Publicação
Sample preparation using three-phase partitioning systems for improved detection of Pentraxin-3 in human serum
| Resumo: | Lung cancer is the primary cause of cancer-related deaths around the world mainly due to late diagnosis [1,2]. To expedite diagnosis, the detection and quantification of biomarkers in human serum, such as the protein pentraxin-3 (PTX-3), is a promising approach [3,4]. However, biomarker detection and quantification in serum is a complex task and lacks accuracy and precision due to the high concentration of human serum albumin (HSA) and immunoglobulin G (IgG) that can interfere with the analysis [5]. To overcome this limitation, a sample preparation step to deplete the high-abundance proteins and concentrate biomarkers of interest is often required. This work aims to develop an alternative sample pretreatment technique resorting to three-phase partitioning (TPP) based on aqueous biphasic systems (ABS) to improve the quantification of PTX-3. The integration of TPP and ABS concepts is beneficial to achieve high-abundance proteins depletion and PTX-3 concentration in one step under protein-friendly conditions. Systems composed of polymers and citrate buffered salt (K3C6H5O7/C6H8O7, pH ≈ 7) were investigated using the homopolymers polypropylene glycol 400 g·mol-1 (PPG 400), polyethylene glycol 400 g·mol-1 (PEG 400), 600 g·mol-1 (PEG 600), 1000 g·mol-1 (PEG 1000) and 2000 g·mol-1 (PEG 2000), or the copolymers Pluronic PE6200, PE6400 and L35 (PEG-block-PPG-block-PEG-block) with 1750-1900 g.mol-1, and UCON (PEG-ran-PPG-ran-PEG) with 2500 g·mol-1. Firstly, the systems were studied regarding HSA and IgG depletion efficiency. The most efficient TPP systems were formed by PPG 400, PEG 1000, PEG 2000 and UCON with depletion efficiencies above 80% for both proteins; particularly, those formed by PEG 1000 and UCON reached depletion efficiencies of 100% for both high-abundance proteins. The previously referred systems were then applied in the extraction of PTX-3 spiked in a human serum sample. ELISA assays showed that among all TPP systems, the one composed of PEG 1000 allowed the complete extraction of PTX-3 towards the polymer-rich phase while the complete depletion of IgG and HAS was preserved. By comparing the values of PTX-3 quantified in both TPP-pretreated and no-pretreated samples, it was possible to confirm that TPP systems, if properly designed, reduce the interference of high-abundance proteins leading to more accurate results. |
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| Autores principais: | Mendes, M. S. M. |
| Outros Autores: | Rosa, M. E.; e Silva, F.. A.; Freire, M. G. |
| Assunto: | Aqueous biphasic systems Biomarkers Lung cancer Sample preparation Three-phase partitioning |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | documento de conferência |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | Lung cancer is the primary cause of cancer-related deaths around the world mainly due to late diagnosis [1,2]. To expedite diagnosis, the detection and quantification of biomarkers in human serum, such as the protein pentraxin-3 (PTX-3), is a promising approach [3,4]. However, biomarker detection and quantification in serum is a complex task and lacks accuracy and precision due to the high concentration of human serum albumin (HSA) and immunoglobulin G (IgG) that can interfere with the analysis [5]. To overcome this limitation, a sample preparation step to deplete the high-abundance proteins and concentrate biomarkers of interest is often required. This work aims to develop an alternative sample pretreatment technique resorting to three-phase partitioning (TPP) based on aqueous biphasic systems (ABS) to improve the quantification of PTX-3. The integration of TPP and ABS concepts is beneficial to achieve high-abundance proteins depletion and PTX-3 concentration in one step under protein-friendly conditions. Systems composed of polymers and citrate buffered salt (K3C6H5O7/C6H8O7, pH ≈ 7) were investigated using the homopolymers polypropylene glycol 400 g·mol-1 (PPG 400), polyethylene glycol 400 g·mol-1 (PEG 400), 600 g·mol-1 (PEG 600), 1000 g·mol-1 (PEG 1000) and 2000 g·mol-1 (PEG 2000), or the copolymers Pluronic PE6200, PE6400 and L35 (PEG-block-PPG-block-PEG-block) with 1750-1900 g.mol-1, and UCON (PEG-ran-PPG-ran-PEG) with 2500 g·mol-1. Firstly, the systems were studied regarding HSA and IgG depletion efficiency. The most efficient TPP systems were formed by PPG 400, PEG 1000, PEG 2000 and UCON with depletion efficiencies above 80% for both proteins; particularly, those formed by PEG 1000 and UCON reached depletion efficiencies of 100% for both high-abundance proteins. The previously referred systems were then applied in the extraction of PTX-3 spiked in a human serum sample. ELISA assays showed that among all TPP systems, the one composed of PEG 1000 allowed the complete extraction of PTX-3 towards the polymer-rich phase while the complete depletion of IgG and HAS was preserved. By comparing the values of PTX-3 quantified in both TPP-pretreated and no-pretreated samples, it was possible to confirm that TPP systems, if properly designed, reduce the interference of high-abundance proteins leading to more accurate results. |
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