Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: RA patient plasma preferentially binds PAD4 citrullinated fibrinogen as measured by ELISA. (A) Normal donor and RA patient antibody levels, as measured by ELISA, to unmodified fibrinogen at 1:20 dilution, PAD2 citrullinated fibrinogen at 1:40 dilution and PAD4 citrullinated fibrinogen at 1:40 dilution. (B) Individual RA patient antibody levels to PAD4 citrullinated fibrinogen and PAD2 citrullinated fibrinogen at various dilutions. (C) Ratio of individual RA patient plasma binding to PAD4 or PAD2 citrullinated fibrinogen. Ratio calculated as (OD of PAD4 citrullinated fibrinogen)/(OD of PAD2 citrullinated fibrinogen) of individual patients from (B) at various dilutions. (D) Individual RA antibody levels to autocitrullinated PAD2 and PAD4 at various dilutions. Data represent 1 of 3 independent experiments. CitFib= citrullinated fibrinogen OD= optical density. *** P<0.005, ns=not significant.

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: RA patient plasma preferentially recognizes PAD4 citrullinated fibrinogen on Western blot. Comparison of normalized band intensities at 76 kD and 56 kD of PAD2 citrullinated fibrinogen and PAD4 citrullinated fibrinogen resolved on SDS-page and probed with individual RA patient plasma diluted 1:1000. Normalized band intensity indicates band intensity of RA patient plasma divided by load control anti-fibrinogen antibody. Data represent 1 of 2 independent experiments. ** P<0.01

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Western Blot, Comparison, SDS Page, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: There is no difference in RA patient antibody avidity to PAD4 or PAD2 citrullinated fibrinogen. Avidity ELISA of RA patient plasma at various titers, incubated with either PAD2 citrullinated fibrinogen or PAD4 citrullinated fibrinogen with increasing concentrations of sodium thiocyanate. Avidity index represents OD of RA patient plasma for a given concentration of sodium thiocyanate divided by the OD without sodium thiocyanate. * P<0.05, *** P<0.001. Data presented are from 1 RA patient and are representative of 3 patients tested.

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: Fibrinogen beta arginine sites 44–74 are a hotspot of citrullination that are uniformly modified by PAD2 and intermittently modified by PAD4. Unmodified, PAD2, or PAD4 citrullinated fibrinogen were enzymatically digested and analyzed by mass spectrometry. (A) Summed signal (PAD2 and PAD4) of peptides containing a citrullinated arginine are plotted as a function of amino acid residue number for fibrinogen beta chain. Green bar below depicts residues covered based on peptides matched with a false discovery of <1%. (B) Relative abundance of identified peptide signals carrying modifications at R44 and R47 in PAD2 and PAD4 citrullinated fibrinogen. Unreacted fibrinogen beta is presented as a control. (C) Relative abundance of identified peptide signals carrying modifications at sites R53, R60, R72 and R74 in PAD2 and PAD4 citrullinated fibrinogen. Unreacted fibrinogen beta is presented as a control.

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Modification, Mass Spectrometry, Residue, Control

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: Intensity based mass spectrometry quantitation of citrullination of PAD2 and PAD4 modified fibrinogen at various arginine sites.

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Mass Spectrometry, Quantitation Assay, Modification

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: High titer rheumatoid arthritis antibodies preferentially bind fibrinogen citrullinated by peptidyl arginine deiminase 4.

doi: 10.1002/art.40035

Figure Lengend Snippet: RA antibodies preferentially bind PAD4 citrullinated fibrinogen because it contains a broader array of citrullinated epitopes. (A) ELISA ODs of RA patient and normal donor (NL) antibodies to citrullinated peptides (1:40). Data represent test peptide minus irrelevant beta-galactosidase peptide. Technical duplicates are presented. cfc-1 = citrullinated filaggrin peptide-1, cfc-0 = noncitrullinated filaggrin peptide-1, FGB= fibrinogen peptides (B) Band intensity of PAD2 or PAD4 citrullinated fibrinogen on Western blot of RA plasma preadsorbed with increasing concentrations of PAD2 citrullinated fibrinogen. Data represent 1 of 3 experiments. (C) Western blots of citrullinated fibrinogen or plasmin cleaved citrullinated fibrinogen. Top panel: probed with polyclonal anti-fibrinogen antibody. Bottom panel: probed with RA patient plasma. Data represent 1 of 3 experiments. Right Panel: Summary of Western blot results presented in (C). Data represents relative band intensity of fragment D divided by relative band intensity of total fibrinogen beta from 10 RA patient samples. (D) Band intensity of citrullinated fibrinogen beta probed with RA patient plasma samples preadsorbed with native peptides or citrullinated peptides from the hotspot region of FGB. Native peptides: FBG34–54, FBG41–61, FBG45–65, FBG66–86, FBG285–305, Citrullinated peptides: FBG41–61(cit 44,47,53), FBG41–61(cit 44, 47), FBG45–65(cit 53, 60), FBG45–65 (cit 53), FBG45–65(cit 60), FBG66–86(cit 72, 74), FBG66–86(cit 72)

Article Snippet: Membranes were probed with antibodies: monoclonal anti-citrullinated fibrinogen (1:1000) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-PAD2 antibody (1:500) ( 16 ) (Abnova, #H00011240-M01, Taipei City, Taiwan), anti-PAD4 antibody (1:1000) ( 17 ) (Abcam #ab128086, Cambridge, MA), monoclonal anti-fibrinogen (1:500) (Abcam #ab10066, Cambridge, MA), or patient plasma diluted 1:1000 in 5% non-fat milk in TBS containing 0.1% Tween 20.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot