It had been previously shown by the Microbial Adherence Immobilization Assay (MAIA) that Borrelia burgdorferi sensu stricto, type strain B31 was clumped, immobilized and killed in vitro by sensitizing antibodies that activated the classical complement pathway and the complement-killing of live borrelia. In the present study, the target antigens and sensitizing antibodies responsible for the complement-killing of borrelia were investigated, using MAIA as a selective identification tool. It was found that the fractions containing the 31 and 34 kDa outer surface proteins from strain B31 were the unique antigens producing sensitizing antibodies in rabbits that activated the complement-killing of B31. An anti-OspB, but not an anti-OspA, monoclonal antibody did activate the B31 complement-killing in MAIA. From these results, constraints on the effectiveness of OspB and OspA as immunogens for the prevention and control of Lyme borreliosis in humans are discussed.