by Differential Cross-Reactivity of Fluorophore-Labeled Single-Stranded DNA

“…Proteases are associated with most biological processes because of their ability to regulate the physiological functions of proteins via hydrolysis. Because its aberrant activity plays a crucial role in a variety of diseases, including cardiovascular and inflammatory diseases as well as cancer, the detection of proteases is of great importance for diagnosis and medical treatment. − Typically, protease detection is achieved by specificity-based assays, such as enzyme-catalyzed reactions ,− and enzyme-linked immunosorbent assays (ELISAs) based on antigen–antibody reactions. − However, the development of highly specific recognition remains difficult, and the design/production of antibodies or enzyme substrates for binding or reaction visualization are costly and laborious.…”