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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

Schematic workflow illustrating the steps involved in the study. a Selection step, and b validation step. Arthritis was induced in collagen-induced arthritis (CIA) mice by immunization with type II collagen (CII). Immunoglobulin G (IgG) was purified by protein G beads from serum of mice presenting with acute arthritis and naïve mice. A phage display library was used to select mimotopes against purified IgG from CIA mice (three cycles of biopanning), with a subtractive step against IgG from naïve mice. IgG-bound phages from CIA mice were competitively eluted by incubation with total proteins extracted from inflamed joints. Total proteins extracted from inflamed joints were also used for anti-M12 antibody recognition in Western blot. Immunoreactivity of each selected mimotope was tested by phage exyme-linked immunosorbent assay (ELISA). After DNA extraction and in silico and in vitro analysis, the most reactive mimotope (M12) was identified as a peptide that mimics a predicted antigenic site of the human carbonic anhydrase III protein. Validation of the M12 synthetic peptide as a possible rheumatoid arthritis (RA) autoantigen was carried out by ELISA assay
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Fig1: Schematic workflow illustrating the steps involved in the study. a Selection step, and b validation step. Arthritis was induced in collagen-induced arthritis (CIA) mice by immunization with type II collagen (CII). Immunoglobulin G (IgG) was purified by protein G beads from serum of mice presenting with acute arthritis and naïve mice. A phage display library was used to select mimotopes against purified IgG from CIA mice (three cycles of biopanning), with a subtractive step against IgG from naïve mice. IgG-bound phages from CIA mice were competitively eluted by incubation with total proteins extracted from inflamed joints. Total proteins extracted from inflamed joints were also used for anti-M12 antibody recognition in Western blot. Immunoreactivity of each selected mimotope was tested by phage exyme-linked immunosorbent assay (ELISA). After DNA extraction and in silico and in vitro analysis, the most reactive mimotope (M12) was identified as a peptide that mimics a predicted antigenic site of the human carbonic anhydrase III protein. Validation of the M12 synthetic peptide as a possible rheumatoid arthritis (RA) autoantigen was carried out by ELISA assay

Mentions: Arthritis induction was efficient in CIA mice. A total of 80 % of male DBA/1 J mice developed acute arthritis around 60 days after CII immunization. The severity and incidence of arthritis were assessed as described in Methods. No manifestation of arthritis was observed in mice in the control group treated with PBS in CFA. A schematic workflow illustrating the steps employed in this study is shown in Fig. 1.Fig. 1


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)

Schematic workflow illustrating the steps involved in the study. a Selection step, and b validation step. Arthritis was induced in collagen-induced arthritis (CIA) mice by immunization with type II collagen (CII). Immunoglobulin G (IgG) was purified by protein G beads from serum of mice presenting with acute arthritis and naïve mice. A phage display library was used to select mimotopes against purified IgG from CIA mice (three cycles of biopanning), with a subtractive step against IgG from naïve mice. IgG-bound phages from CIA mice were competitively eluted by incubation with total proteins extracted from inflamed joints. Total proteins extracted from inflamed joints were also used for anti-M12 antibody recognition in Western blot. Immunoreactivity of each selected mimotope was tested by phage exyme-linked immunosorbent assay (ELISA). After DNA extraction and in silico and in vitro analysis, the most reactive mimotope (M12) was identified as a peptide that mimics a predicted antigenic site of the human carbonic anhydrase III protein. Validation of the M12 synthetic peptide as a possible rheumatoid arthritis (RA) autoantigen was carried out by ELISA assay
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Schematic workflow illustrating the steps involved in the study. a Selection step, and b validation step. Arthritis was induced in collagen-induced arthritis (CIA) mice by immunization with type II collagen (CII). Immunoglobulin G (IgG) was purified by protein G beads from serum of mice presenting with acute arthritis and naïve mice. A phage display library was used to select mimotopes against purified IgG from CIA mice (three cycles of biopanning), with a subtractive step against IgG from naïve mice. IgG-bound phages from CIA mice were competitively eluted by incubation with total proteins extracted from inflamed joints. Total proteins extracted from inflamed joints were also used for anti-M12 antibody recognition in Western blot. Immunoreactivity of each selected mimotope was tested by phage exyme-linked immunosorbent assay (ELISA). After DNA extraction and in silico and in vitro analysis, the most reactive mimotope (M12) was identified as a peptide that mimics a predicted antigenic site of the human carbonic anhydrase III protein. Validation of the M12 synthetic peptide as a possible rheumatoid arthritis (RA) autoantigen was carried out by ELISA assay
Mentions: Arthritis induction was efficient in CIA mice. A total of 80 % of male DBA/1 J mice developed acute arthritis around 60 days after CII immunization. The severity and incidence of arthritis were assessed as described in Methods. No manifestation of arthritis was observed in mice in the control group treated with PBS in CFA. A schematic workflow illustrating the steps employed in this study is shown in Fig. 1.Fig. 1

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