| Alzheimer's disease(AD) is a progressive degenerative disorder of the central nervous system.AD is characterized pathologically by extracellular deposits of beta-amyloid(Aβ) in senile plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated tau and synaptic and neuronal cell loss.So far there is no specific therapy for AD.The accumulation of Aβis one of the most important hallmarks of the progression of AD,and has been considered to be the main cause for neuronal injury and impairment of memory.In murine models of AD,both active and passive immunization against Aβinduce a marked reduction in amyloid brain burden and an improvement in congnitive functions.Immunotherapy especially passive immunotherapy has become the effective strategy for AD.Anti-Aβmonoclonal antibody(McAb) is valuable tool for AD therapy.Our work included:(ⅰ) Preparation and characterization of McAb against AβOligomer;(ⅱ) Therapeutic effect study of McAb both in vitro and in vivo;(ⅲ) Cloning and analyzing the sequence of variable region of the McAb.1.Anti-AβMcAb preparation and characterizationThe Aβ1-42 oligomer mixture was assembled with synthetic Aβ1-42 peptide in vitro, and then the resulting mixture was employed to vaccinate mice.The spleen cells were obtained from the mouse with the highest serum titer and fused with myeloma cells. Hybridoma subclones were screened by indirect ELISA after subcloning by the limiting dilution technique.McAb was purified through protein A affinity chromatography.The specificity and affinity of the McAb were analyzed by ELISA and western blot.One hybridoma cell line(A8) secreting McAb specific for the Aβoligomer was established after subcloning.Then,the biological characteristics of McAb A8 were clarified.The purity of McAb A8 was 95%.McAb A8 displayed much higher affinity to Aβoligomer of 16.5-25 KD,The isotype of McAb A8 is IgG3.The epitope of McAb A8 was Aβ1-6 and the Kd of McAb A8 was 10-8 mol/L.2.Biological activity of anti-AβMcAb in vitro and in vivoIn order to study the protective role of McAb A8 against cell damage induced by Aβoligomer,human neuroblastoma SH-SY5Y cell line was exploited and cell viability was measured by CCK-8 assay.The cell viability of SH-SY5Y was improved significantly in McAb A8(1μmol/L) and Aβoligomer(20μmol/L) treated group,compared with single Aβoligomer(20μmol/L) group(p＜0.05).SAMP8 mice were administered(500μg/mouse) intraperitoneally with McAb A8. Then Morris water maze trial,immunohistochemistry and western blot methods were used to analyze cognitive behaviour and the level of Aβplaque and P-tau,etc. Compared to the mice of control groups,there was an improvement in cognitive ability in the McAb treated mice.In the meantime,McAb A8 reduced Aβaccumulation and inhibited phosphorylated tau(P-tan) levels in the brain of SAMP8 mice,but the expression of total tau was not affected.3.Cloning and analyzing the sequence of variable region of the MeAb A8Mouse originated McAb is difficult to be used in humans unless it is humanized. Therefore,we amplified the genes of light chain variable region(VL) and heavy chain variable region(VH) of McAb A8 by using 5' RACE PCR technique.Then,the PCR products were sequenced,analyzed and cloned into PMD18-T vector.The VH gene contained 450 bp and encoded 150 amino acid residues;the VL gene contained 429 bp and encoded 143 amino acid residues.They were homologous with the sequences of variable region of mouse IgG,published in Genebank.This work was important for further humanized performace.In this study,a strain of monoclonal antibody targeting Aβoligomers has been successfully developed.The isotype and the epitope of McAb A8 is IgG3 and Aβ1-6, respectively.The affinity(kD) of McAb A8 was 10-8 mol/L.McAb A8 can inhibit the neurotoxicity of Aβoligomer in vitro and displays therapeutic effect in AD mouse model.At the same time,we have successfully cloned and analyzed the variable region of McAb A8.In a word,the above results pave the way for the further research on AD's therapy and diagnosis.
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