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An antibody capture enzyme-linked immunosorbent assay was developed to measure directly intrathecal immunoglobulin (Ig) G, A, and M synthesis specific for Borrelia burgdorferi. Purified, biotin-avidin-peroxidase-labeled B. burgdorferi flagella was used as test antigen. Paired cerebrospinal fluid and serum specimens from 100 patients with clinically definite neuroborreliosis and 35 control subjects with neurological diseases were examined. Significant B. burgdorferi-specific intrathecal IgG, A, and M production was found in 89%, 65% and 67% of patients with neuroborreliosis. Local synthesis of specific IgA was only seen in patients with significant local IgG synthesis. Antibody production in cerebrospinal fluid began by 2 weeks after onset of neurological symptoms. At the end of the second week specific IgM, IgG, or both, was detected in 88% of the patients. Specific IgG synthesis was present in all patients by 6 weeks after onset. Specific local IgM synthesis usually disappeared by 3 to 6 months after therapy, whereas specific IgG synthesis persisted after recovery. Even in patients with a severely altered blood-brain barrier, the assay discriminated between intrathecal antibody synthesis and antibody leakage from serum. The assay makes diagnostic measurement of B. burgdorferi-specific intrathecal antibody synthesis reliable, rapid, and accessible as a routine serological test.