P2‐039: Novel tau fragments are present in human CSF
Jere E. MeredithRandall SlemmonValerie GussAnthony J. LanzettiSethu SankaranarayananHolly SoaresKaj BlennowCharlie AlbrightMichael K. Ahlijanian
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Abstract:
CSF Aβ42, Tau and p181Tau are widely accepted diagnostic biomarkers of Alzheimer's disease (AD). Results from numerous studies demonstrate that CSF Tau levels are ∼2X higher in mild-to-moderate AD compared to age-matched controls. In addition, this increase in CSF Tau is detected prior to the onset of clinical symptoms, and thus is predictive of the conversion from predementia (MCI) to clinical disease. Despite the importance as a diagnostic biomarker, the molecular nature of Tau present in CSF is not known. We have employed CSF denaturation coupled with reverse-phase HPLC in order to enrich and concentrate Tau and to further characterize the CSF Tau profile. Individual fractions from pooled AD and control CSF samples were analyzed by SDS-PAGE/Western blot (WB) using various anti-Tau antibodies or by using in-house, research-grade Tau ELISAs. Multiple fragments of Tau, with apparent MW sizes ranging from ∼15 kD to 40 kD, were identified by WB using an antibody that recognizes the mid-domain of Tau, but not an isotype control IgG. A subset of these bands was recognized using an antibody specific for the N-terminal of Tau. In contrast, relatively low levels of C-terminal-containing fragments were detected as demonstrated using a C-terminal specific antibody. Tau ELISA results confirmed the WB data and demonstrated that differences in Tau levels between AD and control CSF are more prominent in specific fractions, corresponding to certain apparent molecular weight Tau fragments. Our results demonstrate the existence of multiple Tau fragments in CSF and suggest that a subset of these fragments are increased in AD CSF. These particular Tau fragments may have utility as disease and/or pharmacodynamic biomarkers.Keywords:
Isotype
Tau protein
Tau immunotherapy has emerged as a promising approach to clear tau aggregates from the brain. Our previous findings suggest that tau antibodies may act outside and within neurons to promote such clearance.We have developed an approach using flow cytometry, a human neuroblastoma cell model overexpressing tau with the P301L mutation, and paired helical filament (PHF)-enriched pathologic tau to effectively screen uptake and retention of tau antibodies in conjunction with PHF.The flow cytometry approach correlates well with Western blot analysis to detect internalized antibodies in naïve and transfected SH-SY5Y cells (r2 = 0.958, and r2 = 0.968, P = .021 and P = .016, respectively). In transfected cells, more antibodies are taken up/retained as pathologic tau load increases, both under co-treated conditions and when the cells are pretreated with PHF before antibody administration (r2 = 0.999 and r2 = 0.999, P = .013 and P = .011, respectively).This approach allows rapid in vitro screening of antibody uptake and retention in conjunction with pathologic tau protein before more detailed studies in animals or other more complex model systems.
Internalization
Tau protein
Cytometry
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Objective To study the preparation and characterization of monoclonal antibody against human.Methods BALB/c mice were immunized with prokaryotically expressed human EGFRv Ⅲ ex protein.The mAb positive hybridoma cells were screened by indirect ELISA.Specificity of mAb was characterized by Western blot,immunofluorescent staining and immunohischemical staining.Results One hybridoma cells secreting mAb was established.The isotype of mAb was IgM.The titer was 10-6.The mAb recognized human EGFRvⅢex protein on EGFRvⅢex-transfected U87 cells and EGFR protein on SPC-A1 cells.Conclusion One anti-EGFRvⅢex mAb has obtained.The characterization of the mAb indicated the fine specificity of mAb by Western blot,immunofluorescent staining and immunohischemical staining,which can provided a powerful method for the tumor therapy.
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To express the human recombinant PCGF1 protein and prepare monoclonal antibody (mAb) against it.The recombinant expression plasmid pET32a-His-PCGF1-128/189 was made and transformed into E.coli (BL21), and then the recombinant fusion protein His-PCGF1-128/189 was expressed and purified. The BALB/c mice were immuned with purified protein His-PCGF1-128/189 as antigen. The mAbs against PCGF1 were prepared by using standard hybridoma technique. The hybridoma cell lines were obtained by ELISA and Western blot screening procedure, the isotype of the mAbs were further identified by immune-double diffusion. Ascites were prepared from one propagated hybridoma cell line and mAbs were purified by using the Kit from Millipore. The valence of mAb was detected by Western blot.The recombinant protein His-PCGF1-128/189 was expressed and purified. Two hybfidmas producing antibodies against PCGF1 were obtained, the isotypes of two mAbs were IgG1, Western blot showed that the antibodies were high sensitive(1:6 000) and high specific for PCGF1.The anti-PCGF1 mAb prepared by using recombinant His-PCGF1-128/189 protein as antigen can be used for detecting PCGF1 proteins which are either endogenous or exogenous.
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Objective:Preparation and characterization of monoclonal antibodies against NSE protein,and establishment of a double-antibody sandwich ELISA assay.Methods:BALB/c mice were immunized by using purified recombinant NSE,and monoclonal antibodies were generated by hydridoma technique.These antibodies were characterized with ELISA,Western blot,Immunofluorescent and Immunohistochemical staining.The isotypes of these antibodies were determined with an antibody isotyping kit.With Horseradish Peroxidase labelled NSE monoclonal antibody,we were able to establish a double-antibody sandwich ELISA to detect NSE protein.Results:Two positive hybridoma cell lines were selected for test,the titers of these two monoclonal antibodies could reach 4.2×107-6.5×107,and their isotypes were IgG2b.Our NSE antibodies could detect not only endogernous NSE protein from cells,but also secreted NSE protein from cells in culture medium by Western blot,in addition,they could be used for immunofluorescent and immunohistochemical staining.The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8.85 ng/ml.Conclusion:Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers,and we established a double-antibody sandwich ELISA assay which could be used for clinical test in future.
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Abstract: The microtubule‐associated protein τ which stimulates the assembly of α‐β tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of τ and abnormally phosphorylated τ were determined in brain homogenates of AD and age‐matched control cases. A radioimmuno‐slot‐blot assay was developed, using a primary monoclonal antibody, Tau‐1, and a secondary antibody, antimouse 125 I‐immunoglobulin G. To assay the abnormally phosphorylated τ, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total τ were about eightfold higher in AD (7.3 ± 2.7 ng/μg of protein) than in control cases (0.9 ± 0.2 ng/μg), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation—not a decrease in the level of τ—is a likely cause of neurofibrillary degeneration in AD.
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Blood-based biomarkers for amyloid beta and phosphorylated tau show good diagnostic accuracies and agreements with their corresponding CSF and neuroimaging biomarkers in the amyloid/tau/neurodegeneration [A/T/(N)] framework for Alzheimer's disease. However, the blood-based neurodegeneration marker neurofilament light is not specific to Alzheimer's disease while total-tau shows lack of correlation with CSF total-tau. Recent studies suggest that blood total-tau originates principally from peripheral, non-brain sources. We sought to address this challenge by generating an anti-tau antibody that selectively binds brain-derived tau and avoids the peripherally expressed 'big tau' isoform. We applied this antibody to develop an ultrasensitive blood-based assay for brain-derived tau, and validated it in five independent cohorts (n = 609) including a blood-to-autopsy cohort, CSF biomarker-classified cohorts and memory clinic cohorts. In paired samples, serum and CSF brain-derived tau were significantly correlated (rho = 0.85, P < 0.0001), while serum and CSF total-tau were not (rho = 0.23, P = 0.3364). Blood-based brain-derived tau showed equivalent diagnostic performance as CSF total-tau and CSF brain-derived tau to separate biomarker-positive Alzheimer's disease participants from biomarker-negative controls. Furthermore, plasma brain-derived tau accurately distinguished autopsy-confirmed Alzheimer's disease from other neurodegenerative diseases (area under the curve = 86.4%) while neurofilament light did not (area under the curve = 54.3%). These performances were independent of the presence of concomitant pathologies. Plasma brain-derived tau (rho = 0.52-0.67, P = 0.003), but not neurofilament light (rho = -0.14-0.17, P = 0.501), was associated with global and regional amyloid plaque and neurofibrillary tangle counts. These results were further verified in two memory clinic cohorts where serum brain-derived tau differentiated Alzheimer's disease from a range of other neurodegenerative disorders, including frontotemporal lobar degeneration and atypical parkinsonian disorders (area under the curve up to 99.6%). Notably, plasma/serum brain-derived tau correlated with neurofilament light only in Alzheimer's disease but not in the other neurodegenerative diseases. Across cohorts, plasma/serum brain-derived tau was associated with CSF and plasma AT(N) biomarkers and cognitive function. Brain-derived tau is a new blood-based biomarker that outperforms plasma total-tau and, unlike neurofilament light, shows specificity to Alzheimer's disease-type neurodegeneration. Thus, brain-derived tau demonstrates potential to complete the AT(N) scheme in blood, and will be useful to evaluate Alzheimer's disease-dependent neurodegenerative processes for clinical and research purposes.
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To express capsid tail protein P11 of T7 bacteriophage and produce mouse monoclonal antibody(mAb) against the protein.P11 protein was cloned and recombinant P11 protein was expressed as a fusion protein with an N-terminal 6-His tag. The purified protein was used to immunize BALB/c mouse. The specificity of mAb was analyzed by ELISA and Western blot.P11 protein was successfully expressed and purified. SDS-PAGE analysis showed that the molecular weight of the expressed protein was approximately 27 kd. One hybridoma cell (2G11) secreting mAb against P11 was developed. The isotype of the mAb was IgG(2b). ELISA detection showed that titers of mAb was 1:8. 1 x 10(5) in ascites.Western blot analysis proved mAb obtained could react specifically to the recombinant p11 protein.Recombinant P11 protein and mAbs were successfully prepared.
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Cerebrospinal fluid tau fragment correlates with tau PET: a candidate biomarker for tangle pathology
Abstract To date, there is no validated fluid biomarker for tau pathology in Alzheimer’s disease, with contradictory results from studies evaluating the correlation between phosphorylated tau in CSF with tau PET imaging. Tau protein is subjected to proteolytic processing into fragments before being secreted to the CSF. A recent study suggested that tau cleavage after amino acid 368 by asparagine endopeptidase (AEP) is upregulated in Alzheimer’s disease. We used immunoprecipitation followed by mass spectrometric analyses to evaluate the presence of tau368 species in CSF. A novel Simoa® assay for quantification of tau368 in CSF was developed, while total tau (t-tau) was measured by ELISA and the presence of tau368 in tangles was evaluated using immunohistochemistry. The diagnostic utility of tau368 was first evaluated in a pilot study (Alzheimer’s disease = 20, control = 20), then in a second cohort where the IWG-2 biomarker criteria were applied (Alzheimer’s disease = 37, control = 45), and finally in a third cohort where the correlation with 18F-GTP1 tau PET was evaluated (Alzheimer’s disease = 38, control = 11). The tau368/t-tau ratio was significantly decreased in Alzheimer’s disease (P < 0.001) in all cohorts. Immunohistochemical staining demonstrated that tau fragments ending at 368 are present in tangles. There was a strong negative correlation between the CSF tau368/t-tau ratio and 18F-GTP1 retention. Our data suggest that tau368 is a tangle-enriched fragment and that the CSF ratio tau368/t-tau reflects tangle pathology. This novel tau biomarker could be used to improve diagnosis of Alzheimer’s disease and to facilitate the development of drug candidates targeting tau pathology. Furthermore, future longitudinal studies will increase our understanding of tau pathophysiology in Alzheimer’s disease and other tauopathies.
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Tau protein
Tau Pathology
Neurofibrillary tangle
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Our laboratory has pioneered targeting pathological tau proteins for clearance with immunotherapies. We have shown that this approach leads to antibody uptake into neurons, clears tau aggregates from the brain and prevents functional impairments, such as cognitive decline, in mouse tauopathy models. To clarify the mechanism of action of tau antibodies, we are using naïve and transfected (0N4R P301L tau) SHSY5Y human neuroblastoma cells. To mimic a pathological state, we have incubated these cells with paired helical filaments (PHF) enriched from Alzheimer's disease (AD) brain for 3 days, and subsequently incubated with tau antibody (Ser396/404 epitope) for 1, 4, or 6 days. Incubation was performed at 10% (normal) and 1% (compromising) FBS media conditions. In parallel, these cells were analyzed using our flow cytometry protocol and western blot analysis for antibody and PHF uptake as well as tau protein profile, respectively. Transfected cells with 1% FBS had more PHF uptake than all other groups at 1, 4, and 6 days (p<0.0001, p=0.0005, and p=0.0003, respectively, n=3). In both controls and sequentially (PHF and tau antibody) treated samples we observed a gradual increase in tau antibody uptake from 1 to 4 days in both cell lines and additional increase from 4 to 6 days in our transfected line. Only at 6 days of tau antibody incubation and in 1% FBS the transfected cells had significantly greater uptake of tau antibody compared to all other groups (26-39%, p= 0.0002, n=3) and a decrease in total tau and phospho Ser199 tau (64%, p=0.0013 and 49%, p=0.0925, respectively, n=3) levels compared to its PHF incubated control. The compromised transfected cells at 1% FBS and 6 days of antibody incubation show the most promising results as antibody uptake increases and tau levels decrease. Also, PHF seems to have limited effect on antibody uptake, but provides a condition for tau degradation. This neuroblastoma flow cytometry protocol may give us a new high throughput method to better understand the underlying cellular mechanisms and further development of this immunotherapy approach to treat AD and related tauopathies.
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Tauopathy
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There is an urgent need to develop biomarkers for the diagnosis and monitoring of disease progression in Alzheimer's disease (AD) and neurodegenerative tauopathies. Tau levels in cerebrospinal fluid (CSF) are being used as a complementary biomarker and the quantification of both total tau and p-tau 181 have been validated by frequent testing of human CSF samples. Recently, multiple laboratories have established that levels of soluble tau oligomers-mainly in the form of dimers/trimers-are elevated in brain tissues from AD compared to age-matched controls. Here we present our findings from multiple independent experiments that validate tau oligomer levels in human CSF as a reliable biomarker for AD. We used novel anti-tau oligomer specific antibodies for biochemical analyses and quantification of tau oligomers in human CSF samples and sera from tau transgenic animals. We performed direct Elisa, Western blot and sandwich Elisa on these biological fluids. Using direct Elisa we found elevated levels of tau oligomers in AD samples compared to samples from prodromal AD and age-matched controls. Western blot analysis confirmed the oligomeric form of tau in CSF. Finally, we performed double-blind analysis of tau oligomers in 171 CSF samples by direct ELISA. The results indicate that the ratio of total tau/tau oligomers is a reliable biomarker and can distinguish between AD, MCI-AD, MCI, and controls. Moreover, we demonstrate that tau oligomer levels in the serum increase shortly after the intravenous administration of an anti-tau oligomer specific antibody to transgenic animals expressing human tau. Our results validate tau oligomers as a reliable biomarker for AD and tauopathies. These assays are suitable for analysis of human CSF samples and may be critical for early diagnosis and the evaluation of AD clinical trials. Moreover, standardization of tau oligomer measurements may be crucial for the guidance and facilitation of drug development.
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Tau Pathology
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