Limits...
Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display.

Araujo GR, Vaz ER, Fujimura PT, Fonseca JE, de Lima LM, Canhão H, Venturini G, Cardozo KH, Carvalho VM, Napimoga MH, Goulart LR, Gonçalves J, Ueira-Vieira C - Arthritis Res. Ther. (2015)

Bottom Line: The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively.The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. galber.araujo@gmail.com.

ABSTRACT

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis.

Methods: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope.

Results: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.

Conclusion: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.

No MeSH data available.


Related in: MedlinePlus

Perfomance of the mimotopes selected by phage display. a Multiple sequence alignment with all selected mimotopes showing their consensus sequences and frequency. b Reactivity obtained though the interaction of the mimotopes selected and pooled sera from rheumatoid arthritis (RA) patients and health controls (HC). aFrequency is defined as the ratio of the number of phage clones expressing a common peptide sequence to that of the total phage clones obtained in the biopanning
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4493817&req=5

Fig2: Perfomance of the mimotopes selected by phage display. a Multiple sequence alignment with all selected mimotopes showing their consensus sequences and frequency. b Reactivity obtained though the interaction of the mimotopes selected and pooled sera from rheumatoid arthritis (RA) patients and health controls (HC). aFrequency is defined as the ratio of the number of phage clones expressing a common peptide sequence to that of the total phage clones obtained in the biopanning

Mentions: The enrichment of phages recovered after each round of biopanning was determined by the output to input ratio. The increase from 8 × 104 in the first round to 4 × 105 after three rounds of affinity selection showed a clear enrichment of phage particles (Table 2). These data indicate a successful affinity selection of phages that specifically recognized IgG present in sera of DBA1/J mice with acute arthritis. A total of 37 randomly selected mimotopes were obtained after three rounds of biopanning using a phage displayed 12-mer random peptide library. From the 37 mimotopes selected, 14 presented different sequences. Alignment analysis revealed some consensus sequence between the mimotopes selected (Fig. 2a), indicating that these motifs were positively selected during the biopanning. The selected mimotopes showed different reactivities when tested against human sera by phage-ELISA, and all of them were able to discriminate RA patients from HCs. The reactivity values were very similar for all tested mimotopes, except for M12, which showed the highest values of absorbance (Fig. 2b) and the highest difference compared to the other clones (p < 0.05). Due to the higher reactivity compared to the other mimotopes, the common motif shared with another five clones, and the highest ratio of RA:HC, we focused on M12 peptide for further investigation. However, the other mimotopes selected will be explored in future studies.Table 2


Improved serological detection of rheumatoid arthritis: a highly antigenic mimotope of carbonic anhydrase III selected in a murine model by phage display.

Araujo GR, Vaz ER, Fujimura PT, Fonseca JE, de Lima LM, Canhão H, Venturini G, Cardozo KH, Carvalho VM, Napimoga MH, Goulart LR, Gonçalves J, Ueira-Vieira C - Arthritis Res. Ther. (2015)

Perfomance of the mimotopes selected by phage display. a Multiple sequence alignment with all selected mimotopes showing their consensus sequences and frequency. b Reactivity obtained though the interaction of the mimotopes selected and pooled sera from rheumatoid arthritis (RA) patients and health controls (HC). aFrequency is defined as the ratio of the number of phage clones expressing a common peptide sequence to that of the total phage clones obtained in the biopanning
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493817&req=5

Fig2: Perfomance of the mimotopes selected by phage display. a Multiple sequence alignment with all selected mimotopes showing their consensus sequences and frequency. b Reactivity obtained though the interaction of the mimotopes selected and pooled sera from rheumatoid arthritis (RA) patients and health controls (HC). aFrequency is defined as the ratio of the number of phage clones expressing a common peptide sequence to that of the total phage clones obtained in the biopanning
Mentions: The enrichment of phages recovered after each round of biopanning was determined by the output to input ratio. The increase from 8 × 104 in the first round to 4 × 105 after three rounds of affinity selection showed a clear enrichment of phage particles (Table 2). These data indicate a successful affinity selection of phages that specifically recognized IgG present in sera of DBA1/J mice with acute arthritis. A total of 37 randomly selected mimotopes were obtained after three rounds of biopanning using a phage displayed 12-mer random peptide library. From the 37 mimotopes selected, 14 presented different sequences. Alignment analysis revealed some consensus sequence between the mimotopes selected (Fig. 2a), indicating that these motifs were positively selected during the biopanning. The selected mimotopes showed different reactivities when tested against human sera by phage-ELISA, and all of them were able to discriminate RA patients from HCs. The reactivity values were very similar for all tested mimotopes, except for M12, which showed the highest values of absorbance (Fig. 2b) and the highest difference compared to the other clones (p < 0.05). Due to the higher reactivity compared to the other mimotopes, the common motif shared with another five clones, and the highest ratio of RA:HC, we focused on M12 peptide for further investigation. However, the other mimotopes selected will be explored in future studies.Table 2

Bottom Line: The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively.The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlândia, Uberlândia, MG, Brazil. galber.araujo@gmail.com.

ABSTRACT

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects around 1% of the human population worldwide. RA diagnosis can be difficult as there is no definitive test for its detection. Therefore, the aim of this study was to identify biomarkers that could be used for RA diagnosis.

Methods: Sera from a collagen-induced arthritis mouse model were used to select potential biomarkers for RA diagnosis by phage display technology. In silico and in vitro analyses were performed to characterize and validate the selected peptides. Samples were classified into three groups: RA; two other immune-mediated rheumatic diseases (systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS)); and healthy controls (HC). Enzyme-linked immunosorbent assay (ELISA) was carried out to determine antibody levels, and diagnostic parameters were determined by constructing receiver operating characteristic curves. Mass spectrometry and Western blot were performed to identify the putative autoantigen that was mimicked by a highly reactive mimotope.

Results: After three rounds of selection, 14 clones were obtained and tested for immunoreactivity analysis against sera from RA and HC groups. The phage-fused peptide with the highest immunoreactivity (M12) was synthesized, and was able to efficiently discriminate RA patients from SLE, AS and HCs (p < 0.0001) by ELISA. The specificity and sensitivity of anti-M12 antibodies for RA diagnosis were 91 % and 84.3 %, respectively. The M12 peptide was identified as one that mimics a predicted antigenic site of the carbonic anhydrase III (CAIII) protein, a ubiquitous biomarker that has been identified in patients with other diseases.

Conclusion: M12 is the first peptide associated with the CAIII protein that may be used as an antigen for antibody detection to aid in RA diagnosis with high sensitivity and specificity.

No MeSH data available.


Related in: MedlinePlus