The RCBPs used in this assay were cloned, expressed, and purified as described previously.14 Briefly, RCBPs containing epitopes from OspA, OspB, OspC, flagellin, and p93 were generated. Portions of DNA of these sequences were cloned in tandem in an expression vector that could produce recombinant fusion proteins. Recombinant proteins OspB-OspC-Fla (B-C-Fla, 64 kd), OspA-p93 (A-93, 97 kd), and 2 OspC (22 kd) proteins from different genospecies (Borrelia afzelii and Borrelia garinii) were used. A protein cocktail composed of a mixture of the RCBPs was made at a concentration of 2.5 mg/mL, which included 10µM B-C-Fla, 12µM A-93, and 9.5µM each OspC (plus 0.1mM dithiothreitol, 1.0mM phenylmethylsulfonyl fluoride (PMSF), and 0.02% sodium azide in 20mM phosphate-buffered saline, pH 7.2). The protein concentration was checked by the Bradford method (Bio-Rad Laboratories, Hercules, Calif) and by direct visualization on a Coomassie-stained sodium dodecyl sulfate–polyacrylamide electrophoresis gel. Then, 100 µg of protein was loaded onto the test zone of a nitrocellulose membrane (Chembio Diagnostics, Medford, NY) on a thin-band layer (Camag Linomat IV; Camag, Muttenz, Switzerland). Staphylococcus aureus protein A antigen (2.0 mg/mL in 50mM Tris-hydrochloride, pH 8.0, and 150mM sodium chloride) was loaded a third of an inch (≈1 cm) away from the B burgdorferi test antigen on the control zone of the membrane. Antibody binding protein conjugated to colloidal gold, which interacts with immunoglobulins, notably IgG, in a nonantibody-type pseudo immune reaction,15 was applied onto the sample zone of the membrane. The assay is polyvalent. The membrane strip was air dried and was packed into a plastic device containing 3 windows labeled sequentially: sample, test, and control (Chembio Diagnostics). The test device was stored at room temperature in a dry area until used (up to 1 year).