Fortunato Ferrara

Looking for celiac disease : Diagnostic accuracy of two rapid commercial assays

BACKGROUND:Early diagnosis and treatment with gluten-free diet reduces mortality and the prevalence of associated disorders in celiac disease (CD). A simple “in the office” test of anti-transglutaminase antibodies might be of great help in first-line screening for CD.AIMS:We evaluated the sensitivity and specificity of two commercial kits based, respectively, on rapid detection of IgA-IgG anti-human-transglutaminase antibodies (anti-h-tTG) in serum and IgA anti-h-tTG antibody in one drop of whole blood. These assays were compared to a well-established enzyme-linked immunosorbent assay technique.METHODS:Serum samples were analyzed from 114 biopsy-confirmed celiacs, 120 healthy controls, 20 first-degree relatives of celiacs, and 75 diseased controls. The whole blood samples were analyzed from 51 biopsy-confirmed celiacs and 100 controls.RESULTS:The serum-based test was positive in all 114 celiacs (sensitivity 100%). Among the controls there were seven healthy blood donors, one first-degree relative, and three diseased controls who tested positive (specificity 94.9%). The blood drop-based assay testing IgA antibodies was positive in 46 of 51 (sensitivity 90.2%), and since three of the five patients testing negative had total IgA deficiency, the sensitivity value can be increased to 95.8%. All 100 controls tested negative (specificity 100%).CONCLUSIONS:The commercial kits described here produce high values of sensitivity and specificity, offering the general practitioner who suspects a possible case of CD the real possibility to look for anti-h-tTG antibodies in his own medical office during a standard visit at a satisfyingly low cost.

Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker

Background Current diagnostic methods for tuberculosis (TB), a major global health challenge that kills nearly two million people annually, are time-consuming and inadequate. During infection a number of bacterial molecules that play a role in the infective process are released and have been proposed as biomarkers for early TB diagnosis. Antigen 85 (Ag85) is the most abundant secreted TB protein, and a potential target for this diagnostic approach. One of the bottlenecks in the direct detection of such bacterial targets is the availability of robust, sensitive, specific antibodies. Methods Using Ag85 as a model, we describe a method to select antibodies against any potential target using a novel combination of phage and yeast display that exploits the advantage of each approach. Results The efficiency of this approach was attested to by the 111 specific antibodies identified in initial screens. These were assessed for binding to the different Ag85 subunits, affinity, and activity in sandwich assays. Conclusions The novelty of this approach lies in the possibility of screening the entire output of a phage antibody selection in a single experiment by yeast display. This can be considered analogous to carrying out a million ELISAs. The monoclonal antibodies (mAbs) identified in this way show high binding affinity and selectivity for the antigens and offer an advantage over traditional mAbs produced by relatively expensive and time consuming techniques. This approach has wide applicability, and the affinity of selected antibodies can be significantly improved, if required.

Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases.

Anti‐transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti‐transglutaminase antibodies. The aim was to measure positive anti‐transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti‐transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty‐two children suffering from infectious diseases were enrolled prospectively along with seven biopsy‐proven coeliacs. Serum samples were tested for anti‐transglutaminase antibodies and anti‐endomysium antibodies; positive samples were tested for coeliac‐related human leucocyte antigen (HLA)‐DQ2/8 and anti‐viral antibodies. Purified anti‐transglutaminase antibodies from the two study groups were tested for urea‐dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell‐cycle in Caco‐2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti‐transglutaminase, one of whom also tested positive for anti‐endomysium antibodies. This patient was positive for HLA‐DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA‐DQ8. Levels of anti‐transglutaminase returned to normal in all subjects, despite a gluten‐containing diet. Purified anti‐transglutaminase of the two study groups induced actin rearrangements and cell‐cycle progression. During an infectious disease, anti‐transglutaminase antibodies can be produced temporarily and independently of gluten. The infection‐triggered anti‐transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in‐vivo potential for damage.

Regulatory T-Cell Function Is Impaired in Celiac Disease

Celiac disease (CD) is characterized by intolerance to gluten and high risk of developing autoimmune phenomena. Possible defects in immune tolerance could have a role in the pathogenesis of the disease. As regulatory T-cells (Tregs) are the main population involved in maintaining peripheral tolerance, we investigated the number of these cells in celiac patients as compared with healthy donors. Moreover, we analyzed the suppressive function of CD4+CD25+ T-cells from celiac disease patients and controls on autologous responder T-cells (CD4+CD25−). The percentage of CD4+CD25+FOXP3+ cells was not different in celiacs and in healthy controls, and among positive cells the level of expression of the two regulatory markers was comparable. However, the suppressor activity of Tregs was significantly impaired in CD patients. These results suggest that a defect in Tregs function could play a role in the pathogenesis of CD and in CD-associated autoimmunity.

The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

Characterizing monoclonal antibody epitopes by filtered gene fragment phage display

In the present paper, we describe a novel approach to map monoclonal antibody epitopes, using three new monoclonal antibodies that recognize h-TG2 (human transglutaminase 2) as an example. The target gene was fragmented and cloned upstream of an antibiotic-resistance gene, in the vector pPAO2, to select for in-frame polypeptides. After removal of the antibiotic-resistance gene by Cre/Lox recombination, an antigen fragment phage display library was created and selected against specific monoclonal antibodies. Using the h-TG2 fragment library, we were able to identify epitopes. This technique can also be broadly applied to the study of protein-protein interactions.

Deep sequencing in library selection projects: what insight does it bring?

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology.

From deep sequencing to actual clones.

The application of deep sequencing to in vitro display technologies has been invaluable for the straightforward analysis of enriched clones. After sequencing in vitro selected populations, clones are binned into identical or similar groups and ordered by abundance, allowing identification of those that are most enriched. However, the greatest strength of deep sequencing is also its greatest weakness: clones are easily identified by their DNA sequences, but are not physically available for testing without a laborious multistep process involving several rounds of polymerization chain reaction (PCR), assembly and cloning. Here, using the isolation of antibody genes from a phage and yeast display selection as an example, we show the power of a rapid and simple inverse PCR-based method to easily isolate clones identified by deep sequencing. Once primers have been received, clone isolation can be carried out in a single day, rather than two days. Furthermore the reduced number of PCRs required will reduce PCR mutations correspondingly. We have observed a 100% success rate in amplifying clones with an abundance as low as 0.5% in a polyclonal population. This approach allows us to obtain full-length clones even when an incomplete sequence is available, and greatly simplifies the subcloning process. Moreover, rarer, but functional clones missed by traditional screening can be easily isolated using this method, and the approach can be extended to any selected library (scFv, cDNA, libraries based on scaffold proteins) where a unique sequence signature for the desired clones of interest is available.

Integrated nanotechnology platform for tumor-targeted multimodal imaging and therapeutic cargo release

Significance The main goal in the emerging field of cancer nanomedicine is to generate, standardize, and produce multifunctional carriers designed to improve the response of drugs against tumors. Here we report the design, development, and preclinical validation of a ligand-directed bioinorganic platform that integrates tumor targeting, receptor-mediated cell internalization, photon-to-heat conversion, and drug delivery. This enabling hydrogel-based technology can accommodate a broad variety of ligands, nanoparticles, and payloads. We show experimental proof-of-concept in mouse models of breast and prostate cancer with molecular imaging and marked reduction of tumor growth. However, with future proof that this technology is translatable, medical applications beyond cancer may also be leveraged. A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.

RECOMBINANT RENEWABLE POLYCLONAL ANTIBODIES

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.