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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

Anti-M12 antibody detection by ELISA. Detection of anti-M12 antibodies in sera from rheumatoid arthritis (RA) patients (n = 172), healthy controls (HC) (n = 113), systemic lupus erythematosus (SLE) patients (n = 19) and ankylosing spondylitis (AS) patients (n = 13) and the respectively receiver operating characteristic (ROC) curve. a Sera from the four groups individually tested for their ability to bind to synthetic M12 peptide. Horizontal line = cut-off value; error bars show mean and standard deviation. b ROC curve constructed based on the control groups. The area under the curve (AUC), sensitivity (Se), specificity (Sp) and corresponding p-value are indicated inside the graph
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Fig6: Anti-M12 antibody detection by ELISA. Detection of anti-M12 antibodies in sera from rheumatoid arthritis (RA) patients (n = 172), healthy controls (HC) (n = 113), systemic lupus erythematosus (SLE) patients (n = 19) and ankylosing spondylitis (AS) patients (n = 13) and the respectively receiver operating characteristic (ROC) curve. a Sera from the four groups individually tested for their ability to bind to synthetic M12 peptide. Horizontal line = cut-off value; error bars show mean and standard deviation. b ROC curve constructed based on the control groups. The area under the curve (AUC), sensitivity (Se), specificity (Sp) and corresponding p-value are indicated inside the graph

Mentions: The M12 synthetic peptide was tested by ELISA with individual sera obtained from 172 RA patients, 113 HCs, 19 SLE patients and 13 AS patients to evaluate its potential as a diagnostic. The cut-off point was determined as the value of the parameter corresponding to the highest sensitivity without lowering the specificity. Of 172 RA patients, 145 (84.3 %) were positive to anti-M12 antibodies. The synthetic molecule was able to efficiently discriminate sera from RA patients and HCs (p < 0.0001), SLE (p < 0.0001) and AS (p < 0.0001) (Fig. 6a). The ROC curve analysis constructed based on the control groups was significant (p < 0.0001; AUC = 0.946). Based on the determined cut-off point, M12 synthetic peptide presented a specificity of 91 % and sensitivity of 84.3 % (Fig. 6b). For humoral immune response evaluation, 55 (32 %) serum samples from RA patients were simultaneously positive for RF, antiCCP and anti-M12 antibodies, while 45 (26.2 %) samples were positive only for anti-M12 and 12 (7 %) were negative for the three antibodies (Table 3).Fig. 6


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)

Anti-M12 antibody detection by ELISA. Detection of anti-M12 antibodies in sera from rheumatoid arthritis (RA) patients (n = 172), healthy controls (HC) (n = 113), systemic lupus erythematosus (SLE) patients (n = 19) and ankylosing spondylitis (AS) patients (n = 13) and the respectively receiver operating characteristic (ROC) curve. a Sera from the four groups individually tested for their ability to bind to synthetic M12 peptide. Horizontal line = cut-off value; error bars show mean and standard deviation. b ROC curve constructed based on the control groups. The area under the curve (AUC), sensitivity (Se), specificity (Sp) and corresponding p-value are indicated inside the graph
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Anti-M12 antibody detection by ELISA. Detection of anti-M12 antibodies in sera from rheumatoid arthritis (RA) patients (n = 172), healthy controls (HC) (n = 113), systemic lupus erythematosus (SLE) patients (n = 19) and ankylosing spondylitis (AS) patients (n = 13) and the respectively receiver operating characteristic (ROC) curve. a Sera from the four groups individually tested for their ability to bind to synthetic M12 peptide. Horizontal line = cut-off value; error bars show mean and standard deviation. b ROC curve constructed based on the control groups. The area under the curve (AUC), sensitivity (Se), specificity (Sp) and corresponding p-value are indicated inside the graph
Mentions: The M12 synthetic peptide was tested by ELISA with individual sera obtained from 172 RA patients, 113 HCs, 19 SLE patients and 13 AS patients to evaluate its potential as a diagnostic. The cut-off point was determined as the value of the parameter corresponding to the highest sensitivity without lowering the specificity. Of 172 RA patients, 145 (84.3 %) were positive to anti-M12 antibodies. The synthetic molecule was able to efficiently discriminate sera from RA patients and HCs (p < 0.0001), SLE (p < 0.0001) and AS (p < 0.0001) (Fig. 6a). The ROC curve analysis constructed based on the control groups was significant (p < 0.0001; AUC = 0.946). Based on the determined cut-off point, M12 synthetic peptide presented a specificity of 91 % and sensitivity of 84.3 % (Fig. 6b). For humoral immune response evaluation, 55 (32 %) serum samples from RA patients were simultaneously positive for RF, antiCCP and anti-M12 antibodies, while 45 (26.2 %) samples were positive only for anti-M12 and 12 (7 %) were negative for the three antibodies (Table 3).Fig. 6

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