VLP-based indirect ELISA for AKAV antibody detection

Stages

1. 1.
   Chimeric VLP design and production(library_build)

   To generate the engineered VLP that serves as the central immunogen and diagnostic antigen in the study.

   Selection: Incorporation of the neutralizing epitope 1134SVQSFDGKL1142 into a recombinant HBcAg scaffold to produce a novel VLP.

2. 2.
   VLP verification(confirmatory_validation)

   To confirm that the engineered VLP expressing the AKAV epitope was successfully constructed before biological evaluation.

   Selection: Confirmation of successful VLP construction by SDS-PAGE, Western blot, and TEM.

3. 3.
   Mouse immunogenicity evaluation(functional_characterization)

   To determine whether the chimeric VLP elicits antibodies against the AKAV Gc antigen.

   Selection: Detection of antibody titer targeting the AKAV Gc antigen in sera from VLP-immunized mice.

4. 4.
   In vitro neutralization and replication inhibition testing(secondary_characterization)

   To test whether the induced antibodies are functionally antiviral rather than only antigen-binding.

   Selection: Neutralizing activity against AKAV and inhibition of viral replication in BHK-21 cells by antisera from VLP-immunized mice.

5. 5.
   Indirect ELISA establishment and preliminary diagnostic testing(confirmatory_validation)

   To evaluate whether the same engineered VLP can serve as an effective coating antigen for AKAV serological detection.

   Selection: Ability of the VLP-based indirect ELISA to identify AKAV antibody-positive serum with reported sensitivity up to 1:1600 serum dilution.

Steps

1. 1.
   Insert the conserved AKAV Gc neutralizing epitope into the recombinant HBcAg scaffold to produce a chimeric VLPengineered construct

   Create a VLP displaying the conserved neutralizing AKAV epitope.

   The engineered VLP must be created before it can be verified, used for immunization, or applied as an ELISA antigen.

2. 2.
   Verify successful VLP construction by SDS-PAGE, Western blot, and TEMvalidated construct

   Confirm that the engineered VLP expressing the AKAV epitope was successfully constructed.

   Verification is performed before biological testing to ensure the intended particle was obtained.

3. 3.
   Immunize mice with the verified VLP and measure antibody responses against AKAV Gcimmunogen

   Assess whether the VLP is immunogenic and induces AKAV Gc-specific antibodies.

   Immunogenicity testing follows construct verification and precedes functional neutralization testing because antibody generation is required for serum-based antiviral assays.

4. 4.
   Test antisera from VLP-immunized mice for AKAV neutralization and replication inhibition in BHK-21 cells

   Determine whether the induced antibodies have functional antiviral activity.

   This step follows antibody generation because neutralization testing requires antisera and provides a higher-fidelity functional readout than antigen-binding alone.

5. 5.
   Use the chimeric VLP as coating antigen to establish a preliminary indirect ELISA for AKAV detectiondiagnostic antigen and assay method

   Evaluate whether the engineered VLP can function as a coating antigen for serological detection of AKAV antibodies.

   After the VLP is shown to be successfully constructed and immunologically relevant, the same antigen is tested for diagnostic utility.