Establishment of a standard 14-3-3 protein assay of cerebrospinal fluid as a diagnostic tool for Creutzfeldt-Jakob disease.
Periodic sharp wave complexes observed on an electroencephalographic recording and the presence of a 14-3-3 protein in the cerebrospinal fluid (CSF) are both included in the diagnostic criteria for the Creutzfeldt-Jakob disease (CJD) supplied by the World Health Organization; however, the presence or absence of the 14-3-3 protein in the CSF is sometimes difficult to discern on a western blot because of equivocal bands. The goal of this study was to establish a standard 14-3-3 protein assay and to determine the threshold level of a 14-3-3 protein that can be assayed by western blot. We searched for the most suitable isoform of the 14-3-3 protein to test for in protein assays, and the most sensitive antibody among four antibodies with an affinity for 14-3-3. We measured the levels of all 14-3-3 isoforms in 112 patients with CJD and in 100 patients with other diseases. We compared the performances of four different antibodies. We carried out a semi-quantitative analysis of γ-isoform levels using the LAS 3000 system, which was capable of producing a digital image from the luminescence on a western blot. We determined that the most suitable isoform of the 14-3-3 protein for conducting a standardized assay was the γ-isoform. Among the four commercially available antibodies for this protein, the most sensitive and specific was 18647 (IBL, Japan). We report the high repeatability of the detection of the 14-3-3 protein by this antibody to the γ-isoform, showing that western blot can be used for semi-quantitative analysis.
Satoh
Katsuya
K
Department of Internal Medicine of Nagasaki University School of Medicine, Nagasaki, Japan. f1537@cc.nagasaki-u.ac.jp
Tobiume
Minoru
M
Matsui
Yuki
Y
Mutsukura
Kazuo
K
Nishida
Noriyuki
N
Shiga
Yusei
Y
Eguhchi
Katsumi
K
Shirabe
Susumu
S
Sata
Testutaro
T
D048948:14-3-3 Proteins / Q000134:cerebrospinal fluid*; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D007562:Creutzfeldt-Jakob Syndrome / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results
D048948:14-3-3 Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins