Presentation Information
[P04-606]Antibody-incorporated lipid membrane combined with QCM method for early detection of α-synuclein from Parkinson's disease
○Masato Ukawa1, Toshinori Shimanouchi1, Hodaka Yamakado2, Minoru Noda3, Yukitaka Kimura1 (1. Okayama University (Japan), 2. Kyoto University (Japan), 3. Kansai University (Japan))
Keywords:
Early detection,α-synuclein,lipid membrane,antibody,QCM
[Purpose]
Parkinson's disease (PD), a type of amyloidosis, is a neurological disease common in the elderly. The cause of PD is thought to be the accumulation of fibrillary aggregates of α-synuclein (α-Syn), a protein with a molecular weight of 14.5 kDa. Therefore, it is necessary to develop a rapid diagnostic system for early detection.Previous studies, such as ELISA and (Quartz Crystal Microbalance (QCM), have shown that α-Syn aggregates can be detected at pM level. The detectability is determined by the antibody activity on the substrate. Therefore, an intact immobilization method is required. For example, there has been reported to be the introduction of a polymer layer or the use of linker molecules to suppress direct adsorption of the antibody to the solid substrate. In order to create a more convenient method, we investigated a detection system in which the antibody was presented on a bilayer lipid membrane.
[Method]
We prepared 5 kinds of liposomes by mixing 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with DPPC: DOPC = 0: 100, 25: 75, 50: 50, and 75: 25,100: 0. Next, α-Syn accumulation in the lipid membrane was measured using QCM. QCM is a method to measure the mass change of adsorbed and desorbed target substances from the change of the vibration frequency of the lens. The larger the negative frequency change [Hz], the larger the amount of protein adsorbed.
[Results]
The amount of α-Syn adsorbed on DOPC, DPPC, and DOPC/DPPC (25/75) liposomes was compared, and the amount adsorbed on DOPC/DPPC (25/75) was the highest. Furthermore, the detection limit was 100 nM. In order to increase the sensitivity, the amount of α-Syn adsorbed on DOPC/DPPC liposomes was evaluated using anti-α-Syn antibody. At high concentrations, DOPC/DPPC (25/75) showed high adsorption, but at low concentrations, DOPC composition seemed to be better. On the other hand, the detection sensitivity was at least less than 1 nM. Therefore, it was shown that the incorporation of anti-α-Syn antibody into lipid membranes was effective.
[Conclusion]
We combined QCM method with the anti-a-antibody displayed lipid membrane system to detect α-Syn as a target. anti-a-antibody was linked to fatty acids to immobilized into lipid bilayer membranes with keeping its conformation. This system allows us to detect a-syn less than 1 nM, although the further improvement would be required.
Parkinson's disease (PD), a type of amyloidosis, is a neurological disease common in the elderly. The cause of PD is thought to be the accumulation of fibrillary aggregates of α-synuclein (α-Syn), a protein with a molecular weight of 14.5 kDa. Therefore, it is necessary to develop a rapid diagnostic system for early detection.Previous studies, such as ELISA and (Quartz Crystal Microbalance (QCM), have shown that α-Syn aggregates can be detected at pM level. The detectability is determined by the antibody activity on the substrate. Therefore, an intact immobilization method is required. For example, there has been reported to be the introduction of a polymer layer or the use of linker molecules to suppress direct adsorption of the antibody to the solid substrate. In order to create a more convenient method, we investigated a detection system in which the antibody was presented on a bilayer lipid membrane.
[Method]
We prepared 5 kinds of liposomes by mixing 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with DPPC: DOPC = 0: 100, 25: 75, 50: 50, and 75: 25,100: 0. Next, α-Syn accumulation in the lipid membrane was measured using QCM. QCM is a method to measure the mass change of adsorbed and desorbed target substances from the change of the vibration frequency of the lens. The larger the negative frequency change [Hz], the larger the amount of protein adsorbed.
[Results]
The amount of α-Syn adsorbed on DOPC, DPPC, and DOPC/DPPC (25/75) liposomes was compared, and the amount adsorbed on DOPC/DPPC (25/75) was the highest. Furthermore, the detection limit was 100 nM. In order to increase the sensitivity, the amount of α-Syn adsorbed on DOPC/DPPC liposomes was evaluated using anti-α-Syn antibody. At high concentrations, DOPC/DPPC (25/75) showed high adsorption, but at low concentrations, DOPC composition seemed to be better. On the other hand, the detection sensitivity was at least less than 1 nM. Therefore, it was shown that the incorporation of anti-α-Syn antibody into lipid membranes was effective.
[Conclusion]
We combined QCM method with the anti-a-antibody displayed lipid membrane system to detect α-Syn as a target. anti-a-antibody was linked to fatty acids to immobilized into lipid bilayer membranes with keeping its conformation. This system allows us to detect a-syn less than 1 nM, although the further improvement would be required.
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