Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

Abstract

Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.     

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

Use of 14-3-3 in the diagnosis of Creutzfeldt-Jakob disease

The transmissible spongiform encephalopathies include human diseases such as Creutzfeldt-Jakob disease (CJD) and kuru as well as animal diseases such as scrapie and bovine spongiform encephalopathy (BSE). The emergence of variant CJD, which is causally related to BSE, has generated much interest in the development of rapid and sensitive diagnostic tests for the pre-mortem diagnosis of CJD. In 1986 two proteins were detected in the cerebrospinal fluid (CSF) of patients with sporadic CJD. These proteins were later demonstrated to be members of the 14-3-3 family, and tests for the detection of CSF 14-3-3 were developed. A number of studies have shown that the detection of CSF 14-3-3 is an accurate test for sporadic CJD, although the results with variant CJD are less promising.     

14-3-3 and enolase abundances in the CSF of Prion diseased rats

ABSTRACT

Creutzfeldt-Jakob disease (CJD) is characterized by an extended asymptomatic preclinical phase followed by rapid neurodegeneration. There are no effective treatments. CJD diagnosis is initially suspected based upon the clinical presentation of the disease and the exclusion of other etiologies. Neurologic symptoms are assessed in combination with results from cerebrospinal fluid (CSF) biomarker abundances, electroencephalography (EEG), magnetic resonance imaging (MRI), and in some countries, real-time quaking-induced conversion (RT-QuIC). Inconsistencies in sensitivities and specificities of prion disease biomarker abundance in CSF have been described, which can affect diagnostic certainty, but the utility of biomarkers for prognosis has not been fully explored. The clinical presentation of CJD is variable, and factors such as prion protein polymorphic variants, prion strain, and other genetic or environmental contributions may affect the disease progression, confounding the appearance or abundance of biomarkers in the CSF. These same factors may also affect the appearance or abundance of biomarkers, further confounding diagnosis. In this study, we controlled for many of these variables through the analysis of serial samples of CSF from prion-infected and control rats. Prion disease in laboratory rodents follows a defined disease course as the infection route and time, prion strain, genotype, and environmental conditions are all controlled. We measured the relative abundance of 14-3-3 and neuron-specific enolase (NSE) in CSF during the course of prion infection in rats. Even when disease-related, environmental and genetic variables were controlled, CSF 14-3-3 and NSE abundances were variable. Our study emphasizes the considerable diagnostic and prognostic limitations of these prion biomarkers.     

Altered Prion protein expression pattern in CSF as a biomarker for Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression.     

Analysis of the advantage features of Beijing surveillance network for Creutzfeldt-Jakob disease

ABSTRACT. Since 2006, China has conducted the surveillance program for Creutzfeldt-Jakob disease (CJD) and other subtypes of prion diseases covering 12 provinces. In this study, the characteristics and special role of Beijing CJD surveillance network in the national CJD surveillance system were analyzed. Based on the registered permanent resident places, all reporting suspected CJD cases and diagnosed CJD cases via Beijing CJD surveillance network between 2006 and 2013 were grouped as the cases from Beijing and from outside of Beijing. Both numbers of the suspected and diagnosed CJD cases via Beijing CJD surveillance network constantly increased along with the years, totally 532 reporting cases and 192 diagnosed CJD cases were obtained in the past 8 y. About 75% of suspected and diagnosed CJD cases via Beijing CJD surveillance network came from other provinces, mainly from neighboring provinces. Altogether, 46 different hospitals in the Beijing region have reported suspected CJD cases to the CJD surveillance system during the period from 2006 to 2013. Five hospitals continually reported suspected CJD cases during those 8 y and 5 other hospitals had reported cases except for 1 to 2 y. Additionally, we found that the diagnosed CJD patients from Beijing region had less numbers of hospital transfer and shorter interval from the disease onset to the final diagnosis than those outside of Beijing. It indicates that as the most important component, Beijing CJD surveillance network functions more actively, which supplies the special medical services not only for Beijing residents but also for patients from all of China.     

Analysis of the advantage features of Beijing surveillance network for Creutzfeldt-Jakob disease

ABSTRACT. Since 2006, China has conducted the surveillance program for Creutzfeldt-Jakob disease (CJD) and other subtypes of prion diseases covering 12 provinces. In this study, the characteristics and special role of Beijing CJD surveillance network in the national CJD surveillance system were analyzed. Based on the registered permanent resident places, all reporting suspected CJD cases and diagnosed CJD cases via Beijing CJD surveillance network between 2006 and 2013 were grouped as the cases from Beijing and from outside of Beijing. Both numbers of the suspected and diagnosed CJD cases via Beijing CJD surveillance network constantly increased along with the years, totally 532 reporting cases and 192 diagnosed CJD cases were obtained in the past 8 y. About 75% of suspected and diagnosed CJD cases via Beijing CJD surveillance network came from other provinces, mainly from neighboring provinces. Altogether, 46 different hospitals in the Beijing region have reported suspected CJD cases to the CJD surveillance system during the period from 2006 to 2013. Five hospitals continually reported suspected CJD cases during those 8 y and 5 other hospitals had reported cases except for 1 to 2 y. Additionally, we found that the diagnosed CJD patients from Beijing region had less numbers of hospital transfer and shorter interval from the disease onset to the final diagnosis than those outside of Beijing. It indicates that as the most important component, Beijing CJD surveillance network functions more actively, which supplies the special medical services not only for Beijing residents but also for patients from all of China.     

High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease

Background  

The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.     

Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease

The definite diagnosis of Creutzfeldt-Jakob disease (CJD), the most common form of human prion diseases, relies upon neuropathological data usually obtained at autopsy. In living patients, the diagnosis, based on suggestive clinical features and EEG abnormalities, can be aided by the detection of altered levels of isoforms of the 14-3-3 protein in the cerebrospinal fluid (CSF). However, the validity of this test has been recently challenged and the search for other, more reliable biomarkers for CJD remains highly desirable. The present study describes the identification of a new potential surrogate marker in the CSF of CJD-affected patients. A preliminary study employing surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) technology highlighted a protein at 13.4 kDa in a small group (n = 8) of CJD-affected patients. Further analysis aimed at identifying this protein using cationic exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed it to be cystatin C. Additional immunoblot assays confirmed that the level of cystatin C was significantly increased (p 相似文献   

The epidemiological, clinical, and laboratory features of sporadic Creutzfeldt-Jakob disease patients in China: surveillance data from 2006 to 2010

Background

Creutzfeldt-Jakob disease (CJD) is a rare, rapidly progressive fatal central nervous system disorder, which consists of three main catalogues: sporadic, familial, and iatrogenic CJD.

Methodology/Principal Findings

In China, the surveillance for CJD started in 2006, covering 12 provincial Centers for Disease Control and Prevention (CDCs) and 15 hospitals. From 2006 to 2010, 624 suspected patients were referred to China CJD surveillance. The epidemiological, clinical and laboratory features of sporadic CJD (sCJD) were analysed. Both groups of probable and possible sCJD showed highest incidences in the population of 60 to 69 year-olds. The most common presenting symptoms were progressive dementia and mental-related symptoms (neurological symptoms including sleeping turbulence, depression, anxiety and stress). Among the four main clinical manifestations, myoclonus was more frequently observed in the probable sCJD patients. About 2/3 of probable sCJD cases showed positive 14-3-3 in CSF and/or periodic sharp wave complexes (PSWC) in electroencephalography (EEG). The presence of myoclonus was significantly closely related with the appearance of PSWC in EEG. Polymorphisms of codon 129 in PRNP of the notified cases revealed a highly predominant M129M genotype in Han Chinese. Among 23 genetic human prion diseases, ten were D178N/M129M Fatal familial insomnia (FFI) and five were T188K genetic CJD (gCJD), possibly indicating a special distribution of gCJD-related mutations in Han Chinese.

Conclusion

From the period of 2006 to 2010, 261 patients were diagnosed as sCJD and 23 patients were diagnosed as genetic human prion diseases in China. The epidemiological, clinical and laboratory analysis data were consistent with the characteristics of sporadic CJD, which provide insight into the features of CJD in China.     

Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease

So far only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2-D differential gel-electrophoresis (2-D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood- and brain-derived proteins, and an improved 2-D DIGE protocol was established. Using this method in combination with protein identification by MALDI-TOF-MS, several known surrogate markers of sCJD like 14-3-3 protein, neuron-specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF-optimized 2-D DIGE will find broad application in the search for other brain derived proteins in CSF.     

Ubiquitin as potential cerebrospinal fluid marker of Creutzfeldt–Jakob disease

Until today, a definite diagnosis of Creutzfeldt–Jakob disease (CJD) can only be made neuropathologically. At lifetime the early and differential diagnosis is often a problem. With SELDI we analyzed cerebrospinal fluid (CSF) from 32 CJD patients, 32 patients having other dementive diseases and 31 non‐demented control subjects for diagnosis‐dependent protein pattern differences. In a screening set of patients, peaks that discriminate best between groups were identified. These peaks were subsequently analyzed using an independent validation set of patients. Diagnostic accuracies were compared with established markers like tau protein and 14‐3‐3‐protein. Potential marker proteins were purified and identified by LC‐MS/MS. In the validation set only one peak of 8.6 kDa out of ten in the screening set could be confirmed. This protein was identified to be ubiquitin and increased levels in CSF (but not in serum) of CJD patients were confirmed by Western blot. Ubiquitin allows the correct diagnoses of that CJD cases missed by tau protein or 14‐3‐3‐protein. We conclude that ubiquitin is a promising additional CSF biomarker for diagnosis of CJD, especially in differential diagnostically difficult cases. The selective increase of ubiquitin in CSF of CJD patients might point to an involvement of ubiquitin in pathophysiological process.     

The Prepared Tau Exon-Specific Antibodies Revealed Distinct Profiles of Tau in CSF of the Patients with Creutzfeldt-Jakob Disease

Background

The diagnostic value of CSF tau for Creutzfeldt-Jakob disease (CJD) has been widely evaluated, showing a markedly disease-relative manner. However, the profiles of tau isoforms in CSF of CJD patients remain unknown. Here, we prepared the exon-specific antibodies against the peptides encoded by exon-2, exon-3 and exon-10 of human tau protein and evaluated the reactive profiles of tau in CSF samples from the patients with probable CJD.

Methodology/Principal Findings

Sequences encoding exon-2, exon-3 and exon-10 of human tau protein were cloned into a prokaryotic expression vector pGEX-2T. Using recombinant fusion proteins GST-E2, GST-E3 and GST-E10, three tau exon-specific antibodies were elicited. Reliable specificities of the prepared antibodies were obtained after a serial of purification processes, not only in recognizing the tau peptides encoded by exon-2, -3 and -10, but also in distinguishing six recombinant tau isoforms by Western blot and ELISA. Three predominant tau-specific bands were observed in CSF samples with the exon-specific and the commercial tau antibodies, respectively, showing different reactive profiles between the groups of probable CJD and non-CJD. A 65 KD band was detected only in the CSF samples from probable CJD patients, especially with the antibodies against exon-2 (Anti-tE2) and exon-10 (Anit-tE10). The appearances of 65 KD band in CSF correlated well with positive 14-3-3 in CSF and typical abnormality in EEG. Such band was not observed in the CSF samples of six tested genetic CJD patients.

Conclusions/Significance

Three exon-specific polyclonal antibodies were successfully prepared. Based on these antibodies, different CSF tau profiles in Western blots were observed between the groups of probable CJD and non-CJD. A disease-specific tau band emerged in the CSF samples from probable sporadic CJD, which may supply a new biomarker for screening sporadic CJD.     

Rare V180I mutation in PRNP gene of a Chinese patient with Creutzfeldt-Jakob disease

Here, we reported a Chinese case of Creutzfeldt-Jakob disease (CJD) with a rare mutation in the prion protein gene (PRNP) leading to an exchange of amino acid from valine (V) to isoleucine (I) at codon 180 (V180I). The 72 year-old Chinese women started with gradual memory loss. On admission, she did not present special abnormality during clinical examinations except bradykinesia in her lower extremities. Myoclonic jerks and increased muscle tone were noticed 3 months after the onset. No periodic activity was recorded at electroencephalography (EEG) and 14-3-3 protein was negative in the cerebrospinal fluid (CSF) sample. Brain diffusion weighted images (DWI) demonstrated high signal intensities in bilateral frontal, parietal, temporal and occipital cortices, especially on the left hemisphere, and high signal intensities were also seen in the left caudate nucleus and the putamen. The patient had no family history of similar symptoms. Her general condition was gradually deteriorative, but the patient was still alive when we performed the follow-up 12 months after onset.     

Lack of association between 14-3-3 beta gene (YWHAB) polymorphisms and sporadic Creutzfeldt-Jakob disease (CJD)

14-3-3 proteins are highly abundant in brain tissue. The presence of 14-3-3 at elevated levels in the cerebrospinal fluid has been considered as a biomarker for sporadic Creutzfeldt-Jakob disease (CJD). Recent studies showed that 14-3-3 beta protein interacts with the N-terminal amino acids 1–38 and with the central hydrophobic amino acids 106–126 of prion protein. This interaction may indicate a role of 14-3-3 beta in the biological function of PrP and in the pathogenesis of prion disease. An association between the polymorphisms of 14-3-3 beta gene (YWHAB) and prion disease has not been reported previously. In order to investigate whether YWHAB polymorphisms are associated with sporadic CJD in the Korean population, we compared genotype distribution and allele frequencies of six YWHAB polymorphisms in 244 sporadic CJD patients and 219 healthy Koreans. Of six polymorphisms identified, four single nucleotide polymorphisms (SNPs) were known previously (c.60A>C, c.685-120G>A, c.685-89G>A, 92G>A) and two SNPs were novel (c.185T>A and c.377A>C). Two novel polymorphisms were identified within 3′-untranslated region of exon 6. We could not find significant differences in genotype and allele frequencies of the six YWHAB polymorphisms between the controls and sporadic CJD patients. These results indicate that these six YWHAB polymorphisms are not associated with the genetic susceptibility to sporadic CJD. This is the first genetic association study of YWHAB in sporadic CJD.     

Magnetic resonance spectroscopy and measurement of tau epitopes of autopsy proven sporadic Creutzfeldt-Jakob disease in a patient with non-specific initial EEG, MRI and negative 14-3-3 immunoblot

Limited potential of electroencephalogram (EEG), magnetic resonance images (MRI) and cerebrospinal fluid (CSF) test for 14-3-3 protein in the clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) resulted in developments in diagnostic premortem tehniques. Recent studies provided evidence that magnetic resonance spectroscopy (MRS) and measurement of total-tau (T-tau) and phospho-tau (P-tau) may be useful to identify patients with CJD. We combined detected metabolic changes in the brain by MRS and measured T-tau and tau-pT181 by ELISA, and tau-pT231 by Westernblot in a patient with autopsy proven sCJD. Our results show that in contrast to negative CSF 14-3-3 protein, nonspecific EEG and MRI, MRS revealed metabolic alterations in regions of the brain that has appeared normal on MRI, and tau tests has shown measurable levels of phosphorylated and non-phosphorylated isoforms in CSF. We conclude that rapidly progressive dementia with negative 14-3-3 test and non-specific initial EEG and MRI must still be considered in the differential diagnosis of the sCJD. Combination of serial functional MRI along with MRS study and measurement of tau ratio could improve the early diagnosis of sCJD. The current case is the first attempt to study results of the use of MRS and tau tests in a case of sCJD with diagnostic dilemma.     

Analysis of the compliance and the related influence factors in the follow-up process of surveillance for Creutzfeldt-Jakob disease in China

Creutzfeldt-Jakob disease (CJD) is a kind of rare, rapidly progressive fatal central nervous system disorders. In China, the surveillance for CJD has started since 2006. As one of the major issues in CJD surveillance, the follow-up process via telephone plays important role in CJD diagnosis and surveillance. Although the follow-up process was conducted by the experiential staffs from CJD surveillance center in China CDC, it is frequently encountered that some interviewed family members do not cooperate well during follow-up. To screen the possible factors influence on the compliances of the interviewees during CJD follow-up, 11 independent variables from patient aspect and 4 variables from interviewee aspect were selected and a questionnaire was prepared. Based on 199 suspected sporadic CJD cases reported to CJD surveillance center in 2013, a telephone-inquiring was conducted and the degree of compliances of the interviewees were given as good, fair or poor. After screened with univariate analysis and evaluated ordinal logistic regression analysis, several indictors, such as the patient gender, CJD diagnosis, numbers of clinical symptoms, continual medical treatment after diagnosis, medical treatment mode, as well as the relationship with the patient and CJD knowledge of the interviewees, showed influence on the compliance in CJD follow-up process significantly. The data here provide for the first time the factors related with the compliances of the interviewed family members of the suspected CJD patients during follow-up process, which supplies useful clue for us to improve CJD follow-up process and increase the capacity of CJD surveillance.     

A novel binding assay for detection of 14-3-3 protein in cerebrospinal fluid from Creutzfeldt-Jakob patients.

Here we describe a simple and fast microplate assay for 14-3-3 protein, which is based on the binding of 14-3-3 protein to a chemically synthesized peptide containing the phosphorylated recognition sequence of this protein. The peptide is covalently bound to maleimide-activated microplates through reaction of the sulfhydryl group of a N-terminal cysteine residue linked to the peptide. The peptide- 14-3-.3 complexes formed are detected using--as a second recognition principle--an anti-14-3-3 protein antibody. This assay was used for measurement of 14-3-3 protein in brain (bovine) and in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease (CJD) and control cases with diseases other than CJD. This novel assay allowed also the detection of low 14-3-3 protein levels present in cerebrospinal fluid, without further concentration, in contrast to conventional immunoblotting or immunoassay procedures.     

Human 14-3-3 Protein: Radioimmunoassay, Tissue Distribution, and Cerebrospinal Fluid Levels in Patients with Neurological Disorders

Abstract: An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, ¹²⁵I- 14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.m1⁻¹. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3-3 protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 μg. mg⁻¹ soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 μg. mg⁻¹ soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics as authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.m1⁻¹) and in CSF (5-130 ng.ml⁻¹). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be o f diagnostic value.