Lyme Crónico y su complejo de infecciones asociadas

B. burgdorferi WB.

The IgG antibody response to B. burgdorferi B31 was further characterized by Western blot assay (WB), using commercial prepared blots and the Euroblot automated WB instrument according to the manufacturer's protocols (Euroimmun, Boonton, NJ). Briefly, nitrocellulose strips containing electrophoresis-separated B. burgdorferi B31 proteins were blocked and then incubated with 1.5 ml of diluted serum sample (1:50) for 30 min. Membrane strips were washed and incubated with alkaline phosphatase (AP)-conjugated anti-human IgG antibody for 30 min. Bound antibodies were detected using the nitroblue tetrazolium chloride–5-bromo-4-chloro-3′-indolylphosphate (NBT-BCIP) staining system (Euroimmun). Quantitative analysis of bands on each blot was carried out using the EUROLineScan software (Euroimmun). Accurate background correction and determination of cutoff values for positivity were carried out by the software for the p18, p25 (OspC), p28, p30, p31 (OspA), p34 (OspB), p39 (BmpA), p41 (FlaB), p45, p58, p66, p93, and recombinant VlsE borrelial protein bands. 

Determination of IgG-positive serology for Lyme disease was based on the - 1º- CDC criteria, according to which an - 2º- IgG immunoblot can be considered positive if 5 or more of the following 10 bands are positive: 18 kDa, 25 kDa (OspC), 28 kDa, 30 kDa, 39 kDa (BmpA), 41 kDa (Fla), 45 kDa, 58 kDa, 66 kDa, and 93 kDa (1, 5).