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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

Western blot analyses. a The Ponceau staining shows 1 μg of total proteins extracted from inflamed joints of CIA mice (§) separated by 10 % SDS-PAGE. b The same membrane stained with Ponceau was probed with anti-M12 antibodies purified from pooled sera of RA patients (†) and HCs (‡). Anti-M12 antibodies displayed strong reactivity with a protein extracted from inflamed joints of CIA mice presenting with mass of approximately 29 kDa (arrow). Carbonic anhydrase III (CAIII) was identified as the protein target that is mimicked by the M12 mimotope. The membrane was cropped to allow differential incubation with anti-M12 antibodies purified from RA patients and HCs
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Fig3: Western blot analyses. a The Ponceau staining shows 1 μg of total proteins extracted from inflamed joints of CIA mice (§) separated by 10 % SDS-PAGE. b The same membrane stained with Ponceau was probed with anti-M12 antibodies purified from pooled sera of RA patients (†) and HCs (‡). Anti-M12 antibodies displayed strong reactivity with a protein extracted from inflamed joints of CIA mice presenting with mass of approximately 29 kDa (arrow). Carbonic anhydrase III (CAIII) was identified as the protein target that is mimicked by the M12 mimotope. The membrane was cropped to allow differential incubation with anti-M12 antibodies purified from RA patients and HCs

Mentions: SDS-PAGE fractionated proteins from inflamed joints of CIA mice were transferred to a nitrocellulose membrane. Western blot revealed that purified IgG from RA patients against the M12 mimotope recognized a protein with a molecular weight of approximately 29 kDa (Fig. 3).Fig. 3


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)

Western blot analyses. a The Ponceau staining shows 1 μg of total proteins extracted from inflamed joints of CIA mice (§) separated by 10 % SDS-PAGE. b The same membrane stained with Ponceau was probed with anti-M12 antibodies purified from pooled sera of RA patients (†) and HCs (‡). Anti-M12 antibodies displayed strong reactivity with a protein extracted from inflamed joints of CIA mice presenting with mass of approximately 29 kDa (arrow). Carbonic anhydrase III (CAIII) was identified as the protein target that is mimicked by the M12 mimotope. The membrane was cropped to allow differential incubation with anti-M12 antibodies purified from RA patients and HCs
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Western blot analyses. a The Ponceau staining shows 1 μg of total proteins extracted from inflamed joints of CIA mice (§) separated by 10 % SDS-PAGE. b The same membrane stained with Ponceau was probed with anti-M12 antibodies purified from pooled sera of RA patients (†) and HCs (‡). Anti-M12 antibodies displayed strong reactivity with a protein extracted from inflamed joints of CIA mice presenting with mass of approximately 29 kDa (arrow). Carbonic anhydrase III (CAIII) was identified as the protein target that is mimicked by the M12 mimotope. The membrane was cropped to allow differential incubation with anti-M12 antibodies purified from RA patients and HCs
Mentions: SDS-PAGE fractionated proteins from inflamed joints of CIA mice were transferred to a nitrocellulose membrane. Western blot revealed that purified IgG from RA patients against the M12 mimotope recognized a protein with a molecular weight of approximately 29 kDa (Fig. 3).Fig. 3

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