Myasthenia Gravis (MG), is an autoimmune disease, characterized by impairment inneurotransmission at the neuromuscular junction level. The diagnosis of MG is based onclinical symptomatology, assisted by pharmacological, electrophysiological and imagingtests and is usually confirmed by serological detection of autoantibodies to components ofthe neuromuscular junction. The available diagnostic tests are usually able to detect thepresence of antibodies in 85-90% of myasthenic patients with generalized MG and 50% ofpatients with ocular MG. This gap in the diagnosis of MG creates a major problem in mostcases during diagnosis because a negative result (which is the most common result inordinary laboratory diagnosis of MG) leaves an ambiguity regarding the existence ofMG. Even a very low titer of specific antibodies, would be sufficient to explain the presenceof myasthenia, since there has been no significant correlation between the titer of antibodiesand the severity of disease among patients. Therefore, there is increasing need for thedevelopment of highly sensitive diagnostic methods for the unambiguous confirmation of thedisease. The most sensitive diagnostic test so far, is the radiological immunoprecipitationassay (Radioimmunoprecipitation, RIPA) and the most important limiting factor fordetecting low antibody titer, is the inability to use large volumes of serum (maximum is ~ 5-20 ml), which would require the subsequent use of high-volume of anti-serum, which willresult in excessively large immuno-sediments, which in turn are associated withunacceptable levels of nonspecific background values.With the development of two-step RIPA assay in this study we have largelyovercome these problems. This test is based on the immobilization of target antigen to aninsoluble substrate, followed by incubation with large volume (eg 0,1-1ml) of the test serumand release of bound antibodies with small volume of low pH solution, which are thendetected and titrated with simple RIPA using 125I-labeled antigens. We used the principle ofenrichment of serum to a large extent in antigen-specific antibodies by affinity purificationwith no strict but simple and easy way, before the final identification and titration of specificantibodies with standard immunoassays. We attempted the development of this approach inorder to detect antibodies against AChR and MuSK. Numerous efforts have been made to implement this method in order to detect AChR autoantibodies, but although there wassubstantial progress, additional improvements are required for its application in routinediagnosis. With this approach we achieved significant improvement for detection of anti-MuSK autoantibodies. This systematic approach was proved to be at least 20-50 times moresensitive than classical RIPA. All sera which were previously classified as positive ornegative with standard RIPA were also positive and negative respectively, as expected. Inaddition some of the sera which were previously classified as ambiguous were clearlypositive and some others who were classified as negative but were slightly higher than zero,but not statistically significant so as to assess them as higher than the background, werepositive, while others still remain negative.This newly developed test was originally more laborious than the classical RIPA, butwith the development of significant improvements, the process has been simplified andallows the simultaneous testing of large volumes (0,1-1 ml) of multiple serum samples andits use has been made possible in everyday diagnosis. This improved method is currentlyapplied to detect anti-MuSK antibodies and is designed to detect very small amounts ofautoantibodies in the serum of myasthenic patients who are classified as seronegative orambiguous.