COMPARATIVE-STUDIES OF MAGNETIC PARTICLE-BASED SOLID-PHASE FLUOROGENIC AND ELECTROCHEMILUMINESCENT IMMUNOASSAY

Two solid phase immunoassays, an electrochemiluminescent immunoassay ( ECLIA) and a magnetic particle fluorogenic immunoassay (MPFIA) were ev aluated and compared for bacterial detection. Briefly, the ECLIA is ba sed on a redox reaction between ruthenium (II)-trisbipyridyl Ru[(bpy)( 3)](2+) labeled antibody and the excess of tripropylamine, which gener ates photons. The entire reaction is carried on the near surface area between the spherical magnetic beads and an anode electrode. The detec table bacterial spores are at a linear range from 5 x 10(3) to 5 x 10( 5) colony forming units (cfu) of Bacillus subtilis var. niger spores, 10(2) to 10(4) cfu of Bacillus anthrax spores and 10(2) to 10(6) cells of Escherichia coli O157:H7 in ECLIA. The unique MPFIA technique empl oys antibody-coated magnetic beads as solid phase in suspension for ba cterial capture and concentration in a 96-well microplate format. Prim ary capturing antibodies, bacteria form a sandwich with alkaline phosp hatase (AP)-labeled antibodies as reporter followed by a reaction with the AP substrate, AttoPhos(TM) to generate fluorescence for detection . Immunomagnetic separation permits direct isolating and concentrating bacterial cells from the crude samples, such as blood and environment al water. The results of MPFIA for detecting bacteria showed less sens itivity compared with that of ECLIA, however it provides a means for d irect, high throughput screening bacteria from crude biological sample s. Both ECLIA and MPFIA are rapid (less than one hour) and easy to use . (C) 1998 Elsevier Science B.V. All rights reserved.