Establishment And Application Of White Spot Syndrome Virus(WSSV) Sandwich ELISA

White spot syndrome is destructive and infectious desease in shrimp culturecaused by white spot syndrome virus (WSSV), which caused nearly100%mortalityof WSSV-infected shrimp in3~10days. There are no effective methods to cure thisdesease right now, so early detection of WSSV in infected shrimp is very importantthat effective measures can be taken immediately. Around shrimp farm immediatelycan be warned to prevent WSSV spreading if WSSV detected in one shrimp farm.Sandwich enzyme linked immune response (s-ELISA) is very common inpathogens detection. s-ELISA is proved with high specificity, sensitivity andreproducibility. A s-ELISA was described that anti-WSSV serum was as the caputureantibody and anti-WSSV MAbs was as the detection antibody. We generated asigmoidal standard curve in a linear fit via the s-ELISA to measure the serially dilutedpurified WSSV solution. The experimental details will be described as following.（1）Preparation of anti-WSSV rabbit serum. Gills of moribund WSSV-infectedpenaeid shrimp were collected and homogenated in ice-bath. After filtering, thehomogenate subjected differential centrifugation and stepwise sucrose gradientultracentrifugation, purified WSSV were obtained. A big amount of intact WSSV viralparticles coated with integral envelopes were observed by a transmission electronmicroscope (TE). The concentration of purified WSSV was determined as384μg/mL.A2.5kg female New Zealand white rabbit was obtained from Animal researchcenter of Medical College of Shandong University. The rabbit was injectedsubcutaneously with500μg purified WSSV in a1:1ratio with Freund’s CompleteAdjuvant (Sigma) divided between four sites (500μL/site). Two weeks later, therabbit was boosted the first time with500μg purified WSSV in a1:1ratio withFreund’s Incomplete Adjuvant (Sigma) divided between four sites. The rabbit was boosted another two times with500μg purified WSSV at one-week interval.50mLblood was collected and23mL antiserum after recovered from the clot andcentrifuged at2500rpm for5min at4°C to pellet any residual cells.（2）Preparation and purification of MAbs ascitic fluid. WSSV hybridomas2E6and2A3were recovered and cultured in wells of cell cultrue cluster for a week.Hybridomas were elutriated softly from the wells with RPMI1640culture mediumand collected into10mL centrifugal tubes, following by being centrifugated at1000rpm for3min. The hybridomas were elutriated again with RPMI1640culturemedium. After the cell pellet being resuspended with a little RPMI1640culturemedium, the concentrated cells solution (about106/mL) was intraperitoneal injectedinto BALB/c mouse. Ascitic fluid was collected immediately as the abdomen ofBALB/c mouse became very big. Ascitic fluid of MAbs was purified via caprylicacid-ammonium method. The concentration of purified MAbs2E6and2A3were10mg/mL and5mg/mL respectively. SDS-PAGE and Western-blot results proved2E6and2A3were with high purity and concentration and both of two MAbs were IgG.2E6and2A3was proved with high-specificity by indirect immunofluorescence assay.（3）Standard curve of sandwich ELISA. In our optimal WSSV sandwich ELISA,enzyme labeled plate was coated with anti-WSSV serum as the capture antibody, thenserially diluted from7.5to30720ng/mL was added into wells of the plate, thenanti-WSSV MAbs2E6and2A3in a1:1ratio was added into wells as the detectionantibody. We generated a sigmoidal standard curve in a4-parameter logistic fit in aconcentration range of7.5to30720ng/mL, the minimum limit of detection of WSSVsandwich ELISA is120ng/mL. And we generated a linear standard curve in a linearfit in a concentration range of120~7680ng/mL with R2=0.992, the detection limit is60ng/mL. The linear equation is expressed as: y=0.1664x－0.1504; y is the meanOD405of detected samples, x can be caculated input value of y in the linear equation.The recovery of the optimised sandwich ELISA was between0.8483to1.134,Relative error (RE) is between-15.2to13.4(Table2). Intra-assay reproducibility wasdetermined with coefficient of variation (CV) in a range of2.34~7.11%(Table.3),inter-assay reproducibility in a range of2.18~6.9% （4）Application of linear fit standard curve.200healthy WSSV-free crayfisheswere cultured for one week as the temperature of water rised to25°C gradually.Divided crayfishes into two group: WSSV-infected group and WSSV-free group asnegtive control.Each crayfish in WSSV-infected group was fed with WSSV-infectedbaits, while the negtive control group was fed with WSSV-free baits.8crayfishes inboth groups were collected12hours interval in84hours. Gills, hepatopancreas, hearts,gonads, intestine, muscle were collected and homogenized for5min in a1:2(m/V)ratio with0.01mol/L PBS (haemolymph in a1:1ratio with anticoagulant), thencentrifugated at4000rpm for10min at4°C,the supernatant was preserved at-80°Cpreparing for measurement of sandwich ELISA.We measured the quantity of WSSV in these tissues of WSSV-infected crayfishesat different time point. The earlist time to detect WSSV-positive in theWSSV-susceptible tissues (hemolymph, gut and gonad) was at24h, while in thenon-susceptible tissues (gill, hepatopancreas, heart and muscle) at36h. The infectionprocess could be divided into three stages: the early stage (0~36h) virus proliferedfast and no mortality appeared; the medium stage (36~72h) the proliferation rateslowed down obviously and a low level mortality was observed; the later stage whenWSSV propagation went into the plateau phage and massive mortality happened. Inthe infection process, the most favorite replication tissue of WSSV is gut and itreached the maximum value of6,220ng/mL at72h. This article provided importantdata for WSSV infection study.In summary, a linear WSSV standard curve was generated via an optimisedsandwich ELISA method with a detection concentration range of120~7680ng/mL.The specificity, sensitivity and repeatability of the sandwich ELISA is very well.The detection limit of sandwich ELISA compared to real-time PCR or may notallow for the very early detection of infected animals. However, real-time PCR is avery expensive and high-tech professional method, which may not be feasible formany labraries Those labraries can use an sandwich ELISA, a simple and efficientmethod which would satisfy common detection and qualitify antigens. In someresearches, The detection limit of sandwich ELISA can be optimized to5.12ng/mL.The sensitivity of sandwich ELISA still could be improved as the method and techniques to be optimized in the future.