Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: SLA-DR-presented NSP immunopeptides evoke antibody response in vivo. (A) cDNAs of PRRSV NSP immunopeptides from PRRSV strain JXA1 were cloned into the pCAGEN vector and fused with NanoLuc luciferase, followed by transfection of HEK-293T cells for transient expression. Cell lysates from 293T cells with transfection of indicated plasmids were harvested and subjected to Western blotting using rabbit anti-NanoLuc polyclonal antibodies to confirm the expression of NanoLuc-fused NSP immunopeptides. (B) Serum samples collected at 21 days postinoculation (dpi) from PRRSV JXA1-infected pigs (n = 6) and noninfected piglets (n = 6) were subjected to luciferase-linked antibody capture assay (LACA) using NanoLuc luciferase fused with indicated immunopeptides from different NSPs to identify corresponding antibody levels as determined by sample-to-negative (S/N) ratio. (C) Amounts of 1 μg of indicated artificial immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 21-dpi serum from PRRSV JXA1-infected pigs (n = 6) or noninfected piglets (n = 6) (1-to-200 dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. (D) Amounts of 400 ng of indicated KLH-conjugated artificial immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 21-dpi serum from PRRSV JXA1-infected pigs (n = 6) or noninfected piglets (n = 6) (1 to 200 dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. (E) Serum samples collected at 21 dpi from PRRSV SD16- and PRRSV NADC30-like strain-infected pigs (n = 5 for each) and noninfected piglets (n = 5) were subjected to LACA using NanoLuc luciferase fused with indicated immunopeptides from different NSPs (JXA1 strain) and evaluated by S/N ratio. OD 450, optical density at 450 nm. All data are expressed as mean values ± standard deviations and were subjected to Student’s t test. Error bars show standard deviations. Significant differences between indicated groups are marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: In Vivo, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Expressing, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Indirect ELISA

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: SLA-DR-mediated antigen presentation of PRRSV NSPs evokes a nonneutralizing antibody response in vivo. (A) Swine serum (28 dpi) from PRRSV JXA1-infected swine or noninfected swine was used to incubate Sepharose 4B resin conjugated with mixed NSP immunopeptides listed in Table 1, except NSP7α, for 2 h at RT, followed by washing resin with PBS. Next, immunopeptide-specific antibodies were eluted and subjected to SDS-PAGE, and the existence of immunopeptide-specific antibodies was visualized using HRP-labeled goat anti-swine IgG. (B) MARC-145 cells were infected by PRRSV JXA1 at an MOI of 1 for 24 h. After cells were fixed and permeabilized, PRRSV-infected cells were costained by anti-PRRSV N protein MAb PP7EF11 and purified PRRSV NSP immunopeptide (IM-Pep) antibodies, followed by visualization via Alexa Fluor 555-labeled goat anti-mouse IgG(H+L) (red channel) and FITC-labeled goat anti-swine IgG (green channel). (C) MARC-145 cells were infected by PRRSV JXA1 at an MOI of 1 for 24 h and harvested for SDS-PAGE. PVDF membrane was blotted using purified PRRSV NSP immunopeptide swine antibodies or anti-PRRSV N MAb 6D10, followed by visualization via HRP-labeled goat anti-mouse IgG(H+L) or goat anti-swine IgG. (D) PRRSV JXA1 (MOI of 0.1) was preincubated with indicated doses of purified PRRSV NSP immunopeptide swine antibodies at 37°C for 1 h and then used to inoculate MARC-145 cells for 4 h. After removing antibodies and unbound virions by washing cells with PBS, virus-infected cells were incubated at 37°C for another 14 h. Replication of PRRSV was visualized by IFA using anti-PRRSV N MAb PP7EF11 for counting of fluorescent spots. All data are expressed as mean values ± standard deviations from at least three independent experiments.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: In Vivo, Infection, SDS Page, Labeling, Purification, Incubation

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

doi: 10.1128/JVI.01423-20

Figure Lengend Snippet: PRRSV structural protein-derived immunopeptides could be recognized by PRRSV hyperimmune serum but not early serum samples from PRRSV-infected pigs. (A) cDNAs of PRRSV structural protein (SP) immunopeptides from PRRSV strain JXA1 were synthesized and ligated into the pET-28a-His-NanoLuc expression vector to fuse with NanoLuc luciferase, followed by expression in E. coli cells. After dialysis and purification, 10 μg of each recombinant protein was subjected to SDS-PAGE and Western blot analysis using rabbit anti-NanoLuc polyclonal antibodies to confirm the expression. (B) Amounts of 200 ng of indicated NanoLuc-fused immunopeptides were used for coating ELISA plates. After blocking with 5% skim milk, indirect ELISA was conducted using 14-dpi or 21-dpi serum from PRRSV JXA1-infected pigs (n = 4) or PRRSV hyperimmune serum samples (n = 4) (20-fold dilution in PBS), along with visualization by HRP-conjugated goat anti-swine IgG and TMB substrate. OD 450, optical density at 450 nm. All data are expressed as mean values ± standard deviations and were subjected to Student’s t test. Significant differences between indicated groups are marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: Serum samples of immunized mice were evaluated by using the Idexx PRRS X3 enzyme-linked immunosorbent assay (ELISA) kit by replacing the original horseradish peroxidase (HRP)-labeled anti-swine antibodies with HRP-labeled anti-mouse IgG (Thermo Fisher Scientific) in order to determine the serum conversion as previously described ( 68 ).

Techniques: Derivative Assay, Infection, Synthesized, Expressing, Plasmid Preparation, Luciferase, Purification, Recombinant, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Blocking Assay, Indirect ELISA

Journal: Frontiers in Veterinary Science

Article Title: Comparative field efficacy of all-in-one intradermal vaccine based on an inactivated recombinant Mycoplasma hyopneumoniae with an embedded/integrated porcine circovirus type 2 (PCV2) capsid protein and concurrent administration with monovalent of M. hyopneumoniae and PCV2 intradermal vaccine

doi: 10.3389/fvets.2025.1428263

Figure Lengend Snippet: Porcine circovirus type 2 (PCV2) antibodies. (A) Mean values of the ELISA PCV2 antibodies in the farm A. (B) Mean values of the ELISA PCV2 antibodies in the farm B. (C) Mean values of the ELISA PCV2 antibodies in the farm C. Variation is expressed as the standard deviation. Different letters within a sampling point mean statistically significant differences ( p < 0.05).

Article Snippet: The presence of PCV2 and M. hyopneumoniae antibodies was evaluated in serum samples by use of commercially available enzyme-linked immunosorbent assay (ELISA) kits (Porcine Circovirus type 2 Antibody Test, BioChek B.V., Reeuwijk, Holland and M. hyo Ab test, IDEXX Laboratories Inc. Westbrook, Maine, United States) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Sampling

Journal: Frontiers in Veterinary Science

Article Title: Comparative field efficacy of all-in-one intradermal vaccine based on an inactivated recombinant Mycoplasma hyopneumoniae with an embedded/integrated porcine circovirus type 2 (PCV2) capsid protein and concurrent administration with monovalent of M. hyopneumoniae and PCV2 intradermal vaccine

doi: 10.3389/fvets.2025.1428263

Figure Lengend Snippet: Mycoplasma hyopneumoniae antibodies. (A) Mean values of the ELISA M. hyopneumoniae antibodies in the farm A. (B) Mean values of the ELISA M. hyopneumoniae antibodies in the farm B. (C) Mean values of the ELISA M. hyopneumoniae antibodies in the farm C. Variation is expressed as the standard deviation. Different letters within a sampling point mean statistically significant differences ( p < 0.05).

Article Snippet: The presence of PCV2 and M. hyopneumoniae antibodies was evaluated in serum samples by use of commercially available enzyme-linked immunosorbent assay (ELISA) kits (Porcine Circovirus type 2 Antibody Test, BioChek B.V., Reeuwijk, Holland and M. hyo Ab test, IDEXX Laboratories Inc. Westbrook, Maine, United States) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Sampling

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: Well-established CSF diagnostic methods and their application.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Diagnostic Assay, Virus, Isolation, Infection, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: List of representatives of commercially available CSF antibody ELISA kits/reagents.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, Virus, Blocking Assay, Clinical Proteomics, Competitive ELISA, Immunochromatographic Assay, Stripping Membranes

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: Well-established CSF diagnostic methods and their application.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Diagnostic Assay, Virus, Isolation, Infection, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: List of representatives of commercially available CSF antibody ELISA kits/reagents.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, Virus, Blocking Assay, Clinical Proteomics, Competitive ELISA, Immunochromatographic Assay, Stripping Membranes

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: Well-established CSF diagnostic methods and their application.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Diagnostic Assay, Virus, Isolation, Infection, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: List of representatives of commercially available CSF antigen-capture ELISA kits/reagents.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Pathogens

Article Title: Recent Advances in the Diagnosis of Classical Swine Fever and Future Perspectives

doi: 10.3390/pathogens9080658

Figure Lengend Snippet: List of representatives of commercially available CSF antibody ELISA kits/reagents.

Article Snippet: Several commercial CSF antibody ELISA kits are available including those from Biocheck, Boehringer Ingelheim, Cusabio Technology LLC, IDEXX Laboratories, ID VET, Indical Bioscience, iNtRON Biotechnology, Median Diagnostics Inc., Thermo Fisher Scientific, and so on.

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, Virus, Blocking Assay, Clinical Proteomics, Competitive ELISA, Immunochromatographic Assay, Stripping Membranes

Journal: Journal of Clinical Microbiology

Article Title: Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions

doi: 10.1128/JCM.00485-20

Figure Lengend Snippet: Misclassification errors ^a (count) for six Mycoplasma hyopneumoniae antibody ELISAs by day postinoculation in study 1

Article Snippet: Three assays were indirect ELISAs based on the detection of anti-P46 antibodies (SK108 Mhyo, BioChek, Berkshire, UK; M. hyo Ab test, IDEXX Laboratories Inc., Westbrook, ME, USA; and ID Screen Mycoplasma hyopneumoniae Indirect, IDvet, Grabels, France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Microbiology

Article Title: Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions

doi: 10.1128/JCM.00485-20

Figure Lengend Snippet: M. hyopneumoniae DNA and antibody detection by day postinoculation (dpi) in a room housing 1,250 pigs in 46 pens (23 white rectangles by 2 columns) in study 2. Ten pigs in a centrally located pen (yellow rectangle) were intratracheally administered with M. hyopneumoniae (strain 232). Thereafter, one noninoculated pig per pen was sampled/tested at each time point. M. hyopneumoniae DNA and antibody-positive results from noninoculated pigs were represented by “+” for positive PCR in tracheal samples and by squares colored by one of each the three ELISAs (ELISA 1, SK108 Mhyo, BioChek, Berkshire, UK; ELISA 3, M. hyo Ab test, IDEXX Laboratories Inc., Westbrook, ME, USA; ELISA 5, Civtest Suis Mhyo, Laboratorios Hipra, S.A., Girona, Spain) for positive result in serum samples. Results were based on PCR tracheal samples (15 and 28 dpi) and blood samples (15, 28, 42, and 56 dpi) collected from one noninoculated pig at each time point. In the remaining pens, tracheal and blood samples were collected from one pig at 15, 24, 42, 56, 71, 85, and 98 dpi. Serum samples with suspect classification were assumed positive. NS, no sample collected.

Article Snippet: Three assays were indirect ELISAs based on the detection of anti-P46 antibodies (SK108 Mhyo, BioChek, Berkshire, UK; M. hyo Ab test, IDEXX Laboratories Inc., Westbrook, ME, USA; and ID Screen Mycoplasma hyopneumoniae Indirect, IDvet, Grabels, France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Microbiology

Article Title: Performance of Commercial Mycoplasma hyopneumoniae Serum Enzyme-Linked Immunosorbent Assays under Experimental and Field Conditions

doi: 10.1128/JCM.00485-20

Figure Lengend Snippet: Diagnostic sensitivity and specificity ^a of three M. hyopneumoniae antibody ELISAs in study 2

Article Snippet: Three assays were indirect ELISAs based on the detection of anti-P46 antibodies (SK108 Mhyo, BioChek, Berkshire, UK; M. hyo Ab test, IDEXX Laboratories Inc., Westbrook, ME, USA; and ID Screen Mycoplasma hyopneumoniae Indirect, IDvet, Grabels, France).

Techniques: Diagnostic Assay, Enzyme-linked Immunosorbent Assay