Methods for 20S Immunoproteasome and 20S Constitutive Proteasome Determination Based on SPRI Biosensors

Abstract

The 20S proteasome, released into the circulation, is a novel cancer biomarker. It exists in two forms: the constitutive proteasome (20Sc) and the immunoproteasome (20Si), which both have separate diagnostic significance. The aim of this work was to develop new methods for 20Si and 20Sc determination. Five alternative specific biosensors usable with the surface plasmon resonance imaging (SPRI) technique for 20Si determination have been developed. Specific 20Si entrapment on the biosensor surface from an analyzed solution was achieved by means of an immobilized specific 20Si receptor. Four of the biosensors contain newly synthesized specific 20Si receptors, while the fifth contains the inhibitor ONX 0914. A method for 20Sc determination using an SPRI biosensor containing PSI inhibitor has been developed. By the introduction of an inhibitor blocking 20Si, 20Sc is selectively determined. All of the methods developed for 20Si and 20Sc determination exhibit good selectivity and satisfactory precision, recoveries and dynamic response ranges. 20Si and 20Sc were determined in blood plasma samples from healthy donors and patients with acute leukemia. In the case of these patients 20Si was the major component, and its level was more than one order of magnitude higher than in the healthy donors.

Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

“…Only Guillaume et al (6) considered the heterogeneity of 20S subtypes when developing their ELISA assay by using different in-house produced antibodies directed against four different standard and immunocatalytic β subunits. More recently, standard and immunoproteasome subtypes were determined by surface plasmon resonance imaging (SPRI) using specific inhibitors (15). However, the multiplexing capacity of these methods is insufficient to fully assess proteasome heterogeneity in a single assay.…”

Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome

“…The performance of the present immunosensor was compared to previous reports related to detection methodologies of 20S proteasome ( Table 3 ). In relation to the SPR method, which enabled the detection of proteasome through its interaction with inhibitors [ 11 , 12 ], similar detection limits are observed; however, the Au/4-MPBA/Abβ immunosensor allowed a wider dynamic range relevant to proteasome levels in serum. On the other hand, an ELISA method allowed the detection of proteasome in the same concentration range [ 32 ] as the proposed Au/4-MPBA/Abβ electrochemical immunosensor.…”

“…Another proteasome c detection kit uses electro-chemiluminescence, in which the third antibody that allows quantification is labeled so as to generate an electro-chemiluminescent signal, and has been successfully used to detect proteasome c in patients with hepatocellular carcinoma [ 8 ]. Surface plasmon resonance (SPR) imaging has also been applied for 20S detection, based on the interaction of proteasome with immobilized inhibitors [ 11 , 12 ].…”

Electrochemical Immunosensing Platform for the Determination of the 20S Proteasome Using an Aminophenylboronic/Poly-indole-6-carboxylic Acid-Modified Electrode

“…Currently, there have been reports on using antibodies to determine P20S using different techniques, such as surface plasmon resonance imaging (SPRI) and enzyme-linked immunosorbent assay (ELISA). These methods have also been used in kinetic studies drawing on specific inhibitors for P20S. − …”