Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies.
We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.
Jenkins
E C
EC
New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314-6399, USA. ECJenkins@erols.com
Wen
G Y
GY
Kim
K S
KS
Zhong
N
N
Sapienza
V J
VJ
Hong
H
H
Chen
J
J
Li
S Y
SY
Houck
G E
GE
Ding
X
X
Nolin
S L
SL
Dobkin
C S
CS
Brown
W T
WT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002467:Cell Nucleus; D003593:Cytoplasm; D005260:Female; D005315:Fetal Diseases / Q000235:genetics* / Q000276:immunology; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D016601:RNA-Binding Proteins
D000911:Antibodies, Monoclonal; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human