Study of avidity-ELISA: Comparison of chaotropic agents, incubation temperature and affinity maturation after meningococcal immunization

Portilho, Amanda Izeli; Santos, Jessica Silva; Lima, Gabriela Trzewikoswki de; Lima, Gabrielle Gimenes; Gaspari, Elizabeth De

doi:10.1016/j.jim.2022.113387

Cited by 1 publication

(5 citation statements)

References 34 publications

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“…In this sense, aluminum hydroxide as an adjuvant seems to contribute to the presentation of more antigens, the activation of the immune response, and the induction of antibodies with higher binding strength, as observed in previous studies [6,26,33].…”

Section: Immunoblot and Dot-blot For N Meningitidis Omvssupporting

confidence: 64%

“…The sera used in this study was collected 15 days after the 3rd dose [33]. We chose the 3rd dose of the animals' sera because it had a higher antibody titer, and it was expected that the affinity maturation process had already occurred [26].…”

Section: Antigenic Preparations and Serummentioning

confidence: 99%

“…Regarding the main objective of our work, the standardization of the incubation time was proceeded with the sera from the group immunized with OMV+AH, since it was the one that showed the highest antibody titers against OMVs and the highest avidity index with the KSCN chaotropic agent in the ELISA avidity [26,33].…”

Section: Immunoblot and Dot-blot For N Meningitidis Omvsmentioning

confidence: 99%

“…Recently, a study in our laboratory [26] analyzed the effects of using different antigens of Neisseria meningitidis (whole cell suspensions and Outer membrane vesicles), different chaotropic agents (urea and KSCN), and incubation temperatures (4 °C, 25 °C, and 37 °C) to evaluate the avidity of mice serum. However, this study did not evaluate the time of incubation with the chaotropic agents, which we believe is an important parameter.…”

Section: Introductionmentioning

confidence: 99%

“…To accomplish our objectives, we modified the Immunoblot assay into a qualitative in-house Immunoblot avidity assay. We used the potassium thiocyanate (KSCN) 1.5 M as chaotropic agent, like in the modified ELISA [26].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Evaluation of IgG Avidity of Sera from Mice Immunized with RBD of SARS-CoV-2 or OMVs of Neisseria meningitidis by Immunoblot and Dot-Blot

Segati,

Da Costa,

Prudencio

et al. 2023

Preprint

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Several methods can be used to evaluate antibody avidity, one of which is the modified ELISA, in which a chaotropic agent is added to disrupt the binding between the antigen and the antibody. Although the avidity index provides relevant information about the functionality of antibodies, it does not allow us to characterize which antigens the antibodies are strongly bound to. To this end, we adapted the Immunoblot and Dot-Blot techniques by adding potassium thiocyanate (KSCN) after incubating nitrocellulose membranes with serum from mice immunized with Neisseria meningitidis outer membrane vesicles (OMVs) or serum from mice immunized with recombinant receptor-binding domain (RBD) of SARS-CoV-2, with different adjuvants for each antigen. In terms of results, when standardizing the incubation time of sera with KSCN, we observed that 5 minutes was the ideal time for both assays, however, when using different dilutions of serum from mice immunized with RBD, there was an influence on the strength of antigen-antibody binding. For the N. meningitidis assay, the antibodies maintained high avidity for the antigens present in the OMVs, PorA, NadA, and Opa, especially in the presence of the aluminum hydroxide adjuvant. The research showed that standardizing the Immunoblot and Dot-Blot in the same assay optimizes time and resources, allowing qualitative analysis for two different antigens, and resulting in more robust information for immunization studies against these pathogens.

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Section: Immunoblot and Dot-blot For N Meningitidis Omvssupporting

confidence: 64%

Section: Antigenic Preparations and Serummentioning

confidence: 99%

Section: Immunoblot and Dot-blot For N Meningitidis Omvsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Evaluation of IgG Avidity of Sera from Mice Immunized with RBD of SARS-CoV-2 or OMVs of Neisseria meningitidis by Immunoblot and Dot-Blot

Segati,

Da Costa,

Prudencio

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Study of avidity-ELISA: Comparison of chaotropic agents, incubation temperature and affinity maturation after meningococcal immunization

Cited by 1 publication

References 34 publications

Evaluation of IgG Avidity of Sera from Mice Immunized with RBD of SARS-CoV-2 or OMVs of Neisseria meningitidis by Immunoblot and Dot-Blot

Evaluation of IgG Avidity of Sera from Mice Immunized with RBD of SARS-CoV-2 or OMVs of Neisseria meningitidis by Immunoblot and Dot-Blot

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