Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes

Pozsgay, Judit; Szarka, Eszter; Huber, Krisztina; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Sármay, Gabriella

doi:10.1007/978-1-4939-3037-1_17

Cited by 5 publications

(7 citation statements)

References 28 publications

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“…The ACPA content of the acquired serum samples was detected with an indirect ELISA as previously described [ 32 , 46 , 47 ]. Shortly, ELISA plates were pre-coated with 5 μg/mL neutravidine overnight at 4 °C to enhance peptide binding, and then were coated with selected citrulline or arginine-containing biotinylated peptides (see Table 2 for amino acid sequences) at 1 μg/mL concentration for an hour at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

Gyebrovszki

Ács

Szabó

et al. 2022

IJMS

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Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

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Section: Methodsmentioning

confidence: 99%

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

Gyebrovszki

Ács

Szabó

et al. 2022

IJMS

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“…This validation will indicate the potential of the MEV and MPV to elicit the immunogenicity and hence their potential as vaccine candidate can be evaluated (Fig. 6) [75][76][77][78][79].…”

Section: Mevs and Mpvs Allergenicity And Antigenicity Prediction And Physicochemical Analysismentioning

confidence: 98%

“…For dot blot and ELISA-based validations, the standard procedure and earlier mentioned buffers might be optimized (Fig. 6) [75][76][77][78][79]. 1.…”

Section: Molecular Interaction Analysis Of Selected Ctl Epitopes With Tap Transportermentioning

confidence: 99%

“…The constructs are tested for expression in human cell lines, e.g., CHO cells. After the expression trials, protein purification is performed by affinity chromatography and size exclusion chromatography [79,80]. For the purification of the candidate MEV and MPV proteins, the standard affinity and size exclusion chromatography steps mentioned above can be followed or further optimized [79,80].…”

Section: Mevs and Mpvs Allergenicity And Antigenicity Prediction And Physicochemical Analysismentioning

confidence: 99%

“…After the expression trials, protein purification is performed by affinity chromatography and size exclusion chromatography [79,80]. For the purification of the candidate MEV and MPV proteins, the standard affinity and size exclusion chromatography steps mentioned above can be followed or further optimized [79,80]. With the standard dot blot and ELISA-based validation methods, the complex formation tendency of the MEV and MPVs with the patient serum antibodies and with the experimental animal model serum antibodies can be validated.…”

Section: Mevs and Mpvs Allergenicity And Antigenicity Prediction And Physicochemical Analysismentioning

confidence: 99%

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Vaccines Targeting Numerous Coronavirus Antigens, Ensuring Broader Global Population Coverage: Multi-epitope and Multi-patch Vaccines

2021

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Coronaviruses are causative agents of different zoonosis including SARS, MERS, or COVID-19 in humans. The high transmission rate of coronaviruses, the time-consuming development of efficient anti-infectives and vaccines, the possible evolutionary adaptation of the virus to conventional vaccines, and the challenge to cover broad human population worldwide are the major reasons that made it challenging to avoid coronaviruses outbreaks. Although, a plethora of different approaches are being followed to design and develop vaccines against coronaviruses, most of them target subunits, full-length single, or only a very limited number of proteins. Vaccine targeting multiple proteins or even the entire proteome of the coronavirus is yet to come. In the present chapter, we will be discussing multi-epitope vaccine (MEV) and multi-patch vaccine (MPV) approaches to design and develop efficient and sustainably successful strategies against coronaviruses. MEV and MPV utilize highly conserved, potentially immunogenic epitopes and antigenic patches, respectively, and hence they have the potential to target large number of coronavirus proteins or even its entire proteome, allowing us to combat the challenge of its evolutionary adaptation. In addition, the large number of human leukocyte antigen (HLA) alleles targeted by the chosen specific epitopes enables MEV and MPV to cover broader global population.

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Epitope mapping of antibodies in C-reactive protein assay kits by hydrogen-deuterium exchange mass spectrometry explains differential results across kits

et al. 2022

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Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes

Cited by 5 publications

References 28 publications

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis

Vaccines Targeting Numerous Coronavirus Antigens, Ensuring Broader Global Population Coverage: Multi-epitope and Multi-patch Vaccines

Epitope mapping of antibodies in C-reactive protein assay kits by hydrogen-deuterium exchange mass spectrometry explains differential results across kits

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