Establishment Of Array-ELISA To Detect Arboviruses And Respiratory Viruses

Background: Arboviruses viruses are a group of virus transmitted biologically among vertebrate hosts by hematophagous vectors. Arboviruses include more than 500 viruses, belonging to eight viral families and 14 genera. Over 130 arboviruses are of public health importance. Recently global epidemic arboviral activity has dramatically increased due to global warming and human activities. Due to the large area, our country is a natural epidemic focus of many kinds of arboviruses. Arboviruses are now a considerable threat to public health in China. Respiratory viruses are a group of viruses invading human respiratory tract, consist of more than 200 viruses belong to Orthomyxoviridae, Paramyxoviridae, Togaviridae, Picornaviridae, Adenoviridae and Herpesviridae. Respiratory viruses transmit through droplet and aerosol, thus easily causing outbreak. There were several pandemic occurred caused by respiratory viruses in history, imposing severe calamity upon human kind. The World Health Organization ranks respiratory tract infection as the second leading cause of death worldwide for children under 5 years of age. Respiratory viruses take account for 90%~95% clinically acute respiratory infection. Array-ELISA is a newly developed technology invented by Beijing GBI Company, which combines classical ELISA and morden microarray techniques. Array-ELISA derives high specificity and high sensitivity from ELISA and high throughput from microarray. Antigen or antibody is spotted on the bottom of each well in 96-well plate forming detection arrays. Signal intensity is amplified by Electrochemiluminescence, collected by CCD, and recorded by specialised software. 30 n L of capture antibody in sufficient for Array-ELISA, which is 1/3000 the volume required by ELISA. Arboviruses and Respiratory viruses are widespread contagious viruses, which cause diseases present the similar clinic symptoms like fever and headache. Diseases caused by these viruses are difficult to diagnose. The rapid and accurate identification of pathogens enables efficient treatment and control of the disease. This research is aimed to establish a multiplex detection method with high specificity and sensitivity targeting 6 arboviruses and 5 respiratory viruses respectively by means of Array-ELISA.Methods: JEV, TBEV, DENV2, DENV4, SINV, EEEV and IAV, IBV, RSV, ADV, PIV3 were cultured in susceptive cells respectively, titrated by TCID50. Monoclonal antibody ascites were produced by inoculating hybridoma cells to Balb/C mice. Purified MAbs were biotinylated and screened through IFA and ELISA. Capture antibody and detection antibody pairs were optimized by ELISA. Capture antibodies were spotted onto the bottom of 96-well plate, forming capture arrays. The concentration of capture antibodies and detection antibodies were optimized. Specificity and stability were validated using cultured viruses. Sensitivity was determined by detecting diluted cultured viruses.Results: 9 MAbs targeting 6 Arboviruses were obtained from their ascites respectively. Specificity and sensitivity of MAbs were examined. After biotinylation, capture antibody and detection antibody pairs have been optimized. 4D5, 2B5, 2B8, 4F9, 1F1 and 4E11 were selected as both capture antibodies and detection antibodies targeting JEV, TBEV, DENV2, DENV4, SINV and EEEV. The concentration of capture antibodies were 0.025mg/m L, 0.025 mg/m L, 0.05 mg/m L, 0.2 mg/m L, 0.05 mg/m L and 0.05 mg/m L respectively, the concentration of detection antibodies were 0.002 mg/m L, 0.001 mg/m L, 0.012 mg/m L, 0.002 mg/m L, 0.006 mg/m L and 0.008 mg/m L respectively. Array-ELISA exhibited excellent specificity and sensitivity, with the detection limits to JEV, TBEV, DENV2, DENV4, SINV and EEEV as 3.1 ×103TCID50/m L、3.1×103 TCID50/m L、3.1×103 TCID50/m L、1.3×104 TCID50/m L、1.3×104 TCID50/m L and 2.5×104 TCID50/m L respectively. Five pairs of antibodies targeting 5 respiratory viruses have been bought from the companies. Capture antibody and detection antibody pairs were combined according to the providers. After biotinylation of detection antibodies, reaction condition was optimized. The concentration of antibody A108、B12、8C5、8C4 and C65467 M were optimized to 0.025 mg/m L, 0.1 mg/m L, 0.1 mg/m L, 0.05 mg/m L and 0.1 mg/m L respectively. The concentration of detection antibody A245、B27、9C5、1E11 and C65329 M were optimized to 0.005 mg/m L、0.0014 mg/m L、0.02 mg/m L、0.02 mg/m L and 0.024 mg/m L respectively. The established Array-ELISA format showed good specificity and sensitivity, with the detection limits to IAV, IBV, RSV, ADV and PIV3 were 3.1×102 TCID50/m L、1.3×103 TCID50/m L、5×103 TCID50/m L、2.5×103 TCID50/m L and 1.5×104 TCID50/m L.Conclusions: Array-ELISA technology targeting 6 arboviruses and 5 respiratory viruses has been established with good detecting specificity and sensitivity.Array-ELISA is a platform posseses high specificity, high sensitivity and high throughput characteristics which provides an alternative method for multiple pathogen detection.