AbstractA sensitive and specific Enzyme Linked Immunosorbent Assay (ELISA) was developed for the detection of antibodies, primarily autoantibodies, to the enzyme acetylcholinesterase. The working method employed a recombinant human acetylcholinesterase as the capture antigen, and was developed after investigating different antigens, solid phase materials, coating procedures and blocking techniques. The sensitivity of the method was established at 1.8ng/ml of mouse IgG, (AEl) employing a mouse monoclonal antibody specific for human acetylcholinesterase. The specificity of the method was confirmed by using human antibodies to the Yta Cartwright blood group antigen, to act as a positive human control for the assay. These investigations were important, as there was no data on methods previously described, leaving the findings of previous researchers open to criticism on grounds of sensitivity and specificity. The findings of this research required evidential data to prove the methods analytical parameters and support the results produced by the method, so as not to be open to similar criticisms. The possible aetiological role of anti-acetylcholinesterase antibodies was investigated by developing methods capable of detecting the IgG subclass of anti-acetylcholinesterase antibodies (ELISA). Methods were developed with the potential to demonstrate IgG binding to tissue acetylcholinesterase in-vitro (Immunohistochemistry) and the immunological inhibition of acetylcholinesterase catalytic function, employing monoclonal antibodies. The ELISA method was employed to determine its potential role as a technique to detect and grade anti-Yt antibodies. This study also lead to the development of criteria for the classification of positive, negative and equivocal results. The ELISA method was then used to determine the incidence of anti acetylcholinesterase antibodies in patients with autoimmune disease and all forms of Motor Neuron Disease. Positive findings of previous researchers could have been due to poor assay specificity, and / or detection of allogenic anti-AChE antibodies whose characterisation had occurred since much of the early work. These clinical studies produced data that indicated that anti-acetylcholinesterase antibodies are not prevalent in these disorders, which was not consistent with previous findings.
However, the data generated did reveal that IgG autoantibodies of all subclasses and specific to neuronal acetylcholinesterase were present in the only individual with cerebral palsy, included in the study.
|Date of Award||1999|