| Alzheimer's disease (AD) is a high prevalent neurodegenerative disease accompanied by gradual and irreversible behavioral and cognitive impairments. Brain lesions observed during the course of AD involve three main aspects: extracellular amyloid-β(Aβ) deposition as senile plaques and intracellular tau accumulation forming neurofibrillary tangles and promoting cyto-skeletal disorganization, synaptic and neuronal death, anencephalotrophia as well. Many researchers have shown that abnormal folding and aggregation of Aβ42 peptide in the brain is the main causative agent of AD.The C-terminal peptide was buried in the core in formation of Aβoligomers, while the N-terminal peptide exposed on the surface. It has been demonstrated that mAbs against N-terminal region of Aβhave important roles in the treatment and diagonosis of AD.1. The anti-AβN-terminal monoclonal antibodies can protect cells from toxicity induced by Aβ42 oligomers in vitro.In order to study the protective role of mAbs (A8, recognizing amino acid residues 1-6 of Aβ; A6F7, recognizing amino acid residues 1-11 of Aβ; C9, recognizing amino acid residues 4-11 of Aβ) specific to N-terminal region of Aβagainst cell damage induced by Aβoligomers, human neuroblastoma cell line SH-SY5Y was exploited and cell viability was measured by MTT assay. The results were shown as follows:(i) The cell viability of SH-SY5Y had no apparent change before differentiation even if the Aβoligomer was administered in high-dose (20μM). On the contrary, after differentiation was induced with Retinoic acid, the low-dose of Aβoligomers (10μM) had significant toxic effect on SH-SY5Y. Therefore, the induced differentiation plays a vital role in Aβoligomer toxicity to SH-SY5Y. (ii) After adding 0,2,4,6,8,10,12,14,20μM Aβoligomers to the cells, at least 10μM Aβoligomers displayed a significant toxic effect on the cells (p< 0.01). The increasing dose of Aβoligomers made more sever toxicity on the cells. We conclude that the cytotoxicity induced by Aβoligomers is dose-dependent.(iii) Aβoligomer toxicity was reduced significantly in mAbs (A8, A6F7, C9) (1μM) and Aβoligomer (10μM) treated group, compared to only oligomer (10μM) group (p< 0.05).2. Application of anti-AβN-terminal monoclonal antibodies in Aβdetection.In order to establish Sandwich ELISA to analyze Aβ42 in the plasma or cerebrospinal fluid (CSF), we used biotin-labeled A8 as detecting antibody and one of A6F7, C9 or 4G8 as coating antibody. The sensitivity was amplified by avidin-biotin-system (ABS). The optimal analyse condition was decided by the square rank method. The standard curve of ELISA was established by Aβstandard sample. The plasma AP42 level in AD patients and in healthy individuals was detected respectively. The results were as follows:(ⅰ) For AP oligomers'detection system, the optimal dilution of biotin-labeled A8 was 1:800 when 4G8 as coating antibody with dilution of 1:800, which was better than A6F7, C9 or mixture of A6F7 and C9 as coating antibody. (ⅱ) For the established Sandwich ELISA for AP 42, the measurement range was from 0.33 ng/mL to 19.5 ng/mL. (ⅲ) It was found that AP42 in AD patient group was (52.2±11.3) ng/mL, the same age control group was (12.5±9.6) ng/mL. The plasma Aβ42 level of AD patients was 4.16 times higher than the healthy control group.In summary, we confirm that mAbs against N-terminal region of Aβhave an protective effect on cells in vitro, and A8 can be used as detecting antibody to develop ELISA method for analyzing Aβ42 in the samples from AD patients. This research suggests that mAbs against N-terminal region of Aβhave the potential to work in AD diagnosis and therapy.
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