Ag-Ab Complexes Research Articles

A Novel Epitope Dataset: Performance of the MCL-Based Algorithms to Generate Dataset for Graph Learning Model

Naturally, the epitope dataset can be presented as a graph. Dataset preparation in the previous methods is part of model development. There are many graph-based classification and regression methods. Still, it is difficult to identify their performance on the conformational epitope prediction model because datasets in a suitable format are unavailable. This research aims to build a dataset in a suitable format to evaluate kernel graph and graph convolution network. This dataset, which results from graph clustering on graph antigens, can be used to identify the performance of many graph neural network-based algorithms for conformational epitope prediction. The Ag-Ab complexes that meet the criteria for forming a conformational epitope prediction dataset from previous studies were downloaded from the Protein Data Bank. Raw datasets in the form of specific exposed antigen chain residues are labeled as epitope or non-epitope based on their proximity to the paratope. The engineering features in the raw dataset are derived from the structure of the antigen-antibody complex and the propensity score. Aggregating atomic-level interactions into residual levels create an initial graph of the antigen chain. The MCL, MLR-MCL, and PS-MCL are graph clustering algorithms to obtain labeled sub-clusters from the initial graph. A balance factor parameter is set to several values to identify the optimal dataset formation based on minimal fragmentation. The output of the MCL algorithm is used as a baseline. As a result of the fragmentation analysis that occurs, the MLR-MCL algorithm gives the best model performance at a balance factor equal to 2. PS-MCL gives the best performance at a value of 0.9. Based on the minimum fragmentation, the MLR-MCL algorithm provides the best model performance compared to MCL and PS-MCL. The dataset in a format according to benchmarking dataset can be used to identify the characteristics of antigen subgraphs formed from the graph clustering process and to explore the performance of graph-based learning conformational epitope prediction models such as graph convolution networks.

Immunotherapeutic and immunomodulatory potentials of Antigen-Antibody complex vaccines

Immune system battles with deadly pathogens that mostly deteriorate health and cause morbidity and mortality. Antibodies are considered great players in the elimination of pathogens and hence, provide a shield against the future onset of various diseases. The immune complex, also known as an antigen–antibody (Ag-Ab) complex, exhibits the immunomodulatory potential leading to enhanced vaccine efficacy. A deeper understanding of Fc receptors (FcRs) and the interaction of the Fc part of an antibody with the different immune cells lead to in-depth knowledge of using this strategy for the development of immune complex vaccines. The concept of the Ag-Ab complex has been used in the prevention and therapy of various viral and bacterial diseases. Ag-Ab complex is gaining attention in COVID-19 vaccine development too, due to their greater immunoregulatory potential. The present literature highlights the importance of the Ag-Ab complex, the role of Fc receptors in immunomodulation, and their success as vaccines in viral and bacterial diseases of human and animal origin. Moreover, potential areas and lapses are explored to make better use of this prospect as a vaccine candidate. Studies revealed that the immunogenic and immunomodulatory potential of Ag-Ab complex can lead to greater protection. However, there is a dire need of establishing a link between laboratory and clinical findings to make it effective and safe tool for in vivo treatments.

Design of highly sensitive nano-biosensor for diagnosis of hydatid cyst based on gold nanoparticles

Cystic echinococcosis, a zoonotic parasitic infection, is a major public health and economic concern, with worldwide distribution. The development of sensitive diagnostic methods for hydatid disease is important. We designed a highly sensitive nano-biosensor for the diagnosis of hydatid cyst based on gold nanoparticles (AuNPs). AuNPs were synthesized. Echinococcus granulosus antigen was coated on the ELISA microwells. Then, the E. granulosus IgG antibody was added to the microwells. After incubation and washing, the Ag-Ab complex was incubated with a human IgG HRP​-conjugated antibody. Then, the synthesized AuNPs and tetramethylbenzidine (TMB), as a chromogenic substrate of HRP, were added to the reaction. Finally, the absorption rate was measured by spectrophotometry. The results showed that the enzyme peroxide and TMB change the color of the reaction from red to yellow by oxidizing AuNPs. The sensitivity and specificity of the designed method were investigated. The linear equation and regeneration of nanobiosensor designed for red color Y = 0.0312X + 0.649, R2 9962 and for yellow color Y = 0.013X + 0.398, R2 9851 were determined. The limit of detection of the designed nanobiosensor was 0.001 μg mL−1. The results confirmed that the designed nanobiosensor was completely specific for the detection of E. granulosus antibody.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Optimizing heartworm diagnosis in dogs using multiple test combinations

BackgroundVarious heartworm (HW) diagnostic testing modalities detect products of, or reactions to, different life cycle stages of Dirofilaria immitis. Microfilariae (Mf) can be directly visualized in blood, antigen (Ag) from immature and adult heartworms may be detected on commercial assays, and antibody (Ab) tests detect the host immune response to larval stages. Ag and Mf tests are commonly used in dogs, which frequently carry adult HW infections, but Ab tests have only been validated for use in cats. In some HW-infected dogs, Ag is blocked by immune complexing leading to false-negative results. Heat-treatment (HT) to disrupt these complexes can increase the sensitivity of HW Ag tests. The aim of this study was to compare different methods for diagnosing HW infection in dogs at high risk using individual and paired diagnostic tests, including an exploration of using Ab tests designed for cats to test canine samples.MethodsOne hundred stray adult (≥ 2-year-old) dogs in Florida shelters were tested using Mf, HW Ag, and HW Ab tests (feline HW Ab tests currently not commercially validated/approved for use in dogs); two versions of each test platform were used.ResultsFourteen dogs tested positive using point-of-care (POC) Ag tests; an additional 2 dogs tested positive with microtiter well assay, and an additional 12 dogs tested positive using HT Ag testing. For individual tests, Ag test sensitivity/specificity compared to HT Ag was 50–57%/100%, and Ab tests were 46–64%/82–94%. Sensitivity estimates for individual tests were higher when comparing to non-HT Ag. Pairing POC Ag tests with Mf tests improved sensitivity without loss of specificity, while pairing POC Ag and Ab tests modestly increased sensitivity at the expense of specificity.ConclusionsScreening dogs for HW infection using both POC Ag and Mf detection, which is recommended by the American Heartworm Society, improved diagnostic performance in this study compared to single Ag test use, but may have missed more than one in four infected dogs. The need to improve access to highly accurate, rapid, and inexpensive large-scale HW testing for dogs in animal shelters remains largely unmet by current testing availability. The development of practical and validated protocols that incorporate heat or chemical treatment to disrupt Ag-Ab complexes in POC testing or decreasing the cost and time required for such testing in reference laboratories might provide solutions to this unmet need. Similar studies performed in countries where the prevalence of parasites such as D. repens or A. vasorum is different to the USA could potentially yield very different positive predictive values for both HT and non-HT Ag tests.Graphical abstract

A molecular dynamic simulation approach: development of dengue virus vaccine by affinity improvement techniques

This study is about proposing a vaccine for all four strains of dengue virus (DENV) that could be an important approach for reaching the WHO goal of reducing dengue morbidity and mortality. The significance of the DENV envelope proteins III lies in the fact that it elicits an immune response and hence can be a potential vaccine design candidate. This domain appears to play a key role in the host cell receptor binding for viral entry and in inducing long lasting protective immunity against the infection. We used long molecular dynamic simulation and mutagenesis scanning methods to provide the dynamic environment and propose the potential mutation that may result in enhancing the binding specificity and affinity of the antigen–antibody (Ag–Ab) complex. The binding free energetics were also estimated using free energy perturbation method. One charged mutation that is theorinine 93L to arginine interacting with epitopic glutamic acid 368 strongly contributing in increasing the binding affinity as well as specificity, predicted as −9.6 kcal/mol gain in 2H12-Fab with dengue envelope domain III binding free energy relative to the wild-type. In conclusion, the one charged residue that showed theoretically enhances the binding affinity of Ag–Ab complex by making couple of interactions i.e. by substituting theorinine to arginine in the antibody chains and can be considered as potential dengue vaccine candidate. Communicated by Ramaswamy H. Sarma

Enhancement of conformational B-cell epitope prediction using CluSMOTE.

BackgroundA conformational B-cell epitope is one of the main components of vaccine design. It contains separate segments in its sequence, which are spatially close in the antigen chain. The availability of Ag-Ab complex data on the Protein Data Bank allows for the development predictive methods. Several epitope prediction models also have been developed, including learning-based methods. However, the performance of the model is still not optimum. The main problem in learning-based prediction models is class imbalance.MethodsThis study proposes CluSMOTE, which is a combination of a cluster-based undersampling method and Synthetic Minority Oversampling Technique. The approach is used to generate other sample data to ensure that the dataset of the conformational epitope is balanced. The Hierarchical DBSCAN algorithm is performed to identify the cluster in the majority class. Some of the randomly selected data is taken from each cluster, considering the oversampling degree, and combined with the minority class data. The balance data is utilized as the training dataset to develop a conformational epitope prediction. Furthermore, two binary classification methods, Support Vector Machine and Decision Tree, are separately used to develop model prediction and to evaluate the performance of CluSMOTE in predicting conformational B-cell epitope. The experiment is focused on determining the best parameter for optimal CluSMOTE. Two independent datasets are used to compare the proposed prediction model with state of the art methods. The first and the second datasets represent the general protein and the glycoprotein antigens respectively.ResultThe experimental result shows that CluSMOTE Decision Tree outperformed the Support Vector Machine in terms of AUC and Gmean as performance measurements. The mean AUC of CluSMOTE Decision Tree in the Kringelum and the SEPPA 3 test sets are 0.83 and 0.766, respectively. This shows that CluSMOTE Decision Tree is better than other methods in the general protein antigen, though comparable with SEPPA 3 in the glycoprotein antigen.

Benchmarking the PEPOP methods for mimicking discontinuous epitopes

BackgroundComputational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances.ResultsBenchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method.ConclusionThe PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, “optimized” methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.

Establishment of a protein biochip to detect serum IgG antibodies against IL-2 and soluble CD25 in hemophagocytic lymphohistiocytosis

BackgroundInterleukin-2 (IL-2) and soluble CD25 (sCD25) are among the most important cytokines and diagnostic biomarkers in hemophagocytic lymphohistiocytosis (HLH). Detecting serum level of IL-2 and sCD25 is valuable for making clinical diagnosis and treatment decision in HLH. MethodsSince tests showing serum IgG antibody against IL-2 or sCD25 have never been carried out, a new protein biochip, which was modified with cysteine and activated sophorolipid (Cys-SL), was developed. ResultsLimits of detection on the biochip were 78 pg/ml for IL-2 and 39 pg/ml for sCD25, respectively. The data showed that on-chip seroimmunological responses to IL-2 and sCD25 proteins were 20.8% and 83.1% and the seroprevalence of IL-2 and sCD25 IgG antibodies were 45.5% and 57.2%, respectively. Data collection for the seroprevalence of serum antigen-antibody complex of sCD25 was 68.8%. The new biochip model shared similar sensitivity and specificity to chemiluminescent immunoassay (CLIA) in its measuring capacity of serum sCD25. ConclusionsWe addressed and confirmed the involvement of serum IgG antibodies against IL-2 and sCD25 as well as Ag-Ab complex of sCD25 in HLH patients. Therefore, this biochip platform would offer a new technological substitution for clinical serological diagnosis of HLH.

Conformational and dynamical basis for cross-reactivity observed between anti HIV-1 protease antibody with protease and an epitope peptide from it

F11.2.32 is a monoclonal antibody raised against HIV-1 protease and it inhibits protease activity. While the structure of the epitope peptide in complex with the antibody is known, how protease interacts with the antibody is not known. In this study, we model the conformational features of the free and bound epitope peptide and protease-antibody interactions. We find through our simulations, that the free epitope peptide P36–46 samples conformations akin to the bound conformation of the peptide in complex with the Ab, with a β-turn conformation sampled by the 38LPGR41 sequence highlighting the role of inherent conformational preferences of the peptide. Further, to determine the interactions present between the protease and antibody, we docked the protease in its conformation observed in the crystal structure, onto the antibody and simulated the dynamics of the complex in explicit water. We have identified the key residues involved in hydrogen-bond interactions and salt-bridges in Ag-Ab complex and examined the role of CDR flexibility in binding different conformations of the same epitope sequence in peptide and protein antigens. Thus, our results provide the basis for understanding the cross-reactivity observed between the antibody with protease and the epitope peptide from it.

Abstract LB-176: HE4 antigen-autoantibody complexes complement CA125 for detecting early stage ovarian cancer and can be elevated with CA125 in preclinical ovarian cancer

Abstract Early detection of ovarian cancer could significantly improve patient outcomes. Serial monitoring of CA125 with the Risk of Ovarian Cancer Algorithm (ROCA) can attain sufficient specificity for cost effective screening when combined with transvaginal sonography, but not all early stage disease can be detected with CA125 alone. Sensitivity could be improved by combining CA125 with other biomarkers. Different autoantibodies (AAb) have been evaluated including TP53 AAb, but antigen-autoantibody (Ag-Ab) complexes have received less attention. We have measured AAb reactive with the serum biomarker human epididymal protein 4 (HE4) and Ag-Ab complexes containing HE4 in sera from patients with epithelial ovarian cancer at the time of conventional diagnosis and from asymptomatic women with or without ovarian cancer enrolled in the Normal Risk Ovarian Cancer Screening Study (NROSS). We developed a Magplex-based immunoassay to measure autoantibodies against HE4 in human serum. Immune complexes were dissociated in pH3 buffer for 30 min, separated on a desalting spin column and assayed for HE4 AAb. When sera from patients with early stage (I-II) ovarian cancer (n=72) and from healthy women (n=213) in the NROSS trial were assayed for free HE4 autoantibody, 4 cases (5.6%) had autoantibodies against HE4 at 98% specificity using a cut-off of 80 ng/mL. HE4 Ag-Ab complexes were detected in sera from 29 of 72 ovarian cancer cases (40.3%) at early stage (I-II) at the time of conventional diagnosis using a cut-off of 730 ng/mL to achieve a specificity of 98.2%. Complementarity was observed between HE4 Ag-Ab complexes and CA125 levels in early stage cancer cases. Either HE4 Ag-Ab complexes or CA125 were detected in 58 of 72 sera (80.2%) in cancer cases. This result suggests that combining assays for HE4 Ag-Ab complexes and for CA125 might improve detection of early stage ovarian cancer. To determine whether HE4 Ag-Ab complexes could be detected at an earlier interval than CA125, we assayed preclinical serum samples from 16 ovarian cancer patients, 2 women with endometrial cancer and 5 with benign pelvic disease from the NROSS study. Elevated HE4 Ag-Ab complexes were detected in 5 of 16 ovarian cancer cases (31.2%) and exhibited a strong correlation with rising CA125. Free HE4 AAb was found in 2 cases immediately before diagnosis. Consequently, HE4 AAb and Ag-Ab complexes, in contrast to TP53 AAb, appear to arise at the time of CA125 elevation, but can still detect early stage disease, complement CA125 and deserve further evaluation for screening and early detection. Citation Format: Wei-Lei Yang, Zhen Lu, Karen H. Lu, Usha Menon, Makoto Kobayashi, Samir Hanash, Steven Skates, Robert C. Bast. HE4 antigen-autoantibody complexes complement CA125 for detecting early stage ovarian cancer and can be elevated with CA125 in preclinical ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-176.

Antigen Induced Dynamic Conformation Changes of Antibody to Facilitate Recognition of Fc Receptors

Fc-γ Receptors efficiently internalize antigen-antibodies (Ag-Ab) complexes and thus induce processing of antigens into peptides presented by MHC class II molecules. The recognition of p-MHC complex by T-cell receptor (TCR) trigger further immune reactions. It is still unclear how antigen binding in antibody and p-MHC binding in TCR communicate allosterically with their receptor bindings, respectively. The CDRs are responsible for Ag recognition, while the constant domains are crucial to activate effector function. To study the allosteric effects of antigen binding, we simulated four complex structures of free antibody, antigen bound antibody, FcR bound antibody, and antigen-antibody-FcR complex. As the conformation dynamics of antibodies are critical to antibody function, we chose 12 independent initial conformation to enhance the sampling. We reproduced experimental domain distribution density for the apo antibody, with the two Fabs move highly dynamic around the Fc region with Fab and Fc domains constantly forming and breaking contacts. When bind to antigen, the antibody conformation shift to two dominant conformation clusters, with one cluster has Fc-receptor binding site widely open. The final binding of Fc-γ Receptors leads to two conformation clusters, one corresponding to the antigen-free antibody-FcR baseline binding, and one for stronger antigen enhanced antibody-FcR interaction. Structural analysis of the antigen-antibody-FcR complex suggested that the antigen binding signal can transferred from the CDRs to the FcR by two main pathways, i.e. through hinge region and CH1/CL region. The results suggested that the stable contacts between CH1/CL and FcR is important for the transfer of antigen binding signals. Our study revealed the atomic mechanisms of the allosteric communications connection the antibody-antigen recognition and following FcR activation. During these processes, antigen binding shifts the antibody conformations which facilitate the FcR binding as well as allosteric signal transfer.

C1q-Dependent Dendritic Cell Cross-Presentation of In Vivo-Formed Antigen-Antibody Complexes.

Dendritic cells (DCs) are specialized in Ag engulfment via a wide variety of uptake receptors on their cell surface. In the present study we investigated Ag uptake and presentation of in vivo-formed Ag-Ab complexes by i.v. injecting mice with Ag-specific Abs followed by the cognate Ag. We show by this natural Ab-mediated Ag targeting system that uptake by splenic APC subsets is severely hampered in mice lacking complement factor C1q (C1qa-/-). Moreover, no detectable Ag cross-presentation by CD8α+ DCs from C1qa-/- mice was found. On the contrary, Ag uptake was not hampered by APCs in FcγRI/II/III/IV-deficient (FcγR quadruple-/-) mice, and the cross-presentation ability of CD8α+ DCs was not affected. In conclusion, we show that C1q rather than FcγRs controls the Ab-mediated Ag uptake and its presentation by spleen APC subsets to T cells.

Magnetic Printing of a Biosensor: Inexpensive Rapid Sensing To Detect Picomolar Amounts of Antigen with Antibody-Functionalized Carbon Nanotubes.

When an antibody (Ab) is immobilized on its surface, a carbon nanotube (CNT) becomes a biosensor that detects the corresponding antigen (Ag) because Ag-Ab complexes formed on the CNT surface moderate the current flow through it. We synthesized a biological ink containing CNTs that are twice functionalized, first with magnetic nanoparticles and thereafter with the anti-c-Myc monoclonal Ab. The ink is pipetted and dynamically self-organized by an external magnetic field into a dense electrically conducting sensor strip that measures the decrease in current when a sample containing c-Myc Ag is deposited on it. Prototypes are rapidly fabricated materials that cost less than 20 cents (Canadian) for each sensor. With larger current decreases due to real-time specific Ag-Ab binding for higher c-Myc concentrations, the biosensor distinguishes between picomolar c-Myc concentrations within a minute, offering proof of concept of a simple, rapid, economical, and sensitive method to detect specific molecules recognizable by Abs.

Applying Nanosized Gold and Carbon Immunolabels for the Quantitative Detection of Specific Ag–Ab Complexes by Using Atomic Force Microscopy

The application of gold and carbon labels for the detection of antigen molecules using the atomic force microscopy (AFM) method is described in the current study. Direct AFM visualisation made it possible to observe the “antigen–antibody–gold+protein A” and “antigen–antibody– carbon+protein G” complexes. In addition, it was also possible to estimate the morphology and describe the distribution on a surface that used plastic microwell plates as a substrate. The sensitivity of the method for each conjugate was evaluated and compared to an enzyme-linked immunosorbent assay. The results obtained point to a high sensitivity of the approach, especially when using carbon tags. In addition, certain advantages of carbon tags are indicated, when compared with gold tags, because of their simple detection and unambiguous identification on the collected images. Keywords: Antigen–antibody complex, Atomic force microscopy, carbon conjugates, colloidal gold conjugates, molecules identification atomic force microscopy, protein A and protein G.

Design, expression, and characterization of a novel dendritic cell-targeted proteins

In vivo approaches to inducing an effective immune response focus on targeted antigen (Ag) delivery to dendritic cells (DCs). In this study, we developed a new method of targeting plasmid DNA and/or the antigen (Ag)–antibody (Ab) complex to DCs via the DC receptor DEC-205, also known as cluster of differentiation CD205. We cloned and expressed a recombinant protein composed of mouse DEC-205-specific single-chain fragment variable region (mDEC-205-scFv), the streptococcal protein G (SPG) IgG-binding domain and cationic peptide (CP), which named mDEC205-scFv-SPG-CP (msSC). In vitro, the recombinant protein msSC can specifically bind to DCs through the section of mDEC-205-scFv, and bound the Ag–Ab complex via SPG as well as plasmid DNA through electrostatic bonding with CP in vitro. In addition, msSC functioned in a manner similar to anti-DEC-205 monoclonal Ab and bound to mouse bone marrow-derived DCs. It was demonstrated in vivo that msSC can target plasmid DNA to DCs, resulting in efficient uptake and expression. Moreover, msSC can form a complex with pGL3-CMV and transport it to draining lymph nodes when injected in vivo. These results indicate that msSC can be used as a carrier protein for vaccine delivery to DCs via formation of plasmid DNA-Ag–Ab ternary complexes.

Cutting edge: Expression of FcγRIIB tempers memory CD8 T cell function in vivo.

During reinfection, high-affinity IgG Abs form complexes with both soluble Ag and Ag displayed on the surface of infected cells. These interactions regulate cellular activation of both innate cells and B cells, which express specific combinations of activating FcγRs (FcγRI, FcγRIII, FcγRIV) and/or the inhibitory FcγR (FcγRIIB). Direct proof for functional expression of FcγR by Ag-specific CD8 T cells is lacking. In this article, we show that the majority of memory CD8 T cells generated by bacterial or viral infection express only FcγRIIB, and that FcγRIIB could be detected on previously activated human CD8 T cells. Of note, FcγR stimulation during in vivo Ag challenge not only inhibited the cytotoxicity of memory CD8 T cells against peptide-loaded or virus-infected targets, but FcγRIIB blockade during homologous virus challenge enhanced the secondary CD8 T cell response. Thus, memory CD8 T cells intrinsically express a functional FcγRIIB, permitting Ag-Ab complexes to regulate secondary CD8 T cell responses.

Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient’s sera: A novel hypothesis to follow-up of Cystic echinococcosis after surgery

Cystic echinococcosis (CE) is caused by the larval stage of the tapeworm Echinococcus granulosus. Until now, CE does not have an effective follow-up after surgery. To date, CE follow-up is conducted based on either antibody or antigen detection assays by double antibody sandwich ELISA. Unlike antigen detection, antibodies to imply exposure to an Echinococcus infection while their titration could remain for a longer period (1–10years) after surgery. Likewise, antibody respond may be related to the location of a mature hydatid cyst. Antigen detection shows presence of CE infection, it is extremely important and necessary in follow-up of CE after surgery. The circulating antigen (CAg) titration decrease faster than circulating antibody (during 1–3weeks) after operation. Location of hydatid cyst in detecting antigen is affected less than antibody also. Regarding this subject, antigen detection has several limitations that lead to be used less in CE follow-up. Although, AgB 8/1-kDa subunit is considered as a principle and immunogenic CAg but sensitivity of CAg detection compared to with antibody has variable range, between 33% and 85% which owing to formation of circulating immune complexes (CICs) in result of antigen – antibody complex. The another problem is non using specific CAg (AgB 8/1-kDa subunit) for production of specific paratopes (rabbit hyper immune antiserum) against AgB 8/1-kDa which is used as capture (primary) antibody in double antibody sandwich ELISA assay. The designation of synthetic peptides from conserved regions of AgB 8/1-kDa can help to this problem. These regions (motifs) should be selected for allelic, dominant, immunogenic and conserved without any genetic variation. The first part of this hypothesis suggests which patient’s sera should be treated with acidic buffers such as boric acid, acetic acid, glycine/HCl, polyethylene glycol (PEG) or combination of each of them accompanied by boiling patient’s sera which causes breaking Ag-Ab complexes and in result of releasing AgB 8/1. These modifications are effective to releasing CAg from CIC. To date, the synthetic peptides have been widely used in CE serodiagnosis based on circulating antibody detection only. In second part of this hypothesis suggests the using synthetic peptide of p176 derived AgB 8/1-kDa subunit containing conserved specific epitopes for preparation of specific paratopes (rabbit hyper immune antiserum) based on CAg detection. So, there is no need any native antigens for preparation of non-specific rabbit hyper immune antiserum. These novel improvements can help to decrease the cross-reactivity with other parasitic diseases (specificity). Increasing antigen detection could make a chance in sensitivity of patient’s sera and in result of the best and suitable tool for CE follow-up.

Reversion of somatic mutations of the respiratory syncytial virus-specific human monoclonal antibody Fab19 reveal a direct relationship between association rate and neutralizing potency.

The role of affinity in determining neutralizing potency of mAbs directed against viruses is not well understood. We investigated the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in the Ab H chain V region of Fab19, a well-described neutralizing human mAb directed to respiratory syncytial virus. Comparison of the affinity-matured Ab Fab19 with recombinant Fab19 Abs that were variants containing reverted amino acids from the inferred unmutated ancestor sequence revealed the molecular basis for affinity maturation of this Ab. Enhanced binding was achieved through mutations in the third H chain CDR (HCDR3) that conferred a markedly faster on-rate and a desirable increase in antiviral neutralizing activity. In contrast, most somatic mutations in the HCDR1 and HCDR2 regions did not significantly enhance Ag binding or antiviral activity. We observed a direct relationship between the measured association rate (Kon) for F protein and antiviral activity. Modeling studies of the structure of the Ag-Ab complex suggested the HCDR3 loop interacts with the antigenic site A surface loop of the respiratory syncytial virus F protein, previously shown to contain the epitope for this Ab by experimentation. These studies define a direct relationship of affinity and neutralizing activity for a viral glycoprotein-specific human mAb.

A comprehensive lab cookbook for creating carbohydrate modified proteins for therapeutic applications

A comprehensive lab cookbook for creating carbohydrate modified proteins for therapeutic applications