in the lips assay , ia-2 ( amino acids 601979 ) was cloned into the pren2 mammalian expression vector downstream of the renilla luciferase ( ruc ) reporter ( 35 ) . monkey kidney cells ( cos1 ) in a 100 mm dish were transfected , and 48 h later , the cells were sonicated , clarified by centrifugation , and the extract ( enough for 1,500 assays in duplicate ) used without further purification . total luciferase activity in the extract was measured in a luminometer and adjusted so that each reaction contained 1.0  10 light - forming units per 0.1 l of extract . the extract was then incubated with 1.0 l of sera for 1 h at room temperature in a total volume of 100 l , transferred to 96-well filtration plates containing 7 l of 30% suspension of protein a / g beads ( pierce biochem ) , and incubated for an additional hour . the a / g beads with the captured antibody  luciferase labeled ia-2 complexes were washed using a biomek - fx workstation , renilla luciferase substrate ( promega , madison , wi ) was added , and the light - forming units were determined in a microplate luminometer ( berthold technologies , bad wildbad , germany ) . in the rip assay , performed as previously described ( 6 ) , the same intracellular portion of ia-2 used in the lips assay was cloned into a pgbkt7 vector ( clontech , mountain view , ca ) , and the ia-2 protein was produced by in vitro transcription / translation in the presence of s - methionine . ia-2 autoantibodies were detected by liquid - phase radioimmunoprecipitation using 20,000 cpm of radiolabeled ia-2 . a total of 100 sera from patients with newly diagnosed type 1 diabetes who had been previously screened for ia-2 autoantibodies by rip were selected on the basis of the counts precipitated , from very high to moderate and low to negative . a total of 100 sera from age - matched nondiabetic individuals served as the control ( 6 ) . a serum was scored as positive if the precipitated cpm exceeded the mean + 3 sd of the control subjects . sera from diabetic ( n = 100 ) and control subjects ( n = 100 ) were screened for ia-2 autoantibodies by rip assay . only one of the control subject sera fell outside the 3 sd range , whereas 90% of the diabetic subject sera exceeded the 3 sd range of the control subject sera ( fig . the coefficient of variation ( cv ) for duplicate samples of the diabetic subject sera was 5.0% . none of the control subject sera fell outside the 3 sd range , whereas 85% of the diabetic subject sera exceeded the 3 sd range of the control subject sera ( fig . comparison of the rip and lips assays revealed a high correlation , with a 0.805 coefficient of determination ( r ) ( fig . evaluation of the 5 diabetic subject sera that were negative by the lips assay but positive by the rip assay showed that 3 of the 5 negative sera were at the very borderline of positivity ( fig . receiver operating characteristic analysis showed that the areas under the curves for ia-2 by rip and lips were not statistically different ( p = 0.120 ) ( fig . from these experiments , we concluded that there was no significant difference in sensitivity or specificity between the lips and rip assays . first , because it uses luminescence rather than radioactivity , it lends itself better to most clinical laboratories and can be easily automated . second , because the preparation of the protein does not require in vitro transcription / translation , the protein can be prepared at a fraction of the cost of the rip assay . third , because the recombinant protein is fused with a luminescence marker , it does not need to be externally labeled or laboriously separated from the other proteins in the mammalian cell lysate , thereby eliminating the purification steps required for both bacteria - prepared proteins ( 7 ) used in solid - phase enzyme - linked immunosorbent assays . fourth , because the proteins are made in mammalian cells , they may undergo a variety of processing and posttranslational modifications that do not occur when they are prepared via in vitro transcription / translation and , therefore , may more truly reflect the natural state of the autoantigen . it will be of interest to see if this increases detection and/or sensitivity when other autoantigens , prepared in mammalian cells , are screened for autoantibodies . our findings suggest that the lips assay may be of practical value at the clinical level for the detection of autoantibodies not only for diabetes , but also for other autoimmune diseases . however , as with all new assays , lips requires validation with a large number of sera and comparison with rip in a diabetes autoantibody standardization program workshop .