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Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines.

ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) resulted in severe systemic diseases with high morbidity and mortality in Siniperca chuatsi. Vaccination is the primary method for effective prevention and control of these diseases. The development of inactivated ISKNV vaccines made some progress, but the technique of quality evaluation is scarce. Herein, a measurement of the MCP (major capsid protein) antigen concentration for the inactivated ISKNV vaccine was developed by double-antibody sandwich ELISA. Firstly, mouse monoclonal antibodies against ISKNV particles and MCP were generated. Then, a double-antibody sandwich ELISA was developed using the monoclonal antibody 1C8 1B9 as the capture antibody and Biotin-3B12 6B3 as the detection antibody. A standard curve was generated using the MCP concentration versus OD value with the linear range of concentration of 4.69~300 ng/mL. The assay sensitivity was 0.9 ng/mL. The antigen content of three batches of inactivated ISKNV vaccines was quantitatively detected using the double-antibody sandwich ELISA. The results showed that MCP antigen contents of inactivated ISKNV vaccines were positively correlated with the viral titers. The newly established double-antibody sandwich ELISA provided a useful tool for the detection of antigen quality for ISKNV inactivated vaccines.

SUBMITTER: Liang H

PROVIDER: S-EPMC8623861 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEnzyme-linked immunosorbent assays (ELISA) are generally the chosen test for Chagas disease (CD) diagnosis; however, its performance depends on the antigen preparation adsorbed to the solid phase, which may lead to false-positive results and cross-reactions. The use of chimeric recombinant antigens can overcome this limitation. Four chimeric antigens from Trypanosoma cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) were developed and evaluated in phase I, II and III studies using indirect ELISA as diagnostic platform. However, peroxidase-labeled secondary anti-human IgG antibody, which is employed in indirect ELISAs, limits its use for the detection of species-specific and class-specific antibodies. To overcome this limitation, peroxidase-labeled antigens can be utilized, diagnosing both acute or chronic infection, in a species and immunoglobulin class-independent manner, through the use of a double-antigen sandwich ELISA (DAgS-ELISA). We aimed to evaluate and validate the diagnostic performance of the chimeric antigens IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 in the DAgS-ELISA platform.Methodology/principal findingsDAgS-ELISA was optimized by checkerboard titration. In phase I study, 207 positive and 205 negative samples were evaluated. Cross-reactivity to other infections was also assessed using 68 samples. The selected conditions for the tests utilized 25 ng of antigen per well and the conjugate diluted at 1:2,000 for all molecules. In the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with HTLV.Conclusions/significanceDAgS-ELISA is a promising tool for immunodiagnosis, and despite the high AUC values, the performance of this assay was different from the values obtained by our group when using these antigens in the indirect ELISA, for this reason, improvements are being considered to increase the sensitivity of the DAgS-ELISA.

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Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines.

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