Protein l

Antigen strips for Bbsl and RFB immunoblots were prepared as previously described [1 (link),2 (link)]. In brief, purified proteins and control proteins were diluted (7–19 ng protein/line) and sprayed in straight lines on nitrocellulose sheets (Amersham Protran, GE Healthcare Life Science) using a BioDot liquid dispenser (BioDot, Irvine, CA, USA). Protein L (Sigma, St. Louis, MO, USA) and mixed human immunoglobulin M (IgM) and IgG (Sigma, St. Louis, MO, USA) were used as control proteins for detecting the addition of human serum and for detecting the addition of alkaline phosphatase conjugated anti-human antibodies. The sheets were then blocked with 5% non-fat dry milk and sliced into 3-mm-wide strips.

Alkaline phosphatase-conjugated rabbit antibody to the 39/93 kDa LDB antigens (Strategic Biosciences, Stow, MA, USA) diluted in human serum was used as a calibration control in both TBRF and LD IBs and bands with lower intensity than the calibration control were considered to be negative [34 (link)]. The two proteins used as controls in both IBs were a mixture of human IgM and IgG (Sigma, St. Louis, MO, USA) for detecting the addition of alkaline phosphatase conjugated anti-human antibodies (C1), and Protein L (Sigma, St. Louis, MO, USA) for detecting the addition of human serum (C2), as previously described [34 (link)].
Immune rabbit sera for use as a marker control in TBRF IBs were prepared by immunizing individual rabbits separately with each of the recombinant antigens from the different RFB species used in TBRF IBs (Pacific Immunology, Ramona, CA, USA). The rabbit sera were then pooled and used at a dilution of 10⁻⁴ in a single control IB strip. Alkaline phosphatase-labelled goat anti-rabbit whole IgG (KPL, Gaithersburg, MD, USA) was used as the secondary antibody.

Antigen strips were prepared essentially as described for our previously developed IB assays for borreliosis [9 (link), 10 (link)]. Purified antigens diluted to yield approximately 7–19 ng of protein as a line in each 3 mm strip of membrane were sprayed in straight lines onto nitrocellulose membrane (Amersham Protran, GE Healthcare Life Science, Chicago, IL) using a BioDot liquid dispenser (BioDot, Irvine, CA). Human IgG and IgM (Sigma, St. Louis, MO) were applied as controls C1 and C4 respectively in all IB strips for establishing the specificity of antibody class detection and for confirming the addition of alkaline phosphatase conjugated anti-human antibodies. Protein L (Sigma, St. Louis, MO) was used as control C2 for detecting the addition of human serum. A calibration standard C3 was applied on the test strip for use in all IB assays. The membranes were then blocked with 5% dried skim milk and sliced into 3 mm wide strips. The membrane strips containing antigens could be stored for at least 6 months at 2-8 °C before their use for IB assays.