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The SARS-CoV-2 pandemic has provided the stimulus for the rapid development of a variety of diagnostic testing methods. Initially these were deployed as screening tools to evidence spread of the virus within populations. The recent availability of vaccines against the virus and the need to better understand the parameters of post-infection protective immunity requires development of methods, suitable for use in the routine diagnostic laboratory, capable of characterising the viral immune response in greater detail. Such methods need to consider both cellular and humoral immunity. Toward this aim we have investigated use of a commercial multiplex assay (COVID Plus Assay, One Lambda), providing assessment of the SARS-CoV-2 response at structural level, and developed an in-house cell stimulation assay using commercially available viral peptides (Miltenyi). This paper reports our experience in use of these methods in extended investigation of a cohort of healthcare workers with prior screening results indicative of viral infection. The antibody response generated is shown to be both qualitatively and quantitatively different in different individuals. Similarly a recall response to SARS-CoV-2 antigen involving the T cell compartment can be readily demonstrated in recovered individuals but is of variable magnitude.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals., Graphical abstract Image 1

Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike. Thus, neutralization assays are not suitable to irrevocably demonstrate that the examined factors exert their effect via a paracrine mechanism. We were interested in investigating the impact of immunosuppressive factors present in ovarian carcinoma-associated ascites by dissecting paracrine versus autocrine effects of interleukin 10 (IL-10) and prostaglandin E2 (PGE2) on the activation of monocyte-derived dendritic cells (DC). We explored several methods of depletion based on introduction of the neutralizing antibodies bound to beads. Here we describe the pitfalls of the investigated depletion approaches and show the importance of monitoring the presence of residual neutralizing antibodies in the sample upon depletion, which impacts on the suitability of the approach to distinguish paracrine from autocrine effects. Only one of three investigated approaches showed no dislocation of neutralizing antibody from the beads into the sample. This method, which is based on covalently linking antibody to magnetic beads harbouring a reactive group allowed for the complete removal of the investigated factors from ascites and represents an elegant tool to elucidate immunoregulatory or -stimulatory cytokine networks in considerably more depth than the use of neutralizing antibodies in cell cultures alone can contribute.

Parenterally administered rotavirus vaccines may overcome the low efficacy observed in resource-poor regions that use live oral formulations. We have reported work on a trivalent nonreplicating rotavirus vaccine (NRRV) for parenteral administration consisting of the recombinant tetanus toxoid P2 CD4 epitope fused to a truncated VP8* fragment (P2-VP8*) for the P[4], P[6], and P[8] serotypes of rotavirus adjuvanted with aluminum. An essential part of developing this vaccine candidate was devising quantification methods for each antigen in the trivalent NRRV in the presence of aluminum adjuvant. This report describes the development of quantitative inhibition enzyme-linked immunosorbent assays (ELISAs) for in vitro antigenicity determination of the adjuvanted trivalent NRRV using serotype-specific monoclonal antibodies (mAbs) against each of the P2-VP8* antigens. Adjuvanted trivalent vaccine samples are titrated and incubated with a constant concentration of specific mAbs against each NRRV P2-VP8* antigen variant. Unbound mAbs are measured by ELISA to indirectly quantify the amount of each antigen present in the trivalent vaccine. Sensitive, specific, and reproducible inhibition ELISAs were developed and qualified for each antigen and used for final product quantification and release testing without desorption of the vaccine antigen.

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response., Highlights • High-throughput array-based characterization platform for polyclonal antibodies. • Development of a biophysical proxy for antibody effector function. • Antigen and Fc receptor recognition characteristics are captured. • Enables systematic serologic studies of NHP and human antibody samples.

Several lines of evidence indicate that antibody-dependent cellular cytotoxicity (Wren et al., 2013) is important in the pathogenesis of HIV-1 infection. Namely, ADCC is induced during natural HIV-1 infection or in HIV-1 vaccine studies, the latter demonstrated by the RV144 vaccine trial. To expedite the assessment of ADCC in studies of HIV, we have developed a high throughput assay. We have optimized the rapid fluorometric antibody-mediated cytotoxicity assay (RFADCC) by transfecting the EGFP-CEM-NKr cell line to constitutively express SNAP-tagged CCR5. This cell line can then serve as a source of HIV-specific targets when coated with monomeric gp120, spinoculated with inactivated intact virions, infected by cell-free viral diffusion or infected by cell-to-cell transmission of virus. The optimized strategy has two significant advantages over the original RFADCC method: First, the preparation of detectable target cells is less labor intensive and faster as it does not rely on multiple staining and washing steps for target cells. Second, because the target cell markers GFP and SNAP are constitutively expressed, the assay provides highly reproducible data. These strengths make the optimized RFADCC assay suitable not only for studies of HIV-1 specific cytotoxicity but also for studies of cell–cell transmission of virus. In conclusion, this assay provides a new generation T cell line that can expedite large clinical studies as well as research studies in humans or non-human primates.

There is much debate in the pharmaceutical industry on how to translate the current guidelines on immunogenicity testing for biotherapeutics into a testing strategy that suits the specific requirements of individual drug candidates. In this paper, member companies from the European immunogenicity platform (EIP) present a consensus view on the essential requirements for immunogenicity testing of a biotherapeutic throughout the various phases of drug development, to ensure patient safety and to enable successful market entry. Our aim is to open the debate and provoke discussion on this important topic which is unique to biotherapeutic drug development. The scope of this paper is limited to aspects relevant to biotherapeutic drug development and does not include fundamental academic studies of immunogenicity. Here, we propose two pre-defined testing strategies for the detection and characterization of anti-drug antibody (ADA) responses where the different strategies are based on the phase of development for a biotherapeutic, a. without (category 1) and b. with (category 2) the expected potential to elicit ADA mediated severe clinical consequences. The harm of a potential ADA response determines which of the two testing strategies is adopted. Rather than replacing the overall risk assessment which is known to be challenging and multi-factorial, the testing strategy selection is a starting point for immunogenicity testing which adapts throughout drug development as more information becomes available. The scientific rationale on which the "case-by-case" approach advocated in white papers and guidance documents may be translated for each individual drug development program is provided and, underpins the recommendations made here.

VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection.

Mycotoxin is one of the most important natural pollutants, which poses a global threat to food safety. However, the pollution of mold in food production is inevitable. The detection technology of mycotoxins in food production is an important means to prevent the damage of mycotoxins, so rapid detection and screening to avoid pollution diffusion is essential. The focus of this review is to update the literature on the detection of mycotoxins by surface plasmon resonance (SPR) technology, rather than just traditional chromatographic methods. As a relatively novel and simple analytical method, SPR has been proved to be fast, sensitive and label-free, and has been widely used in real-time qualitative and quantitative analysis of various pollutants. This paper aims to give a broad overview of the sensors for detection and analysis of several common mycotoxins.

Monoclonal antibodies have accelerated the availability of treatment options for many diseases in which the molecular mechanism has been elucidated in detail. Therefore, an assay that can universally analyze antibodies for clinical pharmacokinetics and cross-sectional studies would be indispensable. We have developed a universal antibody bioanalysis with a Fab-selective tryptic reaction, named nano-surface and molecular-orientation limited (nSMOL) proteolysis, that collects the specific antibody signature peptides in biological samples. Using the nSMOL method, we have fully validated the bioanalysis of many antibodies, Fc-fusion proteins, and their biosimilars. Inflammatory immune diseases often require long-term clinical management because of the remission and relapse observed. Accurate antibody monitoring in systemic circulation could contribute to the improvement of clinical outcomes. Because several biopharmaceuticals can be selected as practical treatment options, the assay development that quantitates many antibodies simultaneously would be applicable in many theraprutic monitoring. In this study, we have validated the LC-MS bioanalysis method for seven-mixed antibodies (Infliximab, Adalimumab, Ustekinumab, Golimumab, Eculizumab, Etanercept, and Abatacept) using the nSMOL normal reaction condition and two-mixed antibodies (Tocilizumab and Mepolizumab) using the acidified reduction acceleration condition, as reported in our previous papers. Moreover, this multiplexed assay has been verified using clinical patient samples. The nSMOL approach enables the quantitation of several immunosuppressive antibodies simultaneously in human serum, and nSMOL can potentially be applicable to the drug-drug interaction assays or therapeutic antibody monitoring of several inflammatory immune diseases to optimize administration.

Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ± 0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.

The ever-increasing research efforts to develop new antithrombotic therapies have led to the reassessment of the role of alpha-2-plasmin inhibitor (α2-PI) in pathological conditions. In particular, experimental stroke studies have suggested correlation between increased free α2-PI level and mortality. However there are only a small number of well-characterized and specific assays available for the measurements of free α2-PI. In plasma α2-PI undergoes both N- and/or C-terminal cleavages resulting four isoforms with modified susceptibility to FXIII catalyzed cross-linking to fibrin and/or loss of plasmin(ogen) binding. Present paper describes a new sandwich ELISA method for the determination of free total α2-PI in plasma and other body fluids. A newly generated biotinylated monoclonal antibody recognizes and captures all the four N- and/or C-terminally modified isoforms of α2-PI while HRPO-labeled polyclonal anti-α2-PI antibody detects the captured antigen. Performing the 2-step assay in streptavidin-coated microplate can be completed within three hours. The assay is well reproducible, total (within laboratory) imprecision in the normal, pathological and very low ranges were 7.4%, 9.1% and In conclusion, our ELISA enables accurate and fast measurement of total free α2-PI in human body fluids.

B cells are one of the fundamental components of the adaptive immune system and are best known for their ability to produce antibodies. Among the various types of inborn errors of immunity, antibody deficiencies represent the largest group in terms of the number of affected individuals. Not all antibody deficiencies are due to B cell intrinsic defects but investigating B cell number and function are a critical part of the diagnostic process. B cells studies in clinical practice almost always rely on flow cytometry as the main tool of investigation. The advantage of flow cytometry is that it allows absolute and relative counting of B cells, and their phenotypic and functional evaluation at a single-cell level, while allowing the analysis of a large number of cells. Although versatile and broad in its utility, clinical flow cytometry has both theoretical and practical limitations. These include lack of consensus about definitions and classifications, and the use of non-standardized methods. Patients in all age groups, from newborns to the elderly, may require testing, yet B cells show significant changes in both numbers and subset distribution over the lifespan, requiring distinct reference ranges for narrowly defined age brackets for accurate interpretation. Sampling for testing is usually restricted to peripheral blood samples, and the number of markers routinely used are limited. This paper will provide a brief overview of flow cytometry and B cell biology, describe the human peripheral B cell subsets most commonly identified in clinical flow cytometry and discuss their clinical relevance in different settings.

Taenia solium oncosphere protein TSOL18 is the host-protective antigen against porcine cysticercosis. Little attention has been given to use it as target molecule in immunodiagnostic tests. The objective of this paper is to describe the immunodiagnostic potential of monoclonal antibodies (MoAbs) raised against conformational epitopes of TSOL18. Three murine IgG1 MoAbs (25D12C1, 21C2D2, 10H1F2) against three different conformational epitopes of TSOL18 were produced and evaluated with an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. Serum samples from pigs immunized with TSOL18 inhibited the binding of the three MoAbs to TSOL18 antigen in i-ELISA. The highest inhibition of anti-TSOL18 antibodies in immunized pigs was observed with MoAb 25D12C1. Ten field sera (12.19%) from 82 non-vaccinated and non-infected pigs showed anti-oncosphere antibodies inhibiting the binding of MoAb 25D12C1. Anti-oncosphere antibodies in pigs experimentally infected with T. solium eggs inhibited the binding of MoAb 25D12C1 from 2 to 8 week-post infection. It is concluded that MoAb 25D12C1 has excellent immunodiagnostic potentials to detect anti-oncosphere antibodies in the intermediate hosts at early exposure to T. solium eggs. Further investigations on potential use of MoAb 25D12C1 in a capture antigen ELISA for the detection of post-oncospheral antigens in infected pigs cannot be overemphasized., Graphical abstract Unlabelled Image, Highlights • We describe the potential usefulness of the monoclonal antibody 25D12C1 to TSOL18 protein. • Serum samples from pigs immunized with TSOL18 inhibited the binding of 25D12C1 in an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. • Anti-oncosphere antibodies in pigs infected with T. solium eggs inhibited also the binding of 25D12C1. • It is concluded that MoAb i-ELISA using 25D12C1 has the potential to identify animals exposed to T. solium infection.

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori, measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage - 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics.

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products.

Serial limiting dilution (SLD) assays are used in many areas of infectious disease related research. This paper presents SLDAssay, a free and publicly available R software package and web tool for analyzing data from SLD assays. SLDAssay computes the maximum likelihood estimate (MLE) for the concentration of target cells, with corresponding exact and asymptotic confidence intervals. Exact and asymptotic goodness of fit p-values, and a bias-corrected (BC) MLE are also provided. No other publicly available software currently implements the BC MLE or the exact methods. For validation of SLDAssay, results from Myers et al. (1994) are replicated. Simulations demonstrate the BC MLE is less biased than the MLE. Additionally, simulations demonstrate that exact methods tend to give better confidence interval coverage and goodness-of-fit tests with lower type I error than the asymptotic methods. Additional advantages of using exact methods are also discussed.

The engulfment of cancer cells by macrophages is an important cellular process in innate cancer immunity. Antitumor immunotherapy that utilizes the enhanced engulfment of cancer cells by phagocytic cells has attracted much attention. Therefore, there is a growing demand for methods of measuring cancer cell phagocytosis. Quantifying the various stages of phagocytosis is invaluable for elucidating cancer-immune responses during this process. Here, we describe two phagocytosis assays, a flow cytometric assay and a fluorescent microscopic assay; the flow cytometric method utilizing CellTracker dye provides a simple, measurable, and highly reproducible functional assay to measure the phagocytosis efficiency of cancer cells by bone marrow-derived macrophages. As an alternative method of evaluating various states of cancer cell phagocytosis, a fluorescent microscopic method that employs a pH-sensitive dye (pHrodo-SE dye) is also described in this paper. Image-based analysis using this labeling approach enables researchers to measure phagocytic indices that indicate the number of cancer cells engulfed by each macrophage. We have highlighted that these assays can be applied to multiple tumor types and used as selection tools for a variety of phagocytosis agonist types. The results of this study may facilitate a better understanding of the interactions between tumor cells and phagocytes, which could lead to the identification of new therapeutic targets against cancer.

Various experimental model designs have been used to analyze the inflammatory pathways in human gastrointestinal illnesses. Traditionally, analytical techniques and animal models are popular experimental tools to study the inflammation process of intestinal diseases. However, the comparison of results between animal and human models is difficult for the inconsistency of outcomes. Although there are different animal models for studying the intestinal diseases, none of them fully represents the physiological and environmental conditions typical of the human species. Also, there is currently a concerted effort to decrease the experimental use of animals. On the converse, experimental protocols using the culture of gut mucosa had become popular with the advent of endoscopy which allows explanting multiple fragments from the intestine. The peculiar characteristic of this model is the ability to preserve in vitro the features that we found in vivo, thus also the response to various stimuli that differs from person to person. The aim of the present paper is to provide a review of some of the possible uses of the organ intestinal mucosa culture.

Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in developing novel biomarkers for cancer diagnosis and drug development.

T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes.

As a category A toxic, the botulinum toxin(BoNT) is responsible for human botulism with an estimated lethal dose of 1 ng/kg which greatly increases the potential risk of use as bioweapons. Therefore, the development of anti-BoNT antibodies is urgent. In this paper, the HC domain of BoNT/A was purified and immunized with Balb/c mice. Monoclonal antibodies were screened against BoNT/A from 55 stable positive hybridoma cell lines, and one with the strongest neutralizing activity, designated as ML06, was subcloned, sequenced, and classified as IgG1(κ) subclass. The mouse protection assays showed that ML06 can neutralize the toxin of BoNT/A effectively both in vitro and in vivo, in a dose-dependent manner. The therapeutic assays showed that only 20% of mice injected with 4 LD50 BoNT/A can survive another injection of ML06 after 4 h. The prophylaxis assays showed the residual ML06 from mice injected with ML06 two weeks ago can protect mice against 4 LD50 BoNT/A challenge completely. Collectively, our results indicated that ML06 served as a good candidate for further development of immune therapeutics for BoNT/A.

Cut point determination is an important aspect of immunogenicity assay development. The cut point can be influenced by a myriad of factors. Key among those is the analytical variability of the assay itself and biological variation due to test samples. Since a smaller cut point value may result in improved sensitivity, the existing procedures often employ statistical techniques such as outlier removal to produce a conservative cut point. Although such practices are intended to yield acceptable assay sensitivity, they may fail to fully account for biological variability in the data, thus generating higher than expected number of false positive results. In this paper, we introduce the concept of minimum cut point. It is defined as the cut point that is determined in the absence of biological variability. Under the log-normal assumption of the data used for cut point analysis, closed-form formulas are derived for the minimum cut point. This minimum cut point can be used to benchmark whether a cut point derived from a procedure can compromise assay specificity by being too low.

Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDR H3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs.

This paper describes a novel method for following the changes in mouse tumour-infiltrating immune cell populations by repeated sampling of tumours by fine needle aspiration (FNA), followed by flow cytometry. Using this technique we were able to collect samples from P815 mouse mastocytomas, and identify and enumerate six tumour-infiltrating immune cell types at multiple time points for each mouse. We demonstrate good agreement between cell percentages obtained from FNA samples and matched whole tumour digests (WTDs). We also demonstrate that neither survival nor the incidence of liver metastasis is adversely affected by the procedure. Our method has a clear advantage over the common practice of sacrificing mice and collecting tissue at pre-determined time points, as the technique allows 1) repeated sampling of each mouse over time, thus many fewer mice are required, and 2) the correlation of survival data with tumour-infiltrating immune cell types at different time points. This potentially allows immune cell types associated with increased or decreased survival to be identified. Therefore, our technique should greatly facilitate the characterisation of anti-tumour immunity induced in response to cancer therapy in small animal models.

The lymphatic system in mice consists of lymphatic vessels and 22 types of lymph nodes. Metastatic tumor cells in the lymphatic system spread to distant organs through the venous system. However, the communication routes between the lymphatic and venous systems have not been fully elucidated. Here, we identify the communication routes between the lymphatic and venous systems in the axillary and subiliac regions of MXH10/Mo-lpr/lpr inbred mice, which develop systemic swelling of lymph nodes up to 10mm in diameter, allowing investigation of the topography of the lymph nodes and lymphatic vessels. Using a gross anatomy dissection approach, the efferent lymphatic vessels of the proper axillary lymph node were shown to communicate with the subclavian vein. Furthermore, we found that the thoracoepigastric vein, which connects the subclavian vein and inferior vena cava, runs adjacent to the subiliac and proper axillary lymph nodes, and receives venous blood from these lymph nodes routed through small branches. The direction of blood flow in the thoracoepigastric vein occurred in two directions in the intermediate region between the proper axillary lymph node and subiliac lymph node; one to the subclavian vein, the other to the inferior vena cava. This paper reveals the anatomy of the communication between the lymphatic and venous systems in the axillary and subiliac regions of the mouse, and provides new insights relevant to the investigation of the mechanisms of lymph node metastasis and cancer immunology, and the development of diagnostic and treatment methods for lymph node metastasis, including drug delivery systems.

Rats are used as animal models for many human diseases. Cytokines can serve as biomarkers indicative of these diseases or disease states. Techniques for measuring cytokine expression levels often do not provide the sensitivity needed to measure these biomarkers in biological fluids because the concentrations of many cytokines are below the detection limits of conventional methods. In this paper, we present ultra-sensitive digital immunoassays using Single Molecule Arrays (Simoa) for seven rat cytokines: TNF-α, IL-10, IL-17F, GM-CSF, IFN-γ, IL-4, and IL-1α. These ultra-sensitive immunoassays have limits of detection (LODs) in the femtomolar range and provide the ability to measure rat cytokines in serum below the LODs of conventional immunoassays. We also measured these cytokines in healthy rat serum to obtain baseline levels. The ability to measure cytokines present at low concentrations in rat serum will facilitate future studies of disease using rats as animal models.

Photonic ring resonance is a property of light where in certain circumstances specific wavelengths are trapped in a ring resonator. Sensors based on silicon photonic ring resonators function by detecting the interaction between light circulating inside the sensor and matter deposited on the sensor surface. Binding of biological material results in a localized change in refractive index on the sensor surface, which affects the circulating optical field extending beyond the sensor boundary. That is, the resonant wavelength will change when the refractive index of the medium around the ring resonator changes. Ring resonators can be fabricated onto small silicon chips, allowing development of a miniature multiplex array of ring based biosensors. This paper describes the properties of such a system when responding to the refractive index changed in a simple and precise way by changing the ionic strength of the surrounding media, and in a more useful way by the binding of macromolecules to the surface above the resonators. Specifically, a capture immunoassay is described that measures the change of resonant wavelength as a patient serum sample with anti-SS-A autoantibodies is flowed over a chip spotted with SS-A antigen and amplified with anti-IgG. The technology has been miniaturized and etched into a 4×6mm silicon chip that can measure 32 different reactions in quadruplicate simultaneously. The variability between 128 rings on a chip as measured by 2M salt assays averaged 0.6% CV. The output of the assays is the average shift per cluster of 4 rings, and the assays averaged 0.5% CV between clusters. The variability between chips averaged 1.8%. Running the same array on multiple instruments showed that after some improvements to the wavelength referencing system, the upper boundary of variation was 3% between 13 different instruments. The immunoassay displayed about 2% higher variability than the salt assays. There are several outstanding features of this system. The amount of antigen used on the chip for each test is around 200 picograms, only a few microliters of sample is necessary, and the assays take

The clinical effect of patient immune responses to therapeutic antibodies affect product safety and efficacy, which makes the development of valid, sensitive immune assays a key aspect of antibody drug development. In this paper, we reported the generations of mouse monoclonal and Cynomolgus monkey polyclonal antibodies against the anti-CD147 antibody (Metuzumab) as the internal standards and the positive controls. Seven mouse monoclonal antibodies were shown to recognize both (Fab)2 and full length of Metuzumab, but not the control normal human IgGs, and monoclonal anti-Metuzumab, Clone 2D9 was chosen to be used as the internal standard for anti-Metuzumab study. A Bridging ELISA assay was developed by coating the wells with the antibody drug, and the anti-drug antibody (ADA) in the animal sera were detected by enzyme-labeled antibody. Its limit of detection (LOD) was determined to be 0.39ng/ml of anti-Metuzumab antibody (ADA) with linear range between 0.39-50ng/ml and R(2)=0.994. For normal monkey sera, a minimal dilution was determined to be 1:80. However, very different from peptide or other protein drugs, strong interferences from the residual antibody drugs were observed from most of the testing monkey sera in the preclinical study. It was experimentally determined that the concentration of the residual antibody drug in the assay have to be lower than 1μg/ml, so the assays were carried out at 1:100 dilution of the monkey sera. In the pre-clinical study, 32 monkeys were treated with escalating doses of Metuzumab between 0, 10, 50, 200mg/kg for 13 times over 13weeks of time period. 16 of them were terminated right after the last injection, while the other 16 were rested for additional 4weeks before termination. Afraid to miss any positive response to antibody drug, sera samples were collected at six time points, including 2-, 6- and 10-weeks post 1st dose, prior to last dose, and 2-, 4-weeks into recovery. The highest positive rates were seen with the Medium- and High-dose group 2-weeks post the first injection, 6 out 8 monkeys in the High-dose were positive for free ADA. However, no significant pathologic and clinic adversary effect was observed in those monkeys.

Perforin (PFN) is one of the most important protein effectors of the immune system. It is produced by cytotoxic T lymphocytes and natural killer cells and helps with the clearance of virus-infected and tumor cells. PFN is a pore-forming protein that readily binds to the lipid membranes of target cells, oligomerizes at the cell surface and forms transmembrane pores that allow passage of ions and other larger molecules. Its characterization was hindered in the past by a lack of efficient and reliable expression systems that would result in pure and functional product. In this paper we present optimization of PFN expression in a baculovirus expression system. We optimized several parameters of murine PFN (mPFN) expression and purification and showed that the expressed product is pure and hemolytically active and that it forms pores in the plasma membranes of K562 cells. We could also observe circular pores formed on liposome membranes by cryo-electron microscopy (cryo-EM). Our protocol opens the door for further biochemical and biophysical assessment of PFN properties and interactions with small ligands and lipid membranes.

Identifying of B-cell epitopes from antigen is a challenging task in bioinformatics and applied in vaccine design and drug development. Recently, several methods have been presented to predict epitopes. The physicochemical or structural properties are used by these methods. In this paper, we propose a more appropriate epitope prediction method, LRC, that is based on a combination of physicochemical and structural properties. First, we construct a graph from the surface of antigen, then by using the logistic regression, we model the physicochemical and structural properties and weight each vertex of the graph. Finally, we utilize a clustering method, MCL, to cluster the graph. The effectiveness of the proposed method is benchmarked using several antibody-antigen PDB complexes. The results of LRC algorithm are compared with other methods (DiscoTope, SEPPA and Ellipro) in terms of sensitivity, specificity and other well-known measures. Results indicate that applying the LRC algorithm improves the precision of prediction epitopes in comparison with the mentioned methods. Our LRC program and supplementary material are freely available from http://bs.ipm.ir/softwares/LRC/.

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved.