A NEW COST-EFFECTIVE METHOD FOR QUANTIFICATION OF TOTAL APOE IN HUMAN PLASMA SAMPLES

Abstract

Apolipoprotein E (ApoE) is a 34 kDa glycoprotein involved in lipid metabolism. Three common codominant alleles (ε2, ε3 and ε4) encode three ApoE protein isoforms (E2, E3 and E4). The presence of one or two ε4 alleles of the APOE gene is accepted as a reliable biomarker and risk factor of developing late onset Alzheimer's Disease (AD). Recently, we have developed a non-genetic, cost-effective and highly reliable method to detect the presence of apoE4 in human plasma using high-throughput clinical chemistry analysers. The method was based on the use of a proprietary coating agent, which allowed the capture of ApoE4 from plasma samples without using capture antibodies. Recent evidence shows that ApoE plasma levels are associated with increased risk of AD and dementia, independently of APOE genotype; suggesting that plasma ApoE is an easily accessible pre-clinical biomarker of AD. In this work, we explored the use of the same method for the quantification of plasma total ApoE. The nature and stability of ApoE binding to the ELISA plates treated with our proprietary coating solution was explored under a wide range of pH, different buffers and stringent conditions. Additionally, latex particles were used to demonstrate the feasibility of the methodology in suspension. We demonstrated that our methodology successfully bound the ApoE present in plasma without the need of capture antibodies. The binding to the plate is highly stable and not affected by the pH in the range tested (2–10), by the presence of detergents (polysorbate 20, Triton X-100 (from 0–0,5%) or by increasing concentrations of NaCl (0–2.4M). Furthermore, ApoE bound to the ELISA plate could only be released after treatments that destroy the apolipoprotein (enzymatic detergents or sodium hypochlorite). These results suggest that plasma ApoE could be captured and quantified using this methodology. The adaptation of the method to immunoturbidimetry to develop a fast and cost-effective method to quantify ApoE in plasma samples is currently ongoing.