Determination of islet cell antibodies using an ELISA system with a preparation of rat insulinoma (RIN A2) cells

An enzyme-linked immunosorbent assay (ELISA) was established for
the detection of islet cell antibodies in human sera. The antigen
was prepared from rat insulinoma (RIN A2) cells. Cells were
dissociated in lysis buffer and the lysate was centrifuged at
100,000 x g. The supernatant was used to coat microtiter ELISA
plates (10 micrograms protein/ml in PBS pH 7.2). Non-specific
binding sites on the plates were blocked with 2% PBS-BSA. Human
test sera were preabsorbed on separate plates using 2% PBS-BSA and
incubated on precoated plates at an optimal dilution of 1/10 in 60
mM PBS for 60 min at 37 degrees C. Phosphatase-labeled anti-human
IgG serum and phosphatase substrate were applied and the reaction
was stopped by adding 3 M NaOH. Out of 90 sera from type I diabetic
patients, 47 (52.2%) reacted in the new ELISA whereas none of 15
type II diabetics, 50 sera containing non-islet specific antibodies
or 100 normal controls were positive. In the same group of
patients, ICA were positive in 63.3%. When both, the ELISA and
conventional ICA testing were applied, the number of positives was
increased to 83%. The ICA-ELISA with the above described antigen
preparation provides a well standardized and reproducible test
method which is highly specific for type I diabetes. It may
therefore be useful for large screening procedures.