EVALUATION AND OPTIMIZATION OF A LABORATORY-CONSTRUCTED FLOW CHAMBER FOR ON-LINE IMMUNOMAGNETIC SEPARATION

Abstract

ABSTRACT Immunomagnetic separation (IMS) uses magnetic beads to facilitate separation of antibody-bound labeled antigens from free antigens in solution. Off-line immunomagnetic separation is time consuming due to the requirement of washing the magnetizable beads between assays and the multiple steps involved in the separation process. On-line IMS, in the flow injection mode, can overcome the disadvantages of off-line IMS, such as multiple washing steps. The purpose of this work is to develop, optimize, and validate an on-line magnetic separation flow chamber in the flow injection mode, suitable for post-column immunoreaction detection. The ends of the flow chamber were connected to the flow injection system. The on-line magnetic flow chamber was constructed using 1.0 I.D. × 1.58 O.D.mm Teflon tubing that was attached to an electromagnet. The electromagnet was constructed by wrapping a 305 × 25 × 40 mm block of hard steel with fine copper wire which was supplied with direct current (dc). The magnetic field was applied and released by switching the power on and off. The electromagnet was designed to have three terminals with 750 turns (yellow), 1000 turns (blue) and 1250 turns (red), respectively, to generate three different electromagnetic fields. A cooling tube was wound among the copper wire and ice water was used to cool down the core of the electromagnet. Water was pumped into the cooling tube using a VWR peristaltic pump and circulated in a forty-gallon container full of ice water. Various orientations and volumes of the flow chamber, various flow rates, and currents were investigated to obtain the maximum magnetic trapping efficiency. Digoxin was selected as a model analyte and a competitive format was used for immunoassay. Digoxigenin was labeled with R-phycoerythrin (PE-D) as a competing labeled antigen and anti-digoxin antibodies covalently linked biotin through streptavidin coated magnetic beads were used for magnetic separation. The immunoassay mixture was injected into the flow chamber after immunoreaction and immunomagnetic complexes were captured on the wall of the flow chamber using the electromagnet. The maximum trapping efficiency obtained was 98% using a five-fold straight flow chamber at a flow rate of 0.5 mL/min and a current of 1.5 amperes (A). The on-line magnetic separation immunoassay of digoxin in spiked phosphate buffer demonstrated a dynamic range of 0.5–15 ng/mL. A quadratic fit was found to provide the best fit to the data (r=0.9937). The precision for two controls, 4.0 and 12 ng/mL, were 14.05% RSD and 10.75% RSD (n=6) and the accuracy was 5.8% and 3.31% (n=6), respectively. The limit of detection was calculated to be 0.44 ng/mL.