Immunoassay to measure ataxia-telangiectasia mutated protein in cellular lysates.
BACKGROUND
Ataxia-telangiectasia (A-T) is a neurologic disorder caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. A clinical diagnosis of A-T is confirmed by radiosensitivity testing and immunoblotting for ATM protein. Because both of these tests have long turnaround times (> or =3 months), we developed a rapid immunoassay to measure ATM protein and determined its sensitivity and specificity for diagnosing A-T.


METHODS
Recombinant ATM protein was used for standardization. Lysates of lymphoblastoid cell lines (LCLs) and peripheral blood mononuclear cells (PBMCs) from A-T patients, controls, and A-T heterozygotes were tested for ATM protein by immunoassay.


RESULTS
Between-run imprecision (CV) was < or =13%. Nuclear lysates from control LCLs and PBMCs had ATM protein concentrations of 49-610 microg/L and 48-943 microg/L, respectively. ATM protein was not detectable in LCL nuclear lysates from 18 of 21 A-T patients. The three remaining A-T patients had trace amounts of ATM protein, which was confirmed on immuoblots. ATM protein was also detectable in whole-cell lysates from 4 x 10(6) cells at concentrations of 64-463 microg/L and 42-444 microg/L for control LCLs and PBMCs, respectively. A-T heterozygotes had ATM protein concentrations of 52-98 microg/L. ATM protein was stable in PBMCs stored for 1 month at -70 degrees C, but rapidly decreased after 1 day in unprocessed blood.


CONCLUSIONS
This ATM protein immunoassay can be used to confirm a diagnosis of A-T in 2 days on small numbers of PBMCs and can potentially identify A-T carriers and individuals at increased risk for cancer.
Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA. abutch@mednet.ucla.edu
Anthony W
AW
Butch
Helen H
HH
Chun
Shareef A
SA
Nahas
Richard A
RA
Gatti
D001260:Ataxia Telangiectasia / Q000097:blood / Q000175:diagnosis*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000097:blood* / Q000235:genetics; D011994:Recombinant Proteins / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins; D014616:Vaccinia virus / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases