Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display

Nitrotyrosine is a posttranslational protein modification that occurs under oxidative and nitrosative stress, and plays an important role in numerous pathological conditions. To analyse nitrotyrosine formation several commercial monoclonal and polyclonal antibodies reacting with 3-nitrotyrosine have been developed which however do not work properly in all required assays. Here, antibody phage display was used to select recombinant antibodies that specifically react with nitrotyrosine in various protein contexts. Nine initial selections were carried out, using synthetic peptides, peroxynitrite-modified proteins and conjugated proteins as antigens. Four antibodies were isolated that each exhibited a characteristic binding reactivity that greatly depended on the antigens that were used for their selections. In general, the selections using small, synthetic and biotinylated peptides were the most successful approach. Subsequently, antibody 11B1 was affinity matured by error prone mutagenesis, resulting in the isolation of two antibodies, designated 47A7 and 47B1. Competition ELISA and immunoblotting after treatment with sodium dithionite further demonstrated the specificity of antibody 47B1 for nitrotyrosine. The results presented here demonstrate that antibody phage display is a useful method to isolate antibodies against posttranslational modifications, which are powerful tools in the proteomic era

Circulating FABP4 Is a prognostic biomarker in patients with acute coronary syndrome but not in asymptomatic individuals.

OBJECTIVE: Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS: Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS: Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS

Schematic overview of the four different JUP isoforms.

<p>JUP-81 and JUP-63 have an identical N-terminus (the N-terminal 303 amino acids), and JUP-63 further shares its C-terminus with cytokeratin 19 (K1C19). The sequences of JUP-55 and JUP-30 are currently unknown but they contain epitopes that are shared with JUP-81 and are thought to be located in the N-terminal region of native JUP. JUP-30 lacks (at least) the N-terminal 50 amino acids of JUP-81. JUP-81, which is also referred to as gamma-catenin, is furthermore homologous to beta-catenin. Amino acids 1, 303 and 745 of JUP-81 are indicated with arrows.</p

Thrombendarterectomy tissue for secretome production and immunohistochemistry.

<p>a) Tissue sample as it was received from the surgical department. b) The tissue was separated into the plaque part (I) for production of the plaque secretome, and the best preserved part (II) for production of the control secretome.</p

Detection of JUP in plaques by immunohistochemistry and in secretome by immunoblotting.

<p>a) Overview of JUP immunoreactivity on endarterectomised tissue. Strong staining in the atherosclerotic plaque tissue is observed. b) Clockwise from top left (400-fold magnification): H&E staining (1), anti-CD68 (2), negative control (3), and anti-JUP staining (4). c and d) Six plaque secretomes (lanes 1–6), two control secretomes (lanes 7 and 8) and GST-tagged JUP (lane 9, 107 kD) were immunoblotted with anti-JUP mAb 2C9 (c) and scFv 25G5 (d). e) Competition experiment with mAb 2G9 (which replaced 2C9). Western blots containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), and secretome (4.5 µl in lane 3 and 1.9 µl in lane 4) were incubated with mAb 2G9, which was (blot on the right) or was not (blot on the left) pre-incubated with soluble, recombinant GST-tagged JUP protein. f) Competition experiment with scFv 25G5. Western blots containing different amount of atherosclerotic plaque secretome (lane 1∶4.5 µl, lane 2∶1.9 µl, lane 3∶0.9 µl, lane 4∶0.4 µl) were incubated with scFv 25G5, which was (blot on the right) or was not (blot on the left) pre-incubated with soluble, recombinant GST-tagged JUP protein. For all immunoblots: known molecular weights of protein markers are depicted on the left and estimated molecular weights of detected protein bands are depicted on the right of both Western blots.</p

Schematic presentation of the selection strategy.

<p>a) The phage library, containing billions of different phages, was amplified from the bacterial stock. ScFv are fused to minor coat protein III and as such displayed on the phage surface. b) In a subtraction step, the phage library was incubated with secretome from the healthy control tissue. Binders to common proteins were removed in this way. c) Phages that did not bind to the control secretome were incubated with the atherosclerotic secretome in this panning step. d) Unbound phages were washed off, and e) bound phages were eluted and used to infect a suitable <i>E.coli</i> strain (<i>E.coli</i> TG1). f) Bacteria infected with the selected and eluted phages were plated on large agar plates. g) To further enrich for phages that specifically bind to the atherosclerotic secretome, the selection round was repeated. h) Single colonies were induced to produce monoclonal phages. i) Monoclonal phages were analysed for their reactivity with atherosclerotic versus control secretomes in ELISA. In total, six different selections were performed. For each separate selection the control and atherosclerotic secretomes from one individual patient were used. In order not to loose diversity, only two subtractive panning rounds were performed for each selection. To analyse whether enrichment of atherosclerotic secretome-specific binders had taken place, polyclonal phage pools (as obtained in step a) after each subtractive panning round (and of the unselected library as a control) were analysed in ELISA for reactivity with atherosclerotic and control secretomes.</p

Reactivities of ten different commercial anti-JUP antibodies with the four JUP isoforms.

<p>Reactivities of ten different commercial anti-JUP antibodies with the four JUP isoforms.</p

Detection of JUP isoforms in plasma from PAOD patients with atherosclerosis and in plasma from a swine model of myocardial infarction without atherosclerosis and plaque rupture.

<p>a) Western blot containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), control plasma (lane 3) and plasma from four PAOD patients (lanes 4 to 7) were detected with mAb 2G9 (which replaced 2C9). JUP-55 and JUP-30 are clearly detected besides JUP-81. b) Western blot containing recombinant GST-tagged JUP (lane 1, 107 kD), ACS plasma (lane 2), control plasma (lane 3) and plasma samples from swine before ligation (lanes 4 and 7), three hours after ligation (lanes 5 and 8) and three days after ligation (lanes 6 and 9) were detected with mAb 2G9. JUP-81 was not detected in the swine samples, whereas JUP-30 and a protein band with a slightly larger molecular weight were detected with similar intensities before and after ligation.</p