Novel immunoassay technique for rapid measurement of intracellular proteins using paramagnetic particles

Abstract

Magnetic immunoassays have been shown to have similar detection limits to conventional immunoassays, with the advantage of reduced total assay time. The aim of this study was to demonstrate that an integrated lysis and measurement system could be used to quantitatively measure intracellular target molecules using prostate specific antigen as a model analyte.The system described utilises the inherent physical properties of paramagnetic particles for both cell lysis and antigen quantification in the same vessel. This is achieved using ultrasound to energise paramagnetic particles to lyse cells combined with a magnetic immunoassay to measure intracellular protein, synthesised within the cells. Antibody coated paramagnetic particles were energised using pulses of ultrasound energy to penetrate and lyse cells and capture intracellular protein on the particle surface. The particles were drawn to an antibody coated sensor surface, in an applied magnetic field, which bound to captured analyte on the paramagnetic particle. The number of immobilised particles on the sensor surface is quantified by a resonant coil magnetometer. The total assay time was reduced to less than 15min.To demonstrate the utility of the system a model assay for intracellular prostate specific antigen was developed to show that the assay could detect differences in the amount of intracellular protein. This was achieved by exposing LNCaP cells to increasing concentrations of testosterone, which causes an increase in prostate specific antigen production. The rapid intracellular assay was able to demonstrate increasing amounts of intracellular prostate specific antigen resulting from increasing testosterone exposure.The technology could be used to develop rapid diagnostic tests for intracellular biomarkers that are difficult to detect in normal serum samples, for example viral proteins and intracellular cancer proteins.