The Basic Research On Antigen Screening And Serological Diagnosis Of Angiostrongylus Cantonensis’s Functionalized Gold Nanorods

ObjectivesPreparation of crude antigens of Angiostrongylus cantonensis larvae,excretion and secretion of crude antigens,adult crude antigens,larval purified antigens and adult purified antigens functionalized nanorods.To screen the diagnostic antigen and the optimum concentration of the functional nanogold rod of A.cantonensis,and confirm that the functionalized nano-gold rod can detect the serum antibody of A.cantonensis with early or low infection.Methods1.The gold crystal seed polymerization method controls the nanorods with controllable diameter-length ratios.2.The crude antigen and adult crude antigen of larvae of A.cantonensis were prepared by grinding with liquid nitrogen,crude antigen was excreted and secreted by RPMI-1640 culture method,and larval purified antigen and adult purified antigen were prepared by SDS-PAGE.The thiolated crude antigens,purified antigens and nanogold rods were combined to prepare functional nanorods.Scanning by a UV spectrophotometer was used to compare the size of the red peak shift of the surface plasmon resonance absorption peak to optimize the optimum coating concentration of the functionalized nanorods.3.Angiostrongylus cantonensis functionalized nanogold rods were used to detect serum antibodies against different infection durations,different infectivity,and different dilutions.According to the change of the red peak shift of the surface plasmon resonance absorption peak,the dominant diagnostic antigen of Angiostrongylus cantonensis was screened out.4.Comparison of the results of ELISA detection between serum antibody and different infection time,different degree of infection,and different dilution of A.cantonensis functional nanogold rod.Explore the sensitivity and sensitivity of functional nanogold rod assays.Results1.The preparation of crude antigens,adult crude antigens,excreted and secreted crude antigens,larval purified antigens and adult purified antigen-functionalized nanorods were successfully prepared,and preferably its optimum coating concentration and corresponding maximum red shift:The crude antigen coating concentration of larvae was 30 μg/mL,corresponding to the maximum red-peak shift of LSPR absorption peak was 35 nm;the optimum coating concentration of adult crude antigen was 40μg/mL,corresponding to the maximum red-peak shift was 35 nm;excretion and secretion of crude antigens coating concentration 3 μg/mL,the peak of plasmon resonance absorption before and after coating showed a maximum red peak shift of 35 nm;the optimal protein molecular weight of larval purified antigen was 43kD,and when the corresponding antigen concentration was 20 μg/mL,the maximum plasmon resonance absorption peak appeared before and after coating the red shift was 25 nm;the optimum protein molecular weight of adult purified antigen was 45kD,and when the corresponding antigen concentration was 10 μg/mL,the maximum red shift of the peak corresponding to the LSPR absorption peak was 40 nm.2.The crude antigens of A.cantonensis larvae,crude antigens of adults,crude antigens excreted and secreted,larval purified antigens,and adult purified antigen functionalized nano-gold rods can be used to detect serum antibodies with different infection durations and different infectivity.Being positive,the minimum infection was 50 Ⅲ stage larvae.In the case of mild infection,positive serum antibodies were detected 9 days ahead of the rat serum antibody ELISA kit,and positive serum antibodies were detected 2 days earlier than the rat serum antibody ELISA kit in moderate or severe infection.Among them,the nanorods functionalized with adult purified antigens are more sensitive to detect serum antibodies against early rounds of infection with A.cantonensis,and have higher sensitivity than traditional ELISA assays.The maximum dilution of positive serum antibodies can reach 1:600.ConclusionThe gold nanorods with controlled diameter length and high yield were obtained from the seed growth method,and the dominant diagnostic antigen of Angiostrongylus cantonensis was selected as adult purified antigen.The angiostrongylus cantonensis antigen-functionalized nanogold rod antigen has a simple preparation,low cost,good sensitivity and specificity in detection of serum antibodies.Compared with the traditional ELISA method,it has the advantages of early detection of antibodies,high dilution,and low infection,providing new ideas and technical support for the detection of early infection and low-grade infection of angiostrongylus cantonensis infection.It is expected to be applied to the research of dynamic epidemiology and the development of clinical kits.