The Screen,Characterization And Appliacation Of Human Single-Chain Antibodytargeted Atherosclerotic Plaque

Objective Cardiovascular diseases,especially acute myocardial infarction?AMI?,will seriously affect people's health.Atherosclerosis is the basic pathological process of plaque formation in blood vessels.However,there is currently no clinically effective non-invasive diagnostic method for detecting atherosclerotic plaques.I hypothesized that anti-atherosclerosis single-chain antibody conjugated NaNdF4@NaGdF4 nanoparticles can be a non-invasive diagnostic method for clinical detection of atherosclerotic plaques.To achieve this,I screened and characterized single-chain antibodies and synthesized nanoparticles and coupled them to produce a probe for the detection of atherosclerotic plaque.Methods I collected peripheral arterial blood from 50 patients with atherosclerosis diagnosed by coronary angiography from the emergency department of the Affiliated Hospital of Qingdao University,and extracted the total RNA to establish the antibody gene library and antibody phage library.I collected atherosclerosis tissue samples from the Department of Vascular Surgery,Affiliated Hospital of Qingdao University for phage panning.I performed 6 rounds of panning of antibody phage library using phage-ELISA,expressed in vitro using E.coil TOP10 and verified the affinity and specificity of selected single-chain antibodies to atherosclerotic plaque by ELISA and western blot.Then I synthesized NaNdF4@NaGdF4 nanoparticles and conjugated the nanoparticles and single-chain antibody by EDC-NHS reaction to form a dual modality imaging probe,and analyzed the solution dispersion and size of coupled probe by dynamic light scattering and electron microscopy..Finally,ApoE^-/-mice were used to construct an atherosclerosis animal model,and the probe's ability to detect atherosclerosis in magnetic resonance imaging?MRI?and near infrared spectroscopy?NIRS?was verified.Results Screened a novel single-chain antibody called ASA6 against atherosclerotic plaques from 50 patients with myocardial infarction by phage display.Twelve positive clones were identified by Phage-ELISA.ASA6 has the strongest affinity and specificity among the twelve positive clones.The results of western blot and coomassie brilliant blue protein staining showed that the size of single-chain antibody was about 25kd.The results of ELISA and western blot showed that ASA6 had high affinity for oxidized low-density lipoprotein and atherosclerotic tissue.Immunofluorescence and oil red O staining showed that ASA6 has specificity and high affinity for atherosclerotic plaques.Dynamic light scattering and electron microscopy showed that the probe had good dispersion in solution and its diameter was less than 1000 nm.The results of NIRS and MRI showed that the probe has high specificity and sensitivity for atherosclerotic plaques.The results showed that atherosclerotic plaques in animal model are located in the right coronary artery and the left anterior descending branch,and the positive signal of the left anterior descending branch is higher than that of the right coronary artery,which indicates that the degree of atherosclerosis of the left anterior descending branch is higher.It is more severe than the right coronary artery.The liver MRI signal showed a positive signal in the hepatic artery,especially the root of the hepatic artery,indicating the presence of atherosclerotic plaque in the liver.The results of renal imaging showed that there is the positive signal of the plaque in the left renal artery.,and the positive signal density peaked 2 hours after the injection of the probe.Abdominal aorta imaging showed that positive signals appeared at different positions on different cross-sections,indicating that atherosclerosis involves the entire abdominal aorta.These results indicate that the probe can detect the distribution and severity of atherosclerotic plaques in patients at once.Conclusion We screened a novel single-chain antibody that can bind atherosclerotic plaques with high affinity,and developed a novel probe conjugated with Na NdF4@NaGdF4 nanoparticles that can monitor atherosclerotic lesion in real-time and all-round by MRI imaging and NIRS detection.