Beta Amyloid Elisa: Serum Amyloid a Elisa: Amyloid Elisa: Research Papers

Beta Amyloid Elisa: Serum Amyloid a Elisa: Amyloid Elisa: Research Papers
Title:
ELISA analysis of [beta]-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways.

Authors:
Steinhilb, Michelle L 1; Turner, R. Scott 2; Gaut, James R 3

Abstract:
Limiting beta amyloid (A[beta]) production could become an important therapeutic target in Alzheimer's disease (AD). A[beta] is derived by the sequential cleavage of amyloid precursor protein (APP) by [beta]- and [gamma]-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates A[beta]40 and A[beta]42 production. A[beta] production and, therefore, [beta]-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of [beta]-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the [beta]-cleaved product, APPsw[beta]. Using this ELISA, we provide new information regarding the relative amount of [beta]-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the [beta]-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no [beta]-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPsw[beta] and A[beta] secretion, suggesting that protein interactions with this region normally impede [beta]-secretase cleavage. These results suggest that APPsw is cleaved by [beta]-secretase late in the secretory pathway.

Title:
ELISA method for measurement of amyloid-beta levels.

Authors:
Schmidt SD, Nixon RA, Mathews PM.

Abstract:
The neuritic plaque in the brain of Alzheimer's disease (AD) patients consists of an amyloid composed primarily of Abeta, an approx 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Abeta plays a key role in the pathogenesis of the disease, and potential treatments that target Abeta production and/or Abeta accumulation in the brain as beta-amyloid are being aggressively pursued. Methods to quantitate the Abeta peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Abeta in the brains of AD patients and beta-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Abeta peptide. Here we describe methods for the recovery of both soluble and deposited Abeta from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.

Title:
Determination of amyloid beta protein precursors harboring active form of proteinase inhibitor domains in cerebrospinal fluid of Alzheimer's disease patients by trypsin-antibody sandwich ELISA.

Authors: Kitaguchi N, Tokushima Y, Oishi K, Takahashi Y, Shiojiri S, Nakamura S, Tanaka S, Kodaira R, Ito H.

Abstract:
beta-Amyloid protein precursors (APP) having proteinase inhibitor domains (APPI) were quantified by a new sandwich enzyme linked immunosorbent assay for detection of active (free) form of proteinase inhibitors by using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microtiterplate. The concentration of APPs having APPI in cerebrospinal fluid of Alzheimer's disease patients was found, by this method, to be significantly elevated compared with those of multi-infarct dementia.




Title:
Development of a specific ELISA for the quantitative study of amino-terminally truncated beta-amyloid peptides

Authors:
Mohammed El Mouedden, Marc Vandermeeren, Theo Meert and Marc MerckenCorresponding Author Contact Information,

Abstract:
Clinical studies for disease modifying drugs in Alzheimer's disease are in real need for a sensitive biochemical diagnostic and therapeutic marker. Encouraging results have been obtained by measuring levels of pathology related proteins such as amyloid beta (Aβ) peptides and tau proteins in cerebrospinal fluid (CSF) and plasma of patients. We and other research groups have shown that truncated Aβ11–40 and Aβ11–42 is also a potential marker and that it is produced and deposited in the brains of patients and transgenic mouse models. Because of a lack of quantitative methods for specific measurement of the truncated Aβ it has not been possible to evaluate the true value of this potential biomarker. To overcome these limitations we developed a novel monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to measure Aβ11–40 and Aβ11–42 levels in biological materials. For this purpose monoclonal antibodies were produced that react specifically with amyloid precursor protein (APP) products generated by cleavage at position 11 of the Aβ sequence. The assay has the sensitivity, selectivity and dynamic range to allow specific, direct quantitation of truncated Aβ11 peptides in cell culture medium, cerebrospinal fluid and brain tissue extracts.


Title:
Sensitive ELISA detection of amyloid-[beta] protofibrils in biological samples.

Authors:
Englund, Hillevi; Sehlin, Dag ; Johansson, Ann-Sofi ; Nilsson, Lars N. G. ; Gellerfors, Par ; Paulie, Staffan ; Lannfelt, Lars ; Pettersson, Frida Ekholm

Abstract:
Amyloid-[beta] (A[beta]) protofibrils are known intermediates of the in vitro A[beta] aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of A[beta] protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative A[beta] protofibril immunoassay, A[beta] conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of A[beta] protofibrils without interference from A[beta] monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of A[beta] protofibrils in both cell and animal models, proving that A[beta] protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated A[beta] protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of A[beta] protofibrils in AD and has the potential of becoming an important diagnostic assay.

Title: Diagnostic Accuracy of ELISA and xMAP Technology for Analysis of Amyloid ß42 and Tau Proteins
Authors: Thierry S.M. Reijn, Marcel Olde Rikkert, Wieneke J.A. van Geel, Danielle de Jong and Marcel M. Verbeek

Abstract:

Background: Cerebrospinal fluid (CSF) concentrations of amyloid ß42 (Aß42) peptides and tau proteins may serve as biomarkers for Alzheimer disease (AD). Recently, the xMAP technology has been introduced as an alternative to ELISA for measurement of these markers.

Methods: We used xMAP assays and ELISA to analyze CSF concentrations of Aß42, total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau181) in samples from 69 patients with Alzheimer disease, 26 patients with vascular dementia, and 55 controls without neurological disorders.

Results: High CV values (>28%) for the ratio of xMAP:ELISA were observed for each biomarker, indicating that a constant correction factor cannot be applied to recalculate xMAP results into ELISA results. When a combination of CSF markers was used, the sensitivity, specificity, and area under the ROC curves for xMAP assays and ELISAs were not significantly different in differentiating AD patients from vascular dementia patients and controls.

Conclusions: A constant conversion factor cannot be used successfully to recalculate results obtained with xMAP assays to those from the ELISAs. With the use of analysis of a combination of Aß42, t-tau, and p-tau in CSF, however, differentiation of clinical groups is equivalent when either xMAP technology or conventional ELISA is used.



Title:

Methods for sample preparation for direct immunoassay measurement of analytes in tissue homogenates: ELISA assay of amyloid beta-peptides.

Authors:
Hyslop PA, Bender MH.

Abstract:
Use of low abundance analytes in whole tissue homogenates has been realized with the development of assays in which a specific analyte is captured and detected using immunological reagents. One of the many advantages of analyte immunoassay in crude homogenates is its relative simplicity, allowing high throughput analysis of samples. In this unit, some major key determinants in sample and standard preparation and handling are described that have been shown to improve the performance and reliability of these assay systems. The ELISA assay of amyloid peptides from brain tissue is described as an example, since the protocols for this analysis exemplify many of the techniques and problems that are encountered in the development of new assays.

Source: Pub Med