FIG 2.

Detection of Abs to defined B. burgdorferi and A. phagocytophilum antigens. Recombinant proteins (indicated on the left) were generated by PCR amplification of B. burgdorferi B31 and A. phagocytophilum NCH-1 genomic DNA or through gene synthesis (codon optimized; GenScript). The A. phagocytophilum strain Dog2 P44 gene sequence (GenBank accession no. AGR82240.1) was used to generate recombinant P44. All primer sequences, the P44 amino acid sequence, and the p44 codon-optimized gene sequence are provided in Table S1 in the supplemental material. The proteins were expressed from pET-45b(+) (Novagen). All cloning and protein production procedures were done as previously described (15). Dot blots were generated by spotting 125 ng of purified protein onto nitrocellulose. The membranes were air dried overnight and then blocked and screened with each plasma sample as detailed in the legend to Fig. 1. All dot blots were imaged simultaneously.