On-site diagnostic device based on immune-separation of proteins

A membrane immuno-chromatographic system that selectively separates plasma lipopmteins and generates a signal in proportion to the concentration of ch olesterol (HDL-C) within high-density lipoprotein (HDL) was investigated as a point-of-care device for the prognosis of coronary heart disease. The sy stem consists of three functional membrane strip pads connected in a sequen ce for: (from the bottom) immune-separation based on biotinstreptavidin rea ction, catalytic conversion of cholesterol to hydrogen peroxide, and produc tion of a signal. For immunochromatography, a monoclonal antibody, specific to apolipoprotein B100 that is present on the surfaces of low-density lipo proteins (LDL) and very low-density lipoproteins (VLDL), with a high bindin g constant (5x10(10) L/mol) was raised and chemically conjugated to strepta vidin. The conjugate was first reacted with lipoprotein particles, and this mixture was absorbed by thr capillary action into the biotin pad of the sy stem. After being transferred by medium, immuno-capture of LDL and VLDL par ticles onto the biotin pad took place, and ill situ generation of a signal in proportion to HDL-C consecutively occurred. The capture was selective as well as effective (minimum 90% of LDL and VLDL in clinical concentration r anges), and the detection limit of HDL-C was far lower than 20 mg/dL. To co nstruct a user-friendly device, we are currently investigating the automati on of such processes of reactions and separation by adapting a liquid flow- controlling technology that programs the times for the immune reaction and separation. My group further pursues an interdisciplinary study to develop a micro system employing semiconductor-based technologies that will eventua lly enable the handling of sub-micro liter volume of body fluid as a specim en.