| Virus is the most important lethal pathogen that obstructs the development of current aquaculture severely. As there is no effective drug for treatment of viral diseases, detecting virus pathogen at early stage in high efficiency and veracity is no doubt conducive to industry. Hence a novel method for parallel detection of multiple samples or pathogens in a convenient and simple way is urgently needed. Antibody-based microarray, combining the specificity of antigen-antibody reaction and high-density integration of microarray, is a novel proteomic technology that can meet the requirements which is a powerful tool for parallel detection of multiple parameters. It has the advantages of strong specificity, high sensitivity, simple sample handling, and high through-put analysis with minimal sample consumption. The antibody microarray has shown vast prospect on the detection of virus or biomarkers related to cancers in medicine. However, to our best knowledge, no research on protein microarray for pathogen detection of aquatic animals has been reported. In this paper, we developed the antibody microarray system by combining the monoclonal antibody technology, immune markers and antibody microarray technology. Taking white spot syndrome virus (WSSV) and lymphocystis disease virus (LCDV) as model virus, we prepared WSSV/LCDV antibody microarray and optimized the procedure conditions at multiple samples for microarray application. Also we investigated the sensitivity, specificity, accuracy of WSSV/LCDV antibody microarray and utilized the microarrays to detect WSSV/LCDV. Details are as follows.1. Nine kinds of supports (Poly-L-lysine, MPTS, aldehyde, APES, amine, polyacrylamide gel and agarose gel modified slides, Nitrocellulose membrane, PVDF membrane) were compared in the efficiency of immobilizing proteins. The results showed that spots on 1.2% agarose gel-modified slides were round and homogeneous without diffusion tailing on the verge, showing superior size and clear pattern, providing the highest signal value. These characters indicated the highest capacity of protein immobilization. The slides were characterized by atomic force microscope (AFM) and the surface of 1.2% agarose gel-modified slides displayed 3-dimensional structure with many holes and great thickness. Measurement with Nanoscope 5.12 software revealed that the mean roughness of agarose gel was 18.6 nm, which was much greater than other slides (1.45-4.9 nm). These results indicated that rougher mesoporous agarose gel surfaces with aldehyde functions might have higher latent capacity of protein adsorbing and covalent linking in their natural states. Taken together, slides modified with 1.2% agarose gel were chosen as appropriate supports for microarrays.2. Sandwich immunoassay was adopted to develop the antibody microarray for a higher sensitivity and specification. Purified WSSV/LCDV particles were used to immunize New Zealand white rabbits and rabbit anti-WSSV/LCDV antibodies were obtained. After purification we got polyclonal antibodies with high activity and titer. The titers of rabbit anti-WSSV antibody and anti-LCDV antibody were 1:64000 and 1:32000, respectively.Four anti-WSSV monoclonal antibodies (MAbs) (2E6, 2A3, 4G9 and 2D11) and four anti-LCDV MAbs (3G3, 2B6, 1D7 and 2D11), developed previously in our laboratory, were produced in ascites by injection the hybridoma clone into the peritoneal cavity of Balb/c mice individually, and purified with the Ampure PA kit as per the manufacturer’s protocol. Then these MAbs were mixed with equal proportion respectively and labeled with Cy3 according to the manufacturers’instructions. Cy3-conjugated anti-WSSV MAbs or anti-LCDV MAbs was 2400-fold or 3000-fold diluted and used as detection antibody.3. Rabbit anti-WSSV or anti-LCDV polyclonal antibody was diluted and arrayed as capture antibody of the microarray on the agarose gel-modified slides. After immobilization and blocking, the microarray slides were incubated with virus diluents and the antibody-antigen complex was detected by specific Cy3-conjugated anti-WSSV or anti-LCDV MAbs. The results were measured by a laser chipscanner and analyzed with Lab-chipscanner 2.0. To obtain satisfied fluorescence signal intensity, optimal conditions in printing buffer, capture antibody concentration, immobilization, blocking, washing, incubating time and markers were searched. The results illustrated that the optimum conditions were as follows. Rabbit anti-WSSV antibody was diluted to 0.1 mg/ml and rabbit anti-WSSV antibody was 0.5 mg/ml with PBS containing 50% glycerol as printing buffer. After arraying, the microarrays were put in a humid chamber at 37°C for 2 h to immobilize the antibody and then blocked by 3% BSA at 37°C for 1 h. The antibody microarray was washed by rinsing with dH2O, PBST, PBST in sequence for 5 min each and dried by centrifuging, then sealed at low temperature. Incubation was performed for 15~30 min at 37°C saturated humidity, and incubation longer than 45 min would cause a higher background value.4. We investigated influence of the supports, markers (Cy3, Horseradish peroxidase, FITC, colloidal gold and biotin) and storage on antibody microarray sensitivity. The results displayed that the sensitivities of antibody microarray based on agarose gel modified slides with Cy3/HRP conjugated anti-WSSV or anti-LCDV MAbs as detection antibody were 0.31μg/ml for WSSV and 0.55μg/ml for LCDV, which was higher than the ones prepared by other supports or markers. In WSSV concentration range from 0.62 to 9.9μg/ml, LCDV range from 0.55-17.56μg/ml, signal value and logarithmic virus concentration showed a good linear relationship. The correlation coefficients were 0.98925 and 0.9900 respectively. The sensitivity of antibody microarray can be improved to 25 ng/ml or WSSV and 70 ng/ml for LCDV by biotin-streptavidin system to amplify signal value.The signal value of antibody microarray decreased due to the apparent rising of background after 4 months storage at -20°C by using Cy3-conjugated anti-WSSV or anti-LCDV MAbs as detection antibody, which resulted in the sensitivity decrease. When HRP-conjugated anti-WSSV or anti-LCDV MAbs was used as detection antibody, the sensitivity of antibody microarray was still 0.15μg/ml for WSSV while the sensitivity declined to 1.1μg/ml for LCDV after 12-month storage at -20°C.5. Antibody microarrays were applied in samples of diseased shrimp or fish, and results of the antibody microarray with enzyme linked immunosor-bent assay (ELISA) for WSSV/LCDV detection were in 100% concordance and the correlation coefficient (r) was 0.9853/0.9803. These results demonstrate that the antibody microarray can detect the pathogen accurately.6. For the requirements of on-spot detection of aquatic animals’virus, we developed the antibody microarray combining the advantages of the specificity of ELISA, sensitivity and high-throughput of microarray with HRP-conjugated anti-WSSV or anti-LCDV MAbs as detection antibody. The detection results can be read by naked eyes and the detection can be performed on-field conveniently. Detection results of antibody microarray showed 100% concordance with ELISA and >98% concordance with indirect immunofluorescence assay technique (IIFA). In a certain concentration range, signal value and logarithmic virus concentration showed a good linear relationship with which relative quantitative detection of the virus could be performed. The correlation coefficients were 0.9505 and 0.9567 respectively. These merits make it practical in diagnostic and epidemiological studies on WSSV in shrimp or LCDV in fish aquaculture and potentially in other sea animals, in a high efficiency manner.The preparation of antibody microarray system can expand to other aquatic animal virus detection. The antibody microarrays developed for WSSV/LCDV detection can detect multiple samples simultaneously and conveniently. The detection operation with antibody microarray is simple, fast, convenient, accurate, and easy up-scaling to high-throughout at low sample consumption and thus low cost. No expensive equipment was necessary. The antibody microarray provides an effective platform for aquatic animals’pathogen detection and has extensive prospect in disease surveillance and epidemiological studies in aquaculture especially during quarantine inspection on import/export of aquatic goods. |
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