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 .
 objective  insulinoma - associated protein 2 ( ia-2 ) is a major autoantigen in type 1 diabetes , and ia-2 autoantibodies are routinely detected by a liquid - phase radioimmunoprecipitation assay . 
 the present experiments were initiated to develop a new assay that does not require the use of radioisotopes or autoantigens prepared in bacteria or by in vitro transcription / translation.research design and methods  
 ia-2 luciferase fusion protein was expressed in mammalian cells and assayed for autoantibodies by liquid - phase luciferase immunoprecipitation.resultsour study showed that there was no significant difference between the luciferase immunoprecipitation and the radioimmunoprecipitation assays in sensitivity and specificity , and comparison of the two assays revealed a high correlation coefficient ( r2 = 0.805).conclusions  the luciferase system offers a robust , inexpensive , nonradioactive method for the detection of autoantibodies to mammalian cell  prepared ia-2 and could be of practical value at the clinical level . 
RESEARCH DESIGN AND METHODS
RESULTS
CONCLUSIONS