Establishment Of Novel Immunochromatographic Methods And Their Application In The Detection Of Cardiovascular Disease

Cardiac troponin ?(cTn?)is the gold standard for the diagnosis of acute myocardial infarction.Large-scale immunoanalyzers are the mostly used equipments for cTn? detection and the limit of detection can reach 10 pg/m L order of magnitude.However,the professional requirements for operators and high cost of instrument purchase and maintenance restrict their application.Lateral flow immunoassay has also been widely used in the detection of myocardial markers.It has the advantages of simple operation,quick reaction and portable instrument.The main defect of this technology is the low sensitivity.Strategies have been developed to address this problem,such as amplifying colloidal gold's optical signal by gold/silver staining,using more sensitive magnetic nanoprobes,and introducing functional nucleic acid/protein.However,these new approaches often accompany with new issues,including but not limited to longer time,worse background and higher cost.The construction strategy of nanoprobes often determines the detection results.There are some key factors that affect the performance of probes such as the amount,orientation,binding strength and antigen recognition ability of antibodies.Aimed at these factors,we focused on antibody conjugation and orientation,developing several new methods for the preparation of fluorescent quantitative nanoprobes.The main work is as follows:1)Streptavidin-biotin based signal amplification.In this part,nanospheres were used as carrier.Biotin,core streptavidin,fluorescent dye and antibody were conjugated successively.As a result,the loading efficiency of fluorophores and antibodies were increased a lot,which significantly improved the detection sensitivity.The limit of detection for cTn? was 0.049 ng/m L,the with-run precision and between-run precision were both good(CV < 10%),and the linear curve fitted well(R > 0.99).The corresponding lateral flow strips had a good stability during storage.The detection results of human serum samples were consistent with clinical chemiluminescence immune detection system(R > 0.99),showing strong anti-interference capability.2)Antibodies oriented by Protein G and immobilized by crosslinking agent.Protein A and protein G can specifically target the Fc region of Ig G antibodies,which is reversible.Furthermore,the binding strength is related to antibody's animal source and subtype.In this part,we developed a new efficient and robust technology to construct fluorescent nanoprobes with oriented modified antibodies,based on specific binding of the Fc region with protein G on the surface of polystyrene nanospheres and subsequent covalent cross linking at binding sites to firm them.Consequently,antibodies were conjugated in an efficient,oriented and tough way.In addition,heterophilic blocking regent was used to block redundant active sites of protein G for the first time,improving the specificity of detection.The final probes were applied in lateral flow immunoassay to detect cTn?,showing great sensitivity(the limit of detection was 0.032 ng/m L),linearity(R > 0.99),precision(CV < 10%),consistency with immunoanalyzer(R > 0.99)and good stability in thermal acceleration aging test.3)Establishment and mechanism analysis of p H regulated antibody-oriented chemical coupling method.Chemical coupling has been widely used for antibody conjugation.However,the amino groups of antibodies are indiscriminately occupied,which easily causes randomly orientation.When the Fab region heads toward the surface of nanosphere,the antigen-binding sites are very likely to be occupied or be restricted to access antigen due to steric hindrance.Charge distribution and hydrophilicity of antibodies are modifiable in different p H,so we developed and demonstrated a simple antibody-oriented strategy for the lateral flow immunoassay of cTn? by conjugating antibodies onto polystyrene nanospheres at the optimal p H.The binding amount and orientation of antibodies as well as the detection sensitivity were significantly improved.After the coupling p H decreased from 7.8 to 5.8,antibody binding efficiency increased by 30%,the exposure degree of the Fc regions decreased by 60%,and the detection signal intensity increased by 4 times.Although p H regulation has been commonly used to optimize antibody conjugation,this paper illustrates the mechanism of its antibody orientation enhancement ability for the first time and reveals the important influences of the density,the charge distribution and hydrophilicity of the antibody,the control of the velocities of physical adsorption and chemical coupling as well as other factors on antibody orientation.To sum up,probe construction strategies in this study were simple to operate and were applicable to various kinds of antibodies,having great application potential in the detection of antigen markers in disease,environment,food and other fields.