Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods

2018 ◽  
Vol 56 (5) ◽  
pp. 796-802 ◽  
Author(s):  
Sigrun Badrnya ◽  
Tara Doherty ◽  
Ciaran Richardson ◽  
Robert I. McConnell ◽  
John V. Lamont ◽  
...  
Keyword(s):  
Specific Information ◽  
Degenerative Diseases ◽  
Chain Reaction ◽  
Array Technology ◽  
Apoe Protein ◽  
Additional Protein ◽  
Polymerase Chain ◽  
Simultaneous Immunoassay ◽  
Risk Prognosis ◽  
Disease Stages

AbstractBackground:Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of theE4allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous.Methods:Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels.Results:Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for theE4allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity.Conclusions:This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual’sAPOE4status directly from plasma. TheE4genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer’s disease. The biochip’s potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-basedAPOEgenotyping and may deliver additional protein-specific information on a variety of diseases in the future.

scholarly journals Apolipoprotein E promoter genotypes are not associated with white matter hyperintensity development in high-altitude careers

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Richard R. Chapleau ◽  
CharLee A. Martin ◽  
Summer R. Hughes ◽  
James C. Baldwin ◽  
John Sladky ◽  
...  
Keyword(s):  
White Matter ◽  
Apolipoprotein E ◽  
High Altitude ◽  
White Matter Hyperintensities ◽  
White Matter Hyperintensity ◽  
Chain Reaction ◽  
Promoter Regions ◽  
Altitude Exposure ◽  
Apoe Protein ◽  
Polymerase Chain

Abstract Objective This study sought to determine if there is an association between variants in the apolipoprotein E (ApoE) promoter regions and development of white matter hyperintensities (WMH) in military subjects who have been exposed to high altitude. In an earlier study, we found that ApoE status did not correlate with WMH development, and here we hypothesized that regulation of APOE protein expression may be protective. Results Our cohort of 92 subjects encountered altitude exposures above 25,000 feet mean sea level through their occupations as pilots or altitude chamber technicians. Using Taqman-style polymerase chain reaction genotyping and t-tests and two-way analyses of variance we found no significant association between ApoE promoter genotypes and the presence, volume, or quantity of WMHs after high altitude exposure. Taken together, the observations that neither ApoE genotype status nor promoter status are associated with WMH properties, we believe that the mechanism of action for developing WMH does not derive from ApoE, nor would therapies for ApoE-mediated neurodegeneration likely benefit high altitude operators.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

An unusual case of spinal cord restricted mycobacteriosis in a European mink

2013 ◽  
Vol 41 (01) ◽  
pp. 63-66
Author(s):  
D. Schaudien ◽  
C. Flieshardt ◽  
I. Moser ◽  
H. Hotzel ◽  
A. Tipold ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Unusual Case ◽  
Histological Evaluation ◽  
Mustela Lutreola ◽  
Chain Reaction ◽  
European Mink ◽  
Pcr Product ◽  
Granulomatous Lesions ◽  
The Central Nervous System ◽  
Polymerase Chain

SummaryGranulomatous myelitis due to infection with Mycobacterium avium was diagnosed in a 4-year-old male neutered European mink (Mustela lutreola). The causative agent was detected by an acid-fast stain and further characterized by polymerase chain reaction and DNA sequencing of the PCR product. A thorough histological evaluation of the remaining organs revealed no granulomatous lesions or detectable acid-fast organisms. Although minks are generally highly susceptible for mycobacteria, localised infections, especially of the central nervous system, are unusual and may represent an atypical chronic form of the disease.

Detection of the Glanzmann's Thrombasthenia Mutations in Arab and Iraqi-Jewish Patients by Polymerase Chain Reaction and Restriction Analysis of Blood or Urine Samples

1991 ◽  
Vol 66 (04) ◽  
pp. 500-504 ◽  
Author(s):  
H Peretz ◽  
U Seligsohn ◽  
E Zwang ◽  
B S Coller ◽  
P J Newman
Keyword(s):  
Polymerase Chain Reaction ◽  
Base Pair ◽  
Restriction Analysis ◽  
Urine Samples ◽  
Chain Reaction ◽  
Base Pairs ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Base Pair Deletion ◽  
Polymerase Chain

SummarySevere Glanzmann's thrombasthenia is relatively frequent in Iraqi-Jews and Arabs residing in Israel. We have recently described the mutations responsible for the disease in Iraqi-Jews – an 11 base pair deletion in exon 12 of the glycoprotein IIIa gene, and in Arabs – a 13 base pair deletion at the AG acceptor splice site of exon 4 on the glycoprotein IIb gene. In this communication we show that the Iraqi-Jewish mutation can be identified directly by polymerase chain reaction and gel electrophoresis. With specially designed oligonucleotide primers encompassing the mutation site, an 80 base pair segment amplified in healthy controls was clearly distinguished from the 69 base pair segment produced in patients. Patients from 11 unrelated Iraqi-Jewish families had the same mutation. The Arab mutation was identified by first amplifying a DNA segment consisting of 312 base pairs in controls and of 299 base pairs in patients, and then digestion by a restriction enzyme Stu-1, which recognizes a site that is absent in the mutant gene. In controls the 312 bp segment was digested into 235 and 77 bp fragments, while in patients there was no change in the size of the amplified 299 bp segment. The mutation was found in patients from 3 out of 5 unrelated Arab families. Both Iraqi-Jewish and Arab mutations were detectable in DNA extracted from blood and urine samples. The described simple methods of identifying the mutations should be useful for detection of the numerous potential carriers among the affected kindreds and for prenatal diagnosis using DNA extracted from chorionic villi samples.

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

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