Development Of A Low-cost Rapid Peptide-based Chemiluminescence Enzyme Immunoassay (CLEIA) For Diagnosis Of Dengue Virus Infection In Human

Dengue virus(DENV)is a mosquito-borne virus that is widely spread in the world.It is mainly transmitted by Aedes aegypti and Aedes albopictus and can cause dengue fever,dengue haemorrhagic fever and dengue shock syndrome.Thus,DENV pose a huge threat to public health.Currently,there are no DENV vaccines and specific drugs for DENV infections available.Timely and accurate diagnosis is essential for the treatment of DENV infections.At present,the general for detecting DENV infections are PCR and serum methods.Although the PCR method has the advantages of high sensitivity and high specificity,it requires high requirements on experimental conditions and the rate of detection drop rapidly with time.The serum method not only has high sensitivity and specificity,but also has the advantages of easy to use,longer detectable time period,and lower requirements on experimental conditions,thus,compared with PCR,its clinical application is more common.However,due to the current serum method generally uses the DENV recombinant protein with higher cost to detect the antibody in the serum,the detection cost is higher,which limits its use in regions with poor economic conditions..Therefore,this experiment attempts to establish a quick and accurate method that can diagnose DENV at low cost.DENV is mainly composed of nucleoprotein,membrane protein,envelope protein and seven kinds of non-structural proteins.Among them,the envelope protein contains dengue virus-specific antigenic epitopes and antigenic epitopes that can induce protective neutralizing antibodies in the body.Therefore,the screening and detection of specific antigenic epitopes in DENY envelope proteins is crucial for reducing false positive reactions in the diagnosis of DENV infections and the development of vaccines.In order to screen specific epitopes on envelope proteins for use as coating antigens,the surface accessibility,hydrophilicity,antigenicity,plasticity,and secondary structure parameters of the DENV envelope proteins were analyzed using bioinformatics tools.Based on these analysis results,ten potential specific peptides were selected as candidates,namely E1-E10.Then,in this experiment,two peptides with relatively good specificity and sensitivity,namely El and E7,were screened out of the 10 polypeptides to establish the CLEIA method.In order to allow the two peptides to exhibit the best sensitivity and specificity,this experiment optimized the experimental conditions of the CLEIA method.The optimized experimental conditions were:the concentration of the coating antigen was 10 ?g/mL,and incubated at 4? overnight;the blocking solution was incubated with 3%BSA in PBS and incubated at 37 for 2h;The dilution of serum is 1:400,incubated at 37? for 30 min;the concentration of enzyme-conjugated secondary antibody is 1/160000,incubated at 37? for 30 min.In order to determine the cut-off value,sensitivity and specificity of the established CLEIA method,a total of 176 clinical specimens were collected in this study.Among them,120 specimens showed positive for anti-DENV IgM antibody,56 negative specimens showed negative for anti-DENV IgM antibody.These samples included serum from 10 patients with autoimmune disease and 5 patients with malaria.Tests showed that when using E1 as the coating antigen of CLEIA method,the sensitivity and specificity were 82.5%and 94.6%,respectively;when using E7 as the coating antigen of CLEIA method,the sensitivity and specificity were 79.2%and 92.9%,respectively.When E1 and E7 were mixed as the coating antigen,the sensitivity and specificity were 85.0%and 96.4%,respectively.Our experimental results show that the envelope proteins E1 and E7 have better sensitivity and specificity and can better distinguish dengue virus infection from malaria infection and autoimmune diseases.Combining the specific low-cost,stable and easy mass production characteristics of peptides and the high sensitivity of CLEIA method,this method has a good application prospect in the diagnosis of dengue virus infection.In addition,our experiment also give a reference for the research of dengue virus vaccine.