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Porcine aminopeptidase N (pAPN) is a cellular receptor of transmissible gastroenteritis virus (TGEV), a porcine coronavirus. Interaction between the spike (S) protein of TGEV and pAPN initiates cell infection. Small molecules, especially peptides are an expanding area for therapy or diagnostic assays for viral diseases. Here, the peptides capable of binding the pAPN were, for the first time, identified by biopanning using a random 12-mer peptide library to the immobilized protein. Three chemically synthesized peptides recognizing the pAPN showed effective inhibition ability to TGEV infection in vitro. A putative TxxF motif was identified in the S protein of TGEV. Phages bearing the specific peptides interacted with the pAPN in ELISA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays confirmed the protective effect of the peptides on cell infection by TGEV. Moreover, the excellent immune responses in mice induced by the identified phages provided the possibility to develop novel phage-based vaccines.

Introduction Only a limited number of tumor markers for breast cancer are currently available. Antibodies to tumor-associated proteins may expand the number of available tumor markers for breast cancer and may be used together in a serum profile to enhance sensitivity and specificity. Methods In the present study, we interrogated a breast cancer cDNA T7 phage library for tumor-associated proteins using biopan enrichment techniques with sera from normal individuals and from breast cancer patients. The enrichment of tumor-associated proteins after biopanning was tested using a plaque-lift assay and immunochemical detection. The putative tumor-associated phage clones were collected for PCR and sequencing analysis. Unique and open reading frame phage-expressed proteins were then used to develop phage protein ELISAs to measure corresponding autoantibodies using 87 breast cancer patients and 87 normal serum samples. A logistic regression model and leave-one-out validation were used to evaluate predictive accuracies with a single marker as well as with combined markers. Identities of those selected proteins were revealed through the sequence BLAST program. Results We harvested 100 putative tumor-associated phage clones after biopan enrichment. Sequencing analysis revealed that six phage proteins were inframe and unique. Antibodies to these six phage-expressed proteins were measured by ELISAs, and the results showed that three of the phage clones had statistical significance in discriminating patients from normal individuals. BLAST results of the three proteins showed great matches to ASB-9, SERAC1, and RELT. Measurements of the three predictive phage proteins were combined in a logistic regression model that achieved 80% sensitivity and 100% specificity in prediction of sample status, whereas leave-one-out validation achieved 77.0% sensitivity and 82.8% specificity among 87 patient samples and 87 control samples. Receiver operating characteristic curve analysis and the leave-one-out method both showed that combined measurements of the three antibodies were more predictive of disease than any of the single antibodies studied, underscoring the importance of identifying multiple potential markers. Conclusion Serum autoantibody profiling is a promising approach for early detection and diagnosis of breast cancer. Rather than one autoantibody, a panel of autoantibodies appears preferable to achieve superior accuracy. Further refinements will need to be made to further improve the accuracy. Once refined, the assay must be applied to a prospective patient population to demonstrate applicability.

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Background Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). Results In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen. By joining the in vitro immunization and the phage display ten unique phage clones carrying sdAb were selected. Out of ten, seven sdAb showed strong antigen binding ability in phage ELISA. Furthermore, two soluble forms of sdAb were produced and their differential antigen binding affinity was measured with bio-layer interferometry. Conclusion A proposed pipeline has potential to reduce the cost substantially required for maintenance of camelid herd for active immunization. Furthermore, in vitro immunization can be achieved within a week to enrich mRNA copies encoding antigen-specific sdAbs in B cell. This rapid and cost effective pipeline can help researchers to develop efficiently sdAb for diagnostic and therapeutic purposes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Next generation sequencing (NGS) in combination with phage surface display (PSD) are powerful tools in the newly equipped molecular biology toolbox for the identification of specific target binding biomolecules. Application of PSD led to the discovery of manifold ligands in clinical and material research. However, limitations of traditional phage display hinder the identification process. Growth-based library biases and target-unrelated peptides often result in the dominance of parasitic sequences and the collapse of library diversity. This study describes the effective enrichment of specific peptide motifs potentially binding to arsenic as proof-of-concept using the combination of PSD and NGS. Arsenic is an environmental toxin, which is applied in various semiconductors as gallium arsenide and selective recovery of this element is crucial for recycling and remediation. The development of biomolecules as specific arsenic-binding sorbents is a new approach for its recovery. Usage of NGS for all biopanning fractions allowed for evaluation of motif enrichment, in-depth insight into the selection process and the discrimination of biopanning artefacts, e.g., the amplification-induced library-wide reduction in hydrophobic amino acid proportion. Application of bioinformatics tools led to the identification of an SxHS and a carboxy-terminal QxQ motif, which are potentially involved in the binding of arsenic. To the best of our knowledge, this is the first report of PSD combined with NGS of all relevant biopanning fractions.

The development of a rapid and specific assay for 17β-estradiol (E2) will accelerate its in vitro diagnostics and/or environmental pollution control. Here, we employed an open-sandwich (OS) selection scheme to improve the sensitivity for E2 in an OS immunoassay, which is based on antigen-dependent stabilization of the antibody (Ab) variable region, Fv, where the two domains (V(H) and V(L)) dissociated in the absence of an antigen. The V(H) domain of a cloned anti-E2 antibody displayed on M13 phage was randomly mutated, and after three OS biopanning rounds, a mutant that showed higher sensitivity in OS-ELISA for E2 was identified. Interestingly, compared with the wild-type V(H), the cross-reactivity of the mutant was significantly decreased for the analogous steroid testosterone, both in OS and competitive ELISAs. This is the first report concerning selection for an anti-hapten Ab without using any hapten-carrier conjugates, and the method will be especially suitable for selecting Ab fragments that show better performance in hapten OS immunoassays.

Over the past decade, phage display has maturated to be a frequently used method for the generation of monoclonal antibodies of human origin. The essential step of this method is the “biopanning” of phage carrying functional antibody fragments on their surface on an immobilized antigen. The screening of large combinatorial gene libraries with this method usually leads to a set of diverse clones specifically binding to the antigen that need to be characterized further. Beside its specificity, the key parameter to be determined is the affinity of the recombinant antibody fragment to its antigen. Here, we present a mass sensitive microsensor method that allows the estimation of antibody affinity directly from the phage supernatant. Binding of phage antibodies to the antigen immobilized on a quartz crystal microbalance (QCM) induced a mass dependent decrease in frequency. This principle was used to determine the apparent affinity of a single-chain (sc)Fv antibody against the RNA polymerase of Drosophila melanogaster presented on the surface of a filamentous phage (M13) from its association and dissociation rates. The apparent affinity obtained is in accordance with the affinity of the scFv fragment as determined by conventional equilibrium enzyme-linked immunosorbent assay (ELISA) and plasmon resonance methods.

Tumor vasculature expresses a distinct set of molecule signatures on the endothelial cell surface different from the resting blood vessels of other organs and tissues in the body. This makes them an attractive target for cancer therapy and molecular imaging. The current technology using the in vivo phage display biopanning allows us to quickly isolate and identify peptides potentially homing to various tumor blood vessels. Tumor-homing peptides in conjugation with chemotherapeutic drugs or imaging contrast have been extensively tested in various preclinical and clinical studies. These tumor-homing peptides have valuable potential as targeting probes for tumor molecular imaging and drug delivery. In this review, we summarize the recent advances about the applications of tumor-homing peptides selected by in vivo phage display library screening against tumor vasculature. We also introduce the characteristics of the latest discovered tumor-penetrating peptides in their potential clinical applications.

Background Multiple tick saliva proteins, the majority of which are unknown, confer tick resistance in repeatedly infested animals. The objective of this study was to identify the 24-48 h fed Amblyomma americanum tick saliva immuno-proteome. The 24-48 h tick-feeding phase is critical to tick parasitism as it precedes important events in tick biology, blood meal feeding and disease agent transmission. Fed male, 24 and 96 h fed female phage display cDNA expression libraries were biopanned using rabbit antibodies to 24 and 48 h fed A. americanum female tick saliva proteins. Biopanned immuno-cDNA libraries were subjected to next generation sequencing, de novo assembly, and bioinformatic analysis. Results More than 800 transcripts that code for 24-48 h fed A. americanum immuno-proteins are described. Of the 895 immuno-proteins, 52% (464/895) were provisionally identified based on matches in GenBank. Of these, ~19% (86/464) show high level of identity to other tick hypothetical proteins, and the rest include putative proteases (serine, cysteine, leukotriene A-4 hydrolase, carboxypeptidases, and metalloproteases), protease inhibitors (serine and cysteine protease inhibitors, tick carboxypeptidase inhibitor), and transporters and/or ligand binding proteins (histamine binding/lipocalin, fatty acid binding, calreticulin, hemelipoprotein, IgG binding protein, ferritin, insulin-like growth factor binding proteins, and evasin). Others include enzymes (glutathione transferase, cytochrome oxidase, protein disulfide isomerase), ribosomal proteins, and those of miscellaneous functions (histamine release factor, selenoproteins, tetraspanin, defensin, heat shock proteins). Conclusions Data here demonstrate that A. americanum secretes a complex cocktail of immunogenic tick saliva proteins during the first 24-48 h of feeding. Of significance, previously validated immunogenic tick saliva proteins including AV422 protein, calreticulin, histamine release factor, histamine binding/lipocalins, selenoproteins, and paramyosin were identified in this screen, supporting the specificity of the approach in this study. While descriptive, this study opens opportunities for in-depth tick feeding physiology studies. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-518) contains supplementary material, which is available to authorized users.

Background Cancer is a leading cause of death globally and the majority of patients will die from the formation of metastases rather than from their primary tumour as most metastases are resistant to conventional therapies. Based on numerous clinical observations it is clear that different cancers have a propensity to metastasize to specific organs. The central hypothesis for our current study is that an organs vasculature is intimately involved in mediating the interaction between specific cancer cells and the target organ for which they will metastasize to. In this respect we have focused on how melanoma cancer cells metastasize to the liver and lungs, two major sites of metastatic disease within the body, in order to provide insight for the development of new treatments for metastatic cancer. Materials and methods Herein we used an unbiased combinatorial phage in vivo biopanning strategy to isolate a library of peptidedisplaying-phage that home to the liver and lungs of animals treated with a pro-inflammatory stimulus, lipopolysccharaide (LPS). Using a functional screen we isolated a single phage that prevented the recruitment of leukocytes into the liver and lungs of animals following LPS treatment. We investigated if the lung/liver inhibitory phage were also able to inhibit the formation of lung metastasis using a human melanoma metastasis xenograft model. Results Using intravital microscopy, we identified a peptide-displaying-phage, and its corresponding displayed-peptide, that has the ability to inhibit adhesion and recruitment of neutrophils in the liver sinusoids in response to LPS. Excitingly, we also found that animals injected (i.v.) with human melanoma cells (70W) in the presence of the phage, or its corresponding displayed-peptide, showed reduced metastatic lung burden. As the isolated peptide was capable of inhibiting both leukocyte recruitment and melanoma lung metastasis, we asked if leukocytes played a functional role in the establishment of melanoma metastasis. We found that in animals that received Lys6G (Anti Gr-1) antibody to deplete circulating leukocytes there was a dramatic increase in 70W melanoma metastasis to the lungs and that this increase in metastatic state was still blocked by the addition of the peptide. Conclusion

Background Leishmania parasites can cause visceral or cutaneous disease and are found in subtropical and tropical regions of the Old and New World. The pathology of the infection is determined by both host immune factors and species/strain differences of the parasite. Dogs represent the major reservoir of Leishmania infantum (syn. L. chagasi) and vaccines are considered the most cost-effective control tools for canine disease. Methods Selection of immunodominant peptides was performed by Phage Display to identify sequences recognized by L. infantum naturally infected animals. Sera from Leishmania infected animals were used in the biopanning to selection of specific peptides. Serum samples from T. cruzi infected and healthy animals were used as control. After selection, synthetic peptides were produced in membrane (spot-synthesis) in soluble form and blotting and ELISA were performed for validation of serum reactivity. Selected peptide was formulated with aluminum hydroxide and liposomes and immunization was performed in BALB/c mice. Protection was determined by qPCR after challenge infection with virulent L. infantum. Results We reported the selection of Peptide 5 through Phage Display technique and demonstrate its ability to promote a state of immunity against L. infantum infection in murine model after immunization using liposomes as vaccine carrier. Our results demonstrate that immunization with Peptide 5 when formulated with aluminum hydroxide and liposomes is immunogenic and elicited significant protection associated with the induction of mixed Th1/Th2 immune response against L. infantum infection. Conclusion Peptide 5 is a promising vaccine candidate and the findings obtained in the present study encourage canine trials to confirm the effectiveness of a vaccine against CVL.