Comparative studies of magnetic particle-based solid phase fluorogenic and electrochemiluminescent immunoassay.

Abstract

Two solid phase immunoassays, an electrochemiluminescent immunoassay (ECLIA) and a magnetic particle fluorogenic immunoassay (MPFIA) were evaluated and compared for bacterial detection. Briefly, the ECLIA is based on a redox reaction between ruthenium (II)-trisbipyridyl Ru[(bpy)3]2+ labeled antibody and the excess of tripropylamine, which generates photons. The entire reaction is carried on the near surface area between the spherical magnetic beads and an anode electrode. The detectable 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 employs antibody-coated magnetic beads as solid phase in suspension for bacterial capture and concentration in a 96-well microplate format. Primary capturing antibodies, bacteria form a sandwich with alkaline phosphatase (AP)-labeled antibodies as reporter followed by a reaction with the AP substrate, AttoPhos to generate fluorescence for detection. Immunomagnetic separation permits direct isolating and concentrating bacterial cells from the crude samples, such as blood and environmental water. The results of MPFIA for detecting bacteria showed less sensitivity compared with that of ECLIA, however it provides a means for direct, high throughput screening bacteria from crude biological samples. Both ECLIA and MPFIA are rapid (less than one hour) and easy to use.