Nanomaterial-based Double Antibody Sandwich ELISA For Detection Of Nuclear Protein In SFTSV

Sever fever with thrombocytopenia syndrome(SFTS)is a new infectious disease caused by the newly discovered bunyavirus(SFTSV).Nucleocapsid protein(NP)is the most abundant viral antigen of SFTSV and has a highly conserved amino acid sequence.There are no effective treatments or vaccines,so early diagnosis of SFTS is the key to preventing and controlling SFTSV infection.Rapid detection of SFTSV and effective control of the disease are important.With the rapid development of clinical medical diagnosis and treatment technology,many traditional detection technologies can no longer meet the needs of clinical medical development.Therefore,it is urgent to develop ultra-sensitive detection methods for viral antigens.This project has tried a variety of nanomaterials,such as gold nanoparticles(AuNPs),magnetic beads,polystyrene fluorescent microspheres,etc.as the carrier of antibodies and horseradish peroxidase(HRP).The nanomaterials are combined with the sandwich enzyme-linked immunosorbent assay to improve detection sensitivity.At first,we used mouse IgG as the model antigen and established a dual-antibody sandwich ELISA against the model antigen.The purpose was to screen out the most sensitive ELISA system based on these materials.AuNPs with a particle size of about 20 nm were synthesized by sodium citrate reduction method and characterized,which had a characteristic absorption peak at520 nm and a narrow size distribution.We tried various methods to conjugate colloidal gold with antibodies and enzymes,and prepared several colloidal gold probes to reduce the lower limit of detection of mouse IgG to 250 pg/mL.Using magnetic beads and time-resolved fluorescent microspheres,the lower detection limit of mouse IgG can reach 3.9 ng/mL.The sensitivity of ELISA based on these materials was better than that of commercially available ELISA kits.Next,we applied AuNPs-based ELISA with high sensitivity and simple operation to the detection of SFTSV-NP,and established a high-sensitivity detection method with colloidal gold probe modified with HRP-labeled monoclonal antibody(anti-NP McAb).We used HRP to label purified McAb(1H10)with high titer by sodium periodate oxidation.The results showed that the enzyme-labeled monoclonal antibody had good enzyme activity and antibody activity.Then,we determined the appropriate ratio of enzyme-labeled antibodies to AuNPs by gold aggregation experiments,and prepared colloidal gold probes labeled with antibodies for detection of NP.The enhanced optical sensitivity of this method can be attributed to the high loading efficiency of colloidal gold for enzyme-labeled McAb.Compared with conventional ELISA,it can enhance the local concentration of enzyme on each immune sandwich structure.Under the optimized conditions of capture antibody concentration and AuNPs probe dilution,the detection limit of this method is 0.9 pg/mL,which is 100 times lower than that of the traditional method(100 pg/mL),and the coefficient of variation(CV%)is less than 10%.These results indicate that this method has higher sensitivity(the OD value corresponding to the experimental group that detects irrelevant proteins can be controlled below 0.1)and reliable reproducibility.In addition,our method is highly specific for the detection of NP and can be used for the detection of real viruses.The probe is stable for 4 months at 4℃ and 7 days at 37℃.The developed AuNPs-based ELISA for ultra-sensitive detection of NP has high sensitivity and a simple operation process similar to the conventional ELISA,which can be applied in clinical diagnosis.