Borrelia burgdorferi IgM ELISA

Enzyme immunoassay for the qualitative determination of IgM-class antibodies against Borrelia burgdorferi in human serum or plasma (citrate) and CSF (cerebrospinal fluid). Spirochetes are motile bacteria with a periplasmatic axial filament. All pathogenic species belong to the family Treponemataceae, which includes the three genera: Treponema, Borrelia, and Leptospira. The Treponemae are extremely long, flexible, filamentous cells that are usually held in a characteristic spiral, or coiled-spring shape. Borreliae are the largest Treponemataceae with very coarse and irregular spirals. Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted mainly by ticks but probably also by other blood-sucking insects. Habitats are the wooded, humid and temperate regions of North America, Europe, North Africa, Australia and Japan; foresters, farmers, and anybody entering infested forests may be affected. The degree of contamination of ticks amounts to 3-60% dependent on seasonal and regional differences; up to 30% of the population may be infected (about 1500 cases annually in USA, several hundred in Europe). The major constituent of B. burgdorferi flagella is flagellin (41 kDa, p41). Whereas the lipoprotein OspC (22 kDa, p22) within the outer membrane of the spirochete induces an early antibody formation in the ECMphase of Lyme disease. Recombinant p41i is included in the presented antigens to avoid crossreactivity with antibodies of syphilitic sera. The Borrelia burgdorferi IgM-ELISA contains the following recombinant epitopes: OspC of the phylum PKo (B. afzelii) and 20047 (B. garinii) and p41i of the phylum PBi (B. garinii). Microtiter strip wells are pre-coated with recombinant Borrelia burgdorferi antigens. Diluted patient specimens and ready to use controls are added to these wells and antibodies recognizing the immobilized B. burgdorferi antigen bind during the first incubation. After washing the wells to remove all unbound sample and control material horseradish peroxidase labelled anti-human IgM conjugate is added. During a second incubation this conjugate binds to the captured antibodies, and the excess unbound conjugate is removed by a further wash step. The immune complex formed by the bound conjugate is visualized with TMB Substrate Solution which gives a blue reaction product, the intensity of which is proportional to the amount of B. burgdorferi-specific IgM antibody in the patient specimen. Sulphuric acid is added to each well to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader.