ICA512 autoantibody radioassay.
As part of a general program of screening islet expression libraries we have identified a clone from a lambda gt11 human islet expression library that reacts with human diabetic sera and, upon sequencing, was determined to be the neuroendocrine islet autoantigen ICA512 (islet cell antigen 512). In the current communication, we describe the development of a radioassay for autoantibodies to ICA512 (ICA512AA) using in vitro transcribed and translated protein for production of labeled antigen. Our initial results indicate that this radioassay is significantly more sensitive than the enzyme-linked immunosorbant assay, which uses a COOH-terminal fragment of ICA512. The ICA512AA radioassay uses a 96-well format with membrane separation of antibody bound from free antigen and should be readily adaptable to automated large-scale screening. Only 7 microliters of serum is required for triplicate determinations. In order to determine the specificity and sensitivity of this assay and estimate its positive predictive value, we have studied 42 new-onset diabetic patients, 33 first-degree relatives of diabetic patients followed to diabetes, 694 islet cell antibody-negative (ICA-) relatives, and 205 normal control subjects. Thirty-eight percent of new-onset patients and 48% of relatives followed to diabetes express autoantibodies to ICA512 exceeding the 99th percentile of the normal control subjects. In contrast, only 1.4% of ICA- first-degree relatives were positive for ICA512 autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Gianani
R
R
Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver 80262, USA.
Rabin
D U
DU
Verge
C F
CF
Yu
L
L
Babu
S R
SR
Pietropaolo
M
M
Eisenbarth
G S
GS
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005190:Family; D005500:Follow-Up Studies; D005968:Glutamate Decarboxylase / Q000276:immunology; D006801:Humans; D007328:Insulin / Q000276:immunology; D007515:Islets of Langerhans / Q000276:immunology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000276:immunology*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011236:Prediabetic State / Q000097:blood / Q000235:genetics / Q000276:immunology*; D011237:Predictive Value of Tests; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000276:immunology*; D011817:Rabbits; D011865:Radioisotope Dilution Technique; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012016:Reference Values; D012156:Reticulocytes / Q000378:metabolism; D012680:Sensitivity and Specificity; D013462:Sulfur Radioisotopes; D013997:Time Factors
D001323:Autoantibodies; D001324:Autoantigens; D017931:DNA Primers; C096322:ICA512 autoantibody; D007328:Insulin; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C024211:islet cell antibody; D008715:Methionine; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase