Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Immunogenicity assessment of tbo-filgrastim in cancer patients receiving chemotherapy.

AIM: This integrated analysis examined the immunogenicity of tbo-filgrastim and its potential clinical impact in three Phase III randomized studies in patients with breast cancer, lung cancer and non-Hodgkin lymphoma receiving chemotherapy. RESULTS: Treatment-emergent antidrug antibodies (ADA) occurred in 3/213 (1.4%) breast cancer patients, 2/160 (1.3%) lung cancer patients and 1/63 (1.6%) patients with non-Hodgkin lymphoma. None of the treatment-emergent ADA showed cross-reactivity toward native granulocyte-colony stimulating factors or exhibited neutralizing activity against tbo-filgrastim. Among patients with treatment-emergent ADA, there was no treatment-related hypersensitivity or anaphylaxis and no evidence of loss of clinical efficacy. CONCLUSION: Tbo-filgrastim has demonstrated low immunogenicity in cancer patients receiving chemotherapy and ADA response does not impact safety and efficacy in the patients.

Assay signal as an alternative to titer for assessment of magnitude of an antidrug antibody response.

BACKGROUND: Titer methods are commonly used to characterize the magnitude of an antidrug antibody response. Assay S/N is an appealing alternative, but the circumstances under which use of signal-to-noise (S/N) is appropriate have not been well defined. RESULTS: We validated both titer and S/N-based methods for several therapeutics. S/N correlated strongly with titer both in aggregate and when examined on a per subject basis. Analysis of impact of antibody magnitude on pharmacokinetics yielded the same result using either method. Each assay demonstrated excellent precision, good linearity, and adequate drug tolerance. CONCLUSION: Under these circumstances, assay S/N is a valid alternative to titer for assessment of the magnitude of an antidrug antibody response.

Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

Low-cost and highly sensitive immunosensing platform for aflatoxins using one-step competitive displacement reaction mode and portable glucometer-based detection.

Aflatoxins are highly toxic secondary metabolites produced by a number of different fungi and present in a wide range of food and feed commodities. Herein, we designed a simple and low-cost immunosensing platform for highly sensitive detection of mycotoxins (aflatoxin B1, AFB1, used as a model) on polyethylenimine (PEI)-coated mesoporous silica nanocontainers (PEI-MSN). The assay was carried out by using a portable personal glucometer (PGM) as the readout based on a competitive displacement reaction mode between target AFB1 and its pseudo-hapten (PEI-MSN) for monoclonal anti-AFB1 antibody (mAb). To construct such an assay protocol, two nanostructures including mAb-labeled gold nanoparticle (mAb-AuNP) and PEI-MSN were initially synthesized, and then numerous glucose molecules were gated into the pores based on the interaction between negatively charged mAb-AuNP and positively charged PEI-MSN. In the presence of target AFB1, a competitive-type displacement reaction was implemented between mAb-AuNP and PEI-MSN by target AFB1 through the specific antigen-antibody reaction. Accompanying the reaction, target AFB1 could displace the mAb-AuNP from the surface of PEI-MSN, resulting in the release of the loading glucose from the pores due to the gate opened. The released glucose molecules could be quantitatively determined by using a portable PGM. Under optimal conditions, the PGM signal increased with the increment of AFB1 concentration in the range from 0.01 to 15 µg/kg (ppb) with a detection limit (LOD) of 5 ng/kg (5 ppt) at the 3sblank criterion. The selectivity and precision were acceptable. Importantly, the methodology was further validated for assaying naturally contaminated or spiked blank peanut samples, and consistent results between the PGM-based immunoassay and the referenced enzyme-linked immunosorbent assay (ELISA) were obtained. Therefore, the developed immunoassay provides a promising approach for rapid screening of organic pollutants because it is simple, low-cost, sensitive, specific, and without the need of multiple separation and washing steps.

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

Exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization.

Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology.

Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry.

Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by immobilization of one of the binding partners on a biosensor chip, followed by monitoring the binding equilibrium of the other partner. However, for the measurement of high-affinity binding, with dissociation constants in the picomolar range or lower, equilibration times exceed practical limits and one has to resort to the measurement of sorption kinetics. Here we evaluate a new technique, using PEIA(1)-ellipsometry and establishment of equilibrium in solution. Binding parameters are determined for two high-affinity anti-interleukin 6 antibodies, anti-IL6.16 and anti-IL6.8, and compared with values obtained by a bioassay, based on IL6-dependent cell growth, and with values obtained by a standard technique based on SPR.(2) The high affinities of both antibodies as found with the bioassay (5 and 50pM for anti-IL6.8 and anti-IL6.16, respectively), could be conveniently measured by PEIA-ellipsometry. Using SPR, equilibrium measurements indeed proved too time-consuming and analysis of adsorption/desorption kinetics revealed that the binding of the antibodies on the chip caused the appearance of different populations of antibodies with different affinities.

Critical review: assessment of interferon-ß immunogenicity in multiple sclerosis.

This review discusses type I interferon (IFN) immunogenicity with focus on methods of detection of anti-IFN antibodies in patients treated with human recombinant IFN-β. Pitfalls involved in the clinical use of various types of assays for binding antibodies and neutralizing antibodies against IFN-β are presented, and the widely held distinction between binding antibodies and neutralizing antibodies is questioned both in terms of detection and clinical importance. The article also addresses important bioavailability and pharmacokinetic issues occurring with prolonged use of protein drugs. The rationale for individualized or personalized medicine, ie, optimizing therapies according to individual needs rather than using standardized trial-and-error regimens to all patients, is highlighted.

Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

PURPOSE: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO(119-143) region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO(119-143) tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). EXPERIMENTAL DESIGN: We generated tetramers of DRB1*0101 incorporating peptide ESO(119-143) using a previously described strategy. We assessed ESO(119-143)-specific CD4 T cells in peptide-stimulated postvaccine cultures using the tetramers. We isolated DR1/ESO(119-143) tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO(119-143) tetramer(+) T cells ex vivo and characterized them phenotypically. RESULTS: Staining of cultures from vaccinated patients with DR1/ESO(119-143) tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO(123-137) as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO(119-143) tetramer(+) cells using T cell receptor (TCR) ß chain variable region (Vß)-specific antibodies, we identified several frequently used Vß. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. CONCLUSIONS: The development of DR1/ESO(119-143) tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients.

Antidrug antibody assay validation: industry survey results.

Immunogenicity of biopharmaceutical products has attracted considerable attention from the industrial, academia, and regulatory organizations. Many methods exist to detect and characterize level of antidrug antibody response in patients. Still, additional work is required to harmonize various approaches used throughout the industry. This review presents results of a survey sponsored by the American Association of Pharmaceutical Scientists that was designed to collect relevant information and to understand various methods used throughout the bioanalytical field for the detection and evaluation of antidrug antibody responses.

Determination of binding potency of peptidic inhibitors of Grb2-SH2 by using the protein-captured biosensor method.

The growth factor receptor-binding protein 2-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, therefore, peptidic inhibitors of the Grb2-SH2 domain has been chosen as our target for the development of antiproliferative agents. The inhibitory effects of peptide analogs on the Grb2-SH2 domain have been determined by using surface plasmon resonance (SPR) technology developed with the BIACORE biosensor. Recently, we reported the analysis of interactions between peptides and the GST-Grb2-SH2 that was immobilized on the surface of sensor chip by using BIACORE biosensor (the protein-immobilized method). Herein, we analyze interactions of peptides with the GST-Grb2-SH2 that was captured by the anti-GST antibodies immobilized on the surface of sensor chip (the protein-captured method). Results obtained by both methods are in good correlation, indicating the immobilization of GST-Grb2-SH2 on the sensor chip did not significantly affect the binding of Grb2-SH2 with peptides. Both SPR-based assays are very sensitive bioanalytical methods and can be applied in screening inhibitors of target proteins or purifying GST-fusion proteins, however, considering the efficiency and the cost, the GST-Grb2-SH2-immobilized method is suggested for routinely determining the binding potency of inhibitors of Grb2-SH2.

Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation.

Making sound proteomic inferences using ELISA microarray assay requires both an accurate prediction of protein concentration and a credible estimate of its error. We present a method using monotonic spline statistical models (MS), penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict ELISA microarray protein concentrations and estimate their prediction errors. We contrast the MSMC (monotone spline Monte Carlo) method with a LNLS (logistic nonlinear least squares) method using simulated and real ELISA microarray data sets.MSMC rendered good fits in almost all tests, including those with left and/or right clipped standard curves. MS predictions were nominally more accurate; especially at the extremes of the prediction curve. MC provided credible asymmetric prediction intervals for both MS and LN fits that were superior to LNLS propagation-of-error intervals in achieving the target statistical confidence. MSMC was more reliable when automated prediction across simultaneous assays was applied routinely with minimal user guidance.

A direct immunoassay assessment of streptavidin- and N-hydroxysuccinimide-modified biochips in validation of serological TNFalpha responses in hemophagocytic lymphohistiocytosis.

The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH(2) groups to N-hydroxysuccinimide (NHS)-activated surfaces and investigated the potential application of tumor necrosis factor-alpha (TNFalpha) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFalpha antigen and TNFalpha antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients (r) in TNFalpha and TNFalpha antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFalpha-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFalpha, TNFalpha antibody, and TNFalpha-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFalpha, 30.9% (13/42) for TNFalpha antibody, 28.6% (12/42) for TNFalpha-receptor 1 complex, and 26.1% (11/42) for TNFalpha-receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients.

Transfusion-related acute lung injury (TRALI): a review of investigations by the National Tissue Typing Laboratory of cases reported in New Zealand since June 2004.

AIM: To review investigations of reported cases of Transfusion-Related Acute Lung Injury (TRALI) performed by the National Tissue Typing laboratory since 2004. METHOD: Donors associated with reported cases of TRALI are recalled for white cell antibody tests. A donor is implicated if found to have neutrophil or HLA antibodies with specificity against one of the recipient's HLA antigens, or a positive white cell crossmatch. A retrospective review of investigations performed by the Tissue Typing Laboratory on TRALI cases from June 2004 to June 2007 was undertaken. RESULTS: Seventeen cases of TRALI had tests performed by the Tissue Typing Laboratory over the 3-year period. A total of 67 donors were tested. Twenty-nine donors had a positive HLA-antibody screen and the majority of these were female (86%, with fresh frozen plasma (FFP) the commonest component type (41%). In 15 (88%) cases, HLA antibodies were found in donor sera and nine of these had specificity against patient HLA antigen or a positive crossmatch. CONCLUSION: Preliminary data on TRALI investigations concur with overseas studies. Raising awareness of this hazard of transfusion and a consistent approach in investigation of TRALI will allow us to gain further insight into this complication in New Zealand and consequently explore strategies to prevent such adverse transfusion reactions.

Fluorescent resonance energy transfer based detection of biological contaminants through hybrid quantum dot-quencher interactions.

A nanoscale sensor employing fluorescent resonance energy transfer interactions between fluorescent quantum dots (QDs) and organic quencher molecules can be used for the multiplexed detection of biological antigens in solution. Detection occurs when the antigens to be detected displace quencher-labelled inactivated (or dead) antigens of the same type attached to QD-antibody complexes through equilibrium reactions. This unquenches the QDs, allowing detection to take place through the observation of photoluminescence in solution or through the fluorescence imaging of unquenched QD complexes trapped on filter surfaces. Multiplexing can be accomplished by using several different sizes of QDs, with each size QD labelled with an antibody for a different antigen, providing the ability to detect several types of antigens or biological contaminants simultaneously in near real-time with high specificity and sensitivity.

Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

Assessment of recent HIV-1 infection by a line immunoassay for HIV-1/2 confirmation.

BACKGROUND: Knowledge of the number of recent HIV infections is important for epidemiologic surveillance. Over the past decade approaches have been developed to estimate this number by testing HIV-seropositive specimens with assays that discriminate the lower concentration and avidity of HIV antibodies in early infection. We have investigated whether this "recency" information can also be gained from an HIV confirmatory assay. METHODS AND FINDINGS: The ability of a line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) to distinguish recent from older HIV-1 infection was evaluated in comparison with the Calypte HIV-1 BED Incidence enzyme immunoassay (BED-EIA). Both tests were conducted prospectively in all HIV infections newly diagnosed in Switzerland from July 2005 to June 2006. Clinical and laboratory information indicative of recent or older infection was obtained from physicians at the time of HIV diagnosis and used as the reference standard. BED-EIA and various recency algorithms utilizing the antibody reaction to INNO-LIA's five HIV-1 antigen bands were evaluated by logistic regression analysis. A total of 765 HIV-1 infections, 748 (97.8%) with complete test results, were newly diagnosed during the study. A negative or indeterminate HIV antibody assay at diagnosis, symptoms of primary HIV infection, or a negative HIV test during the past 12 mo classified 195 infections (26.1%) as recent (< or = 12 mo). Symptoms of CDC stages B or C classified 161 infections as older (21.5%), and 392 patients with no symptoms remained unclassified. BED-EIA ruled 65% of the 195 recent infections as recent and 80% of the 161 older infections as older. Two INNO-LIA algorithms showed 50% and 40% sensitivity combined with 95% and 99% specificity, respectively. Estimation of recent infection in the entire study population, based on actual results of the three tests and adjusted for a test's sensitivity and specificity, yielded 37% for BED-EIA compared to 35% and 33% for the two INNO-LIA algorithms. Window-based estimation with BED-EIA yielded 41% (95% confidence interval 36%-46%). CONCLUSIONS: Recency information can be extracted from INNO-LIA-based confirmatory testing at no additional costs. This method should improve epidemiologic surveillance in countries that routinely use INNO-LIA for HIV confirmation.

Assessment of the interaction between a synthetic epitope of troponin C and its specific antibody using a label-free faradaic impedimetric immunosensor and alpha-Keggin silicotungstic heteropolyacid as a redox probe.

The development of an immunosensor for the direct probing of the interaction between a cysteine-modified synthetic peptide, which corresponds to the epitope cTnC-89-98 of troponin C, and its specific antibody is described. Following immobilization of the peptide onto gold electrodes through the formation of a self-assembled monolayer, the alteration of the interfacial properties of the electrodes upon peptide-antibody interaction was traced by faradaic electrochemical impedance spectroscopy (EIS) using a silicotungstic heteropolyacid, H(4)SiO(4).12WO(3), as a redox probe. The electrochemical behaviour of the redox probe was evaluated with cyclic voltammetry and EIS. The effect of milk protein or 4-mercaptophenol, which was used as post-blocking agents, on the performance of the immunosensor, was investigated. Treatment with 4-mercaptophenol resulted in immunoeffective electrodes that successfully tested in anti-serum samples. An optimum dilution ratio of the samples, where the effect of the matrix on the measuring signal is negligible, was also determined.