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Anti-idiotypic antibodies: a new approach in prion research.

Colja Venturini A, Bresjanac M, Vranac T, Koren S, Narat M, Popović M, Curin Serbec V - BMC Immunol. (2009)

Bottom Line: Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems.From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope.The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia. anja.venturini@ztm.si

ABSTRACT

Background: In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPSc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPSc-specific monoclonal antibody we have described previously.

Results: The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking.

Conclusion: The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

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Related in: MedlinePlus

The mice immune responses elicited against different antigens. (A) ELISA: immune sera were tested at 10-fold dilution steps starting from a 1:100 dilution. Pre-immune serum was used as the negative control. Each line represents the mean absorbance of 5 individual mice sera ± standard deviation. The antigens used for the immunizations are listed in the upper right corner. (B) Competition assay: V5B2 was pre-incubated with the serially diluted sera of mice immunized with different forms of V5B2 (listed in the upper right corner) and the V5B2 binding to the P1 peptide was measured by ELISA. Each column represents the mean absorbance of 4 individual mice sera with the highest antibody titres ± standard deviation.
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Figure 2: The mice immune responses elicited against different antigens. (A) ELISA: immune sera were tested at 10-fold dilution steps starting from a 1:100 dilution. Pre-immune serum was used as the negative control. Each line represents the mean absorbance of 5 individual mice sera ± standard deviation. The antigens used for the immunizations are listed in the upper right corner. (B) Competition assay: V5B2 was pre-incubated with the serially diluted sera of mice immunized with different forms of V5B2 (listed in the upper right corner) and the V5B2 binding to the P1 peptide was measured by ELISA. Each column represents the mean absorbance of 4 individual mice sera with the highest antibody titres ± standard deviation.

Mentions: In the second experimental model, syngeneic BALB/c mice were challenged with three different forms of the same IgG1 molecule: the whole V5B2 monoclonal antibody, the Fab fragment of V5B2, and the Fab fragment of V5B2 covalently coupled to the highly immunogenic carrier molecule KLH. Ten days after the 3rd immunization, blood was taken from the tail vein and the sera were tested for specific antibodies by indirect ELISA, and for Ab2 antibodies by competitive ELISA, as shown in Fig 2. The mean OD405 values at a 1:100 dilution were 1.11 ± 0.22, 1.40 ± 0.24 and 1.08 ± 0.3 for sera of mice immunized with V5B2, Fab V5B2 and Fab V5B2-KLH, respectively. The anti-V5B2 sera values declined faster with further dilutions, as compared to the anti-Fab V5B2 and anti-Fab V5B2-KLH sera, for which the OD405values were still 0.68 ± 0.24 and 0.35 ± 0.17, respectively, at a dilution 1:10,000. In agreement with these data, the most potent binding inhibition of V5B2 to the P1 peptide in ELISA was seen with the anti-Fab V5B2 and anti-Fab V5B2-KLH sera. More than 90% inhibition was reached with the anti-Fab V5B2 and anti-Fab V5B2-KLH sera diluted to 1:1,000, and more than 50% inhibition with these sera diluted 1:10,000 and 1:5,000, respectively. Since the highest immune response against Fab V5B2 and the strongest competition of Ab2 antibodies in immune sera were seen in mice immunized with Fab V5B2, the two mice with the highest anti-Fab V5B2 antibody titres were chosen and sacrificed for the cell fusion. In addition, the mouse with the highest anti-Fab V5B2 titre from the group of mice immunized with Fab V5B2-KLH was also selected for the cell fusion.


Anti-idiotypic antibodies: a new approach in prion research.

Colja Venturini A, Bresjanac M, Vranac T, Koren S, Narat M, Popović M, Curin Serbec V - BMC Immunol. (2009)

The mice immune responses elicited against different antigens. (A) ELISA: immune sera were tested at 10-fold dilution steps starting from a 1:100 dilution. Pre-immune serum was used as the negative control. Each line represents the mean absorbance of 5 individual mice sera ± standard deviation. The antigens used for the immunizations are listed in the upper right corner. (B) Competition assay: V5B2 was pre-incubated with the serially diluted sera of mice immunized with different forms of V5B2 (listed in the upper right corner) and the V5B2 binding to the P1 peptide was measured by ELISA. Each column represents the mean absorbance of 4 individual mice sera with the highest antibody titres ± standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666643&req=5

Figure 2: The mice immune responses elicited against different antigens. (A) ELISA: immune sera were tested at 10-fold dilution steps starting from a 1:100 dilution. Pre-immune serum was used as the negative control. Each line represents the mean absorbance of 5 individual mice sera ± standard deviation. The antigens used for the immunizations are listed in the upper right corner. (B) Competition assay: V5B2 was pre-incubated with the serially diluted sera of mice immunized with different forms of V5B2 (listed in the upper right corner) and the V5B2 binding to the P1 peptide was measured by ELISA. Each column represents the mean absorbance of 4 individual mice sera with the highest antibody titres ± standard deviation.
Mentions: In the second experimental model, syngeneic BALB/c mice were challenged with three different forms of the same IgG1 molecule: the whole V5B2 monoclonal antibody, the Fab fragment of V5B2, and the Fab fragment of V5B2 covalently coupled to the highly immunogenic carrier molecule KLH. Ten days after the 3rd immunization, blood was taken from the tail vein and the sera were tested for specific antibodies by indirect ELISA, and for Ab2 antibodies by competitive ELISA, as shown in Fig 2. The mean OD405 values at a 1:100 dilution were 1.11 ± 0.22, 1.40 ± 0.24 and 1.08 ± 0.3 for sera of mice immunized with V5B2, Fab V5B2 and Fab V5B2-KLH, respectively. The anti-V5B2 sera values declined faster with further dilutions, as compared to the anti-Fab V5B2 and anti-Fab V5B2-KLH sera, for which the OD405values were still 0.68 ± 0.24 and 0.35 ± 0.17, respectively, at a dilution 1:10,000. In agreement with these data, the most potent binding inhibition of V5B2 to the P1 peptide in ELISA was seen with the anti-Fab V5B2 and anti-Fab V5B2-KLH sera. More than 90% inhibition was reached with the anti-Fab V5B2 and anti-Fab V5B2-KLH sera diluted to 1:1,000, and more than 50% inhibition with these sera diluted 1:10,000 and 1:5,000, respectively. Since the highest immune response against Fab V5B2 and the strongest competition of Ab2 antibodies in immune sera were seen in mice immunized with Fab V5B2, the two mice with the highest anti-Fab V5B2 antibody titres were chosen and sacrificed for the cell fusion. In addition, the mouse with the highest anti-Fab V5B2 titre from the group of mice immunized with Fab V5B2-KLH was also selected for the cell fusion.

Bottom Line: Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems.From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope.The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia. anja.venturini@ztm.si

ABSTRACT

Background: In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPSc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPSc-specific monoclonal antibody we have described previously.

Results: The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking.

Conclusion: The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

Show MeSH
Related in: MedlinePlus