Abstract
The allele ε4 of the apolipoprotein E gene (APOE ε4) is the major genetic risk factor for non-dominantly inherited Alzheimer’s Disease (AD). Current techniques for APOE ε4 carriers identification show good accuracy but have several disadvantages that limit its implementation in a clinical laboratory. These include the need for sample preprocessing, poor automation, low throughput, requirement of additional equipment, and high cost. We followed ISO 13485 guidelines to validate the e4Risk test, a new latex-enhanced immunoturbidimetric blood assay for apolipoprotein E4 (ApoE4) determination in human plasma samples. The test showed high performance in terms of lot to lot variability, precision, interferences, reagents stability, prozone, and detectability. Furthermore, diagnostic accuracy is almost equal (99%) to the gold standard, APOE ε4 genotyping by polymerase chain reaction (PCR). Furthermore, we demonstrated that the e4Risk test can be adapted to any clinical chemistry analyzer, including the high throughput analyzers present in most hospitals and clinical laboratories. The e4Risk test versatility, low cost, and easiness provides an excellent solution for APOE ε4 carriers identification using the same blood sample drawn for biochemical diagnostic work-up of AD patients, which can have important advantages for patient stratification in clinical trials, preventative strategies for AD, and clinical assessment of risk for brain amyloidosis.
Highlights
Apolipoprotein E (ApoE) is a glycoprotein involved in lipid metabolism
This assay represents a fast and cost-effective assay for apolipoprotein E4 (ApoE4) determination (e4Risk test), that can be very implemented within the clinical routine, since it uses the same sample that other routine lab tests, without any further processing, and that can be run in any chemistry analyzer, including the high-throughput clinical chemistry analyzers present in hospitals and clinical laboratories
Four different testing samples were prepared for Limit of Detection (LoD) assays using the same plasmas used in Limit of Blank (LoB) assays but spiked with recombinant ApoE4 to achieve samples with low concentrations of ApoE4
Summary
Apolipoprotein E (ApoE) is a glycoprotein involved in lipid metabolism. It is encoded by the APOE gene, which has three different alleles (ε2, ε3, ε4) that encode for three different ApoE isoforms (ApoE2, ApoE3, and ApoE4). Several effective techniques are described for APOE genotyping, including gene-based analyses such as real-time PCR11 and biochemical (non-genetic) methods such as isoelectric focusing-immunoblotting[12,13], ELISA14 or biochip arrays[15] All of these techniques pose several limitations such as time-consuming, technically complex, need additional sample processing, require the investment on specific instrumentation, lack high-throughput capacity, or lack of fully automation. We recently described an effective ELISA-based technique that could be adapted to immunoturbidimetry[14] in order to eliminate these technical barriers This assay represents a fast and cost-effective assay for ApoE4 determination (e4Risk test), that can be very implemented within the clinical routine, since it uses the same sample that other routine lab tests, without any further processing, and that can be run in any chemistry analyzer, including the high-throughput clinical chemistry analyzers present in hospitals and clinical laboratories. We provide one example for its adaptation to one of the leading high-throughput chemistry analyzer Architect c16000 (Abbott)
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