Ready-to-use ratiometric bioluminescence immunosensor for detection of ochratoxin a in pepper.

Rapid, portable, and accurate detection tools for monitoring ochratoxin A (OTA) in food are essential for the guarantee of food safety and human health. Herein, as a proof-of-concept, this study proposed a ratiometric bioluminescence immunosensor (RBL-immunosensor) for homogeneous detection of OTA in pepper. The construct of the RBL-immunosensor consists of three components, including the large fragment of the split nanoluciferase (NanoLuc)-tagged nanobody (NLg), the small fragment of the split NanoLuc-tagged mimotope peptide heptamer (MPSm), and the calibrator luciferase (GeNL). The specific nanobody-mimotope peptide interaction between NLg and MPSm induces the reconstitution of the NanoLuc, which catalyzes the Nano-Glo substrate and produces a blue emission peak at 458 nm. Meanwhile, GeNL can produce a green emission peak at 518 nm upon substrate conversion via bioluminescent resonance energy transfer (BRET). Therefore, the concentration of OTA can be linked to the variation of the bioluminescence signal (λ458/λ518) measured by microplate reader and the variation of the blue/green ratio measured by smartphone via the competitive immunoreaction where OTA competes with MPSm to bind NLg. The immunosensor is ready-to-use and works by simply mixing the components in a one-step incubation of 10 min for readout. It has a limit of detection (LOD) of 0.98 ng/mL by a microplate reader and an LOD of 1.89 ng/mL by a smartphone. Good selectivity and accuracy were confirmed for the immunosensor by cross-reaction analysis and recovery experiments. The contents of OTA in 10 commercial pepper powder samples were tested by the RBL-immunosensor and validated by high-performance liquid chromatography. Hence, the ready-to-use RBL-immunosensor was demonstrated as a highly reliable tool for detection of OTA in food.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Mimotope peptide modified pompon mum-like magnetic microparticles for precise recognition, capture and biotransformation analysis of rituximab in biological fluids.

Due to low immobilized ligand density, limited binding capacity, and severe interference from serum proteins, developing ideal peptide-based biomaterials for precise recognition and in vivo analysis of biopharmaceuticals remains a huge challenge. In this study, mimotope peptide modified pompon mum-like biomimetic magnetic microparticles (MMPs, 3.8 µm) that mimic the specific functionalities of CD20 on malignant B cells were developed for the first time. Benefit from the numerous ligand binding sites (Ni2+) on the pompon mum-like MMPs, these novel materials achieved ≥10 times higher peptide ligand densities (>2300 mg/g) and antibody binding capacities (1380 mg/g) compared to previous reported biomaterials. Leveraging the high specificity of the mimotope peptide, rituximab can be precisely recognized and enriched from cell culture media or serum samples. We also established an LC‒MS/MS method using the MMPs for tracking rituximab biotransformation in patient serum. Intriguingly, deamidation of Asn55 and Asn33, as well as oxidation of Met81 and Met34 were observed at the key complementarity determining regions of rituximab, which could potentially influence antibody function and require careful monitoring. Overall, these versatile biomimetic MMPs demonstrate superior recognition and enrichment capabilities for target antibodies, offering interesting possibilities for biotransformation analysis of biopharmaceuticals in patient serum.

Computational identification of antibody-binding epitopes from mimotope datasets.

Introduction: A fundamental challenge in computational vaccinology is that most B-cell epitopes are conformational and therefore hard to predict from sequence alone. Another significant challenge is that a great deal of the amino acid sequence of a viral surface protein might not in fact be antigenic. Thus, identifying the regions of a protein that are most promising for vaccine design based on the degree of surface exposure may not lead to a clinically relevant immune response. Methods: Linear peptides selected by phage display experiments that have high affinity to the monoclonal antibody of interest ("mimotopes") usually have similar physicochemical properties to the antigen epitope corresponding to that antibody. The sequences of these linear peptides can be used to find possible epitopes on the surface of the antigen structure or a homology model of the antigen in the absence of an antigen-antibody complex structure. Results and Discussion: Herein we describe two novel methods for mapping mimotopes to epitopes. The first is a novel algorithm named MimoTree that allows for gaps in the mimotopes and epitopes on the antigen. More specifically, a mimotope may have a gap that does not match to the epitope to allow it to adopt a conformation relevant for binding to an antibody, and residues may similarly be discontinuous in conformational epitopes. MimoTree is a fully automated epitope detection algorithm suitable for the identification of conformational as well as linear epitopes. The second is an ensemble approach, which combines the prediction results from MimoTree and two existing methods.

Prevention of bleomycin-induced pulmonary fibrosis by vaccination with the Tocilizumab mimotope.

Mimotope, a kind of peptide vaccine, is developed to bind natural receptor and inhibit the downstream signaling. We have demonstrated that the vaccination of Tocilizumab mimotopes could alleviate the renal fibrosis by interfering with both IL-6 and ferroptosis signaling. However, the effect of the vaccination of Tocilizumab mimotopes on the fibroblast was not investigated in previous study. Thus, we sought to explore the changes in the fibroblast induced by the Tocilizumab mimotopes vaccination. Bleomycin instillation was performed to construct the pulmonary fibrosis model after the immunization of Tocilizumab mimotopes. Lung histological analysis showed that the Tocilizumab mimotopes could significantly reduce the maladaptive repairment and abnormal remodeling. Immunoblotting assay and fluorescence staining showed that Immunization with the Tocilizumab mimotopes reduces the accumulation of fibrosis-related proteins. High level of lipid peroxidation product was observed in the animal model, while the Tocilizumab mimotopes vaccination could reduce the generation of lipid peroxidation product. Mechanism analysis further showed that Nrf-2 signaling, but not GPX-4 and FSP-1 signaling, was upregulated, and reduced the lipid peroxidation. Our results revealed that in the BLM-induced pulmonary fibrosis, high level of lipid peroxidation product was significantly accumulation in the lung tissues, which might lead to the occurrence of ferroptosis. The IL-6 pathway block therapy could inhibit lipid peroxidation product generation in the lung tissues by upregulating the Nrf-2 signaling, and further alleviate the pulmonary fibrosis.

Screening of Deoxynivalenol Cyclic Heptapeptide Mimotope Antigen.

In this study, phage clones that can bind to DON were selected from the phage cyclohepta peptide library by screening through the principle of solid-phase affinity, and mimotope were synthesized to replace the DON toxin standard to establish a green low toxicity detection system. The author conducted four rounds of screening in the phage cyclic heptapeptide library with DON-10a1a monoclonal antibody as the target molecule. Then 38 phage clones were selected and validated, and the results showed that 35 of them could bind to the DON-10a1a monoclonal antibody and were inhibited by DON toxin. Finally, the DNA was extracted and sequenced to obtain 6 different DNA sequences, which were named D1-D6 respectively. The peptides synthesized according to the corresponding amino acid sequences can replace DON toxin to establish a series of green and low toxicity assays.

Alanine Scanning of the Unstructured Region of Ara h 2 and of a Related Mimotope Reveals Critical Amino Acids for IgE Binding.

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.

Combination of nanobody and peptidomimetic to develop novel immunoassay platforms for detecting ochratoxin A in cereals.

Mimotope-based immunoassays for mycotoxins eliminate the requirement for large amounts of mycotoxin standards for the chemosynthesis of artificial antigens. Herein, the nanobody-based magnetic beads were used to screen the mimotope (peptidomimetic) of ochratoxin A (OTA) from the phage-displayed peptide library. The interactions between nanobody and the most sensitive Y4 peptidomimetic were investigated by computer-assisted simulation and compared with those between nanobody and OTA. By combining the nanobody, the phage-displayed Y4 and alkaline phosphatase-tagged Y4 fusion protein as the competing antigens, were used to develop two novel immunoassay platforms (PN-ELISA and APN-ELISA). The two methods are advantageous in the use of nontoxic substitutes of OTA and avoiding the use of monoclonal antibodies. Moreover, good analytical performances of both methods were obtained and confirmed by liquid chromatography tandem mass spectrometry. Therefore, the proposed novel methods based on nanobody and peptidomimetic were demonstrated to be highly reliable for detecting OTA in food.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses.

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.

Integrated in silico-in vitro rational design of oncogenic EGFR-derived specific monoclonal antibody-binding peptide mimotopes.

Human epidermal growth factor receptor (EGFR) is strongly associated with malignant proliferation and has been established as an attractive therapeutic target of diverse cancers and used as a significant biomarker for tumor diagnosis. Over the past decades, a variety of monoclonal antibodies (mAbs) have been successfully developed to specifically recognize the third subdomain (TSD) of EGFR extracellular domain. Here, the complex crystal structures of EGFR TSD subdomain with its cognate mAbs were examined and compared systematically, revealing a consistent binding mode shared by these mAbs. The recognition site is located on the [Formula: see text]-sheet surface of TSD ladder architecture, from which several hotspot residues that significantly confer both stability and specificity to the recognition were identified, responsible for about half of the total binding potency of mAbs to TSD subdomain. A number of linear peptide mimotopes were rationally designed to mimic these TSD hotspot residues in different orientations and/or in different head-to-tail manners by using an orthogonal threading-through-strand (OTTS) strategy, which, however, are intrinsically disordered in Free State and thus cannot be maintained in a native hotspot-like conformation. A chemical stapling strategy was employed to constrain the free peptides into a double-stranded conformation by introducing a disulfide bond across two strand arms of the peptide mimotopes. Both empirical scoring and [Formula: see text]fluorescence assay reached an agreement that the stapling can effectively improve the interaction potency of OTTS-designed peptide mimotopes to different mAbs, with binding affinity increase by [Formula: see text]-fold. Conformational analysis revealed that the stapled cyclic peptide mimotopes can spontaneously fold into a double-stranded conformation that well threads through all the hotspot residues on TSD [Formula: see text]-sheet surface and exhibits a consistent binding mode with the TSD hotspot site to mAbs.

Tumor antigen-unbiased variable epitope library contains mimotopes with antitumor effect in a mouse model of breast cancer.

Breast cancer is one of the leading causes of death that affects the female population worldwide. Despite advances in treatments and a greater understanding of the disease, there are still difficulties in successfully treating patients. Currently, the main challenge in the field of cancer vaccines is antigenic variability which can reduce antigen-specific T- cell response efficacy. The search for and validation of immunogenic antigen targets increased dramatically over the past few decades and, with the advent of modern sequencing techniques, permitting the fast and accurate identification of the neoantigen landscape of tumor cells, will undoubtedly continue to grow exponentially for years to come. We have previously implemented Variable Epitope Libraries (VEL) as an unconventional vaccine strategy in preclinical models and for identifying and selecting mutant epitope variants. Here, we used an alanine-based sequence to generate a 9-mer VEL-like combinatorial mimotope library G3d as a new class of vaccine immunogen. An in silico analysis of the 16,000 G3d-derived sequences revealed potential MHC-I binders and immunogenic mimotopes. We demonstrated the antitumor effect of treatment with G3d in the 4T1 murine model of breast cancer. Moreover, two different T cell proliferation screening assays against a panel of randomly selected G3d-derived mimotopes allowed the isolation of both stimulatory and inhibitory mimotopes showing differential therapeutic vaccine efficacy. Thus, the mimotope library is a promising vaccine immunogen and a reliable source for isolating molecular cancer vaccine components.

ACT001 Relieves NMOSD Symptoms by Reducing Astrocyte Damage with an Autoimmune Antibody.

Neuromyelitis optica spectrum disorder (NMOSD) is a central nervous system inflammatory demyelinating disease, the pathogenesis of which involves autoantibodies targeting the extracellular epitopes of aquaporin-4 on astrocytes. We neutralized the AQP4-IgG from NMOSD patient sera using synthesized AQP4 extracellular epitope peptides and found that the severe cytotoxicity produced by aquaporin-4 immunoglobin (AQP4-IgG) could be blocked by AQP4 extracellular mimotope peptides of Loop A and Loop C in astrocyte protection and animal models. ACT001, a natural compound derivative, has shown anti-tumor activity in various cancers. In our study, the central nervous system anti-inflammatory effect of ACT001 was investigated. The results demonstrated the superior astrocyte protection activity of ACT001 at 10 µM. Furthermore, ACT001 decreases the behavioral score in the mouse NMOSD model, which was not inferior to Methylprednisolone Sodium Succinate, the first-line therapy of NMOSD in clinical practice. In summary, our study showed that astrocytes are protected by specific peptides, or small molecular drugs, which is a new strategy for the treatment of NMOSD. It is possible for ACT001 to be a promising therapy for NMOSD.

Orthogonal threading-through-ß-sheet design of lung cancer EGFR extracellular domain-derived peptidic mimotopes binding to anti-EGFR antibody.

Human epidermal growth factor receptor (EGFR) has been established as a therapeutic target of lung cancer and other diverse tumors. The antibody drug Cetuximab has been developed to target the third subdomain III (TSDIII) of EGFR extracellular domain (ECD) by competitively inhibiting epidermal growth factor binding. In this study, we performed systematic investigation on the crystal complex structure of EGFR ECD domain with Cetuximab to create a residue importance profile for the TSDIII subdomain, based on which a number of U-shaped, double-stranded linear peptides were derived and cyclized to orthogonally thread through most hotspot residues and many responsible residues within the TSDIII ß-sheet plane; they represent mimotopes of the key antibody-recognition site of TSDIII subdomain. Computational analyses revealed that these linear peptides cannot spontaneously fold to the desired conformation in free state due to their intrinsic flexibility. Cell-free assays confirmed that the stapling can considerably improve the binding affinity of linear peptides to Cetuximab by up to 18-fold. The cOrt1 [3-18] cyclic peptide was measured to have the highest affinity in all designed linear and cyclic peptides.

Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide­conjugated BSA.

Tumor­associated (TA) autoantibodies are considered to be promising biomarkers for the early detection of cancer, prior to the development of clinical symptoms. In the present study, a novel TA autoantibody was detected, which may prove to be useful as a diagnostic marker of human HCC using an HBx­transgenic (HBx­tg) hepatocellular carcinoma (HCC) mouse model. Its target antigen was identified as the bromodomain­containing protein 2 (BRD2), a transcriptional regulator that plays a pivotal role in the transcriptional control of diverse genes. BRD2 was upregulated in HCC tissues of the H­ras12V­tg mouse and human subjects, as demonstrated using western blotting or immunohistochemical analysis, with the BRD2 autoantibody. In addition, the truncated BRD2 reactive to the BRD2 autoantibody was detected in tumor cell­derived exosomes, which possibly activated TA immune responses and the generation of autoantibodies. For the detection of the serum BRD2 autoantibody, epitope mimicries of autoantigenic BRD2 were screened from a random cyclic peptide CX7C library with the BRD2 autoantibody. A mimotope with the sequence of CTSVFLPHC, which was cyclized by one pair of cysteine residues, exhibited high affinity to the BRD2 autoantibody and competitively inhibited the binding of the autoantibody to the cellular BRD2 antigen. The use of this cyclic peptide as a capture antigen in human serum enzyme­linked immunosorbent assay allowed the distinction of patients with HCC from healthy subjects with 64.41% sensitivity and 82.42% specificity (area under the ROC curve, 0.7761), which is superior to serum alpha­fetoprotein (AFP; 35.83% sensitivity; 100% specificity; area under the ROC curve, 0.5337) for the diagnosis of HCC. In addition, the detection of the BRD2 autoantibody combined with other autoantibody biomarkers or AFP has increased the accuracy of HCC diagnosis, suggesting that the combinational detection of cancer biomarkers, including the BRD2 autoantibody, is a promising assay for HCC diagnosis.

Screening of sperm antigen epitopes by phage display technique and its preliminary clinical application.

BACKGROUND: At present, there is a lack of standardized preparation methods of sperm antigen for the detection of antisperm antibody (AsAb). To screen sperm antigen mimotopes from a phage display random peptide library and use them to establish an enzyme-linked immunosorbent assay (ELISA) for the detection of AsAb, immunoglobulins were extracted from the sera of rabbits with positive AsAb and negative AsAb, respectively, by the saturated ammonium sulfate method, and a phage display 12-mer peptide library was affinity panned by the extracted immunoglobins coated on the ELISA plate. Then, the obtained positive phage clones were identified by ELISA and sent for sequencing and peptides synthesis. Last, a diagnostic ELISA was established to detect clinical serum and seminal plasma samples. RESULTS: A total of sixty phage clones were chosen by affinity panning, and sixteen of them reacted positively with AsAb in indirect ELISA and sandwich ELISA. Following DNA sequencing and translation, the peptide sequences of the sixteen positive clones were obtained. By comparison in Blast database, four of sixteen positive clones were found to be closely related to male reproduction. Two (#1 and #25) of four mimotopes were synthesized, and an ELISA method was established using the two mimotopes as sperm specific antigens. One hundred and thirty-four serum samples and seventy-four seminal plasma samples from infertile couples were analyzed by the established ELISA with #1 and #25 mimotopes, respectively. The positive rates of AsAb in serum samples were 20.15% (27/134) for #1 and 11.19% (15/134) for #25, respectively, and the coincidence rate between them was 91.04% (122/134). The positive rates of AsAb in seminal plasma samples were 1.35% (1/74) for both #1 and #25, and the coincidence rate was 100%. CONCLUSION: Sperm antigen mimotopes can be obtained successfully by the phage display technique, and can be used as standard sperm specific antigens to establish an ELISA method for the detection of AsAb.

RéSUMé: CONTEXTE: À ce jour, il n'existe pas de méthodes normalisées de préparation d'antigènes spermatiques pour la détection des anticorps anti-spermatozoïdes (ACAS). Dans le but d'élaborer un tel test ELISA (enzyme-linked immunosorbent assay), nous avons extrait de sérum de lapins des anticorps anti-spermatozoïdes humains via la technique du sulfate d'ammonium saturé et en ayant recours à une librairie phagique de peptides (12-mer). Les clones positifs ont été identifiés par ELISA, séquencés à façon et les peptides correspondants ont été synthétisés. In fine, un test ELISA diagnostic a été conçu pour être utilisé avec des échantillons cliniques de sérum et de plasmas séminaux. RéSULTATS: Au total, soixante clones de phages ont été sélectionnés, et seize d'entre eux se sont avérés interagir avec les ACAS en ELISA indirect comme en ELISA sandwich. Les séquences peptidiques de ces seize clones positifs ont été obtenues. Par comparaison avec les bases de données (Blast), quatre de ces seize clones positifs se sont révélés être étroitement liés à la reproduction masculine. Deux des quatre mimotopes (#1 et #25) ont été synthétisés, et un test ELISA a été généré en utilisant ces deux mimotopes comme antigènes spécifiques des spermatozoïdes. Cent trente-quatre échantillons de sérum et soixante-quatorze échantillons de plasma séminal de patients de couples infertiles ont alors été analysés avec ce test ELISA. Respectivement, les échantillons sériques se sont révélés positifs à 20,15% (27/134) pour le mimotope #1 et à 11,19% (15/134) pour le mimotope #25, avec un taux de coïncidence de 91,04% (122/134). Seul un échantillon de plasma séminal (1/74, soit 1, 35%) s'est révélé positif à la fois pour le mimotope #1 et #25 (coïncidence 100%). CONCLUSION: La technique « phage display¼ nous a permis d'identifier des mimotopes d'antigènes spermatiques qui ont pu être utilisés afin de générer un test ELISA pour la détection d'anticorps anti-spermatozoïdes.

Development of a sensitive phage-mimotope and horseradish peroxidase based electrochemical immunosensor for detection of O,O-dimethyl organophosphorus pesticides.

In this work, a green, harmless and signal-amplified electrochemical immunosensor based on phage-mimotope M31 (C-P-D-G-N-H-V-P-F-C) and horseradish peroxidase (HRP) was constructed for detecting O,O-dimethyl organophosphorus pesticides (OPs). The glassy carbon electrode (GCE) was modified by nitrogen and boron doped carbon quantum dots and graphene oxide (NBCQDs@GO) which can provide sufficient surface area and enhance the conductivity of the electrode. The O,O-dimethyl OPs class specific antibody mAb3C9 was assembled onto the NBCQDs@GO and the phage-mimotope M31 competitively bound to mAb3C9 with OPs. Furthermore, large amounts of anti-M13 mAb-HRP were introduced to the electrode through thousands of binding sites on the capsid of phage. HRP can catalyze 4-chloro-1-naphthol (4-CN) to produce insoluble precipitates (Benzo-4-chlorhexanedione, 4-CD). Hence, the concentration of OPs can be quantified by measuring impedance signal with electrochemical impedance spectrum (EIS). Under the optimal detection conditions, the 50% inhibitory concentration (IC50) and limits of detection (LODs) values of 9 O,O-dimethyl OPs were in range of 0.989-4.017 ng/mL and 0.003-0.014 ng/mL, respectively. The recovery rates of spiked OPs in cucumber, cabbage and lettuce were 88.20-112.50% with coefficient of variation from 2.97 to 15.64%. Therefore, the immunosensor showed very good sensitivity and demonstrating potential application for the detection of O,O-dimethyl OPs in food samples.

Harnessing Antibody Polyspecificity for Cancer Immunotherapy.

Targeting the diverse glycan repertoire expressed on tumor cells is considered a viable therapeutic strategy to deal with tumor cell heterogeneity. Inherently polyspecific, natural, glycan-reactive antibodies are purported to be protective in thwarting infections and in cancer immunotherapy. Tumor-associated carbohydrate antigens (TACAs) are related to pathogen glycans, to which nascent or natural antibodies exist and IgM responses are elicited. To capture the polyspecific nature of anticarbohydrate responses, we have focused on the rational design of carbohydrate mimetic peptides (CMPs) cross-reactive with TACA reactive antibodies. In particular, we have focused on the development of CMPs that display reactivity to GD2 and Lewis Y (LeY) reactive monoclonal antibodies. They would serve as templates for pan-immunogens inducing biosimilar polyreactive antibodies. In the design, we relied on structural analyses of CMP's enhanced binding to the templates using molecular modeling. Glycan reactivity patterns of affinity CMP-purified human antibodies further refined specificity profiles in comparison with the immune response to the CMP in clinical trials. In this study, we further define the molecular characteristics for this mimicry by considering the polyspecificity of LeY and GD2 reactive antibodies binding to the lacto-ceramide core Galß(1,4)Glcß(1-1')Cer. Binding to this minimum building block can be capitalized on for cancer therapy and diagnostics and illustrates a new approach in designing cancer vaccines taking advantage of the latent polyspecificity of antibodies and the relevance of natural antibodies in antigen discovery and design.

Enhancing epitope of PEDV spike protein.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80-100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.