Antibody-based System Research Articles

Nanobodies Outperform Antibodies - Rapid Functionalization with Equal In Vivo Targeting Properties.

Highly specific targeting of dendritic cells in vivo is crucial for the development of effective tumor nanovaccines. This group recently presented an antibody-functionalized nanocarrier system able to maintain its targeting properties when transferred from in vitro to in vivo studies. However, producing this system requires long synthesis times and involves high expenses due to the involved site-specific enzymatic multi-step modification procedure of the antibody. Consequently, improving the previously proposed system is necessary in order to accelerate the development. Here, a novel system utilizing nanobodies for the targeting of dendritic cells is presented. A C-terminal cysteine tag facilitates an easy attachment of the nanobody to the nanocarrier via a thiol-maleimide conjugation technique. This reduces the functionalization time from several days to mere hours. Using in vitro and in vivo assays, it is shown that the optimized system possesses equal targeting properties as the antibody-based system. As a result, nanobodies and the coupling chemistry are found to be a superior strategy for the in vivo targeting of dendritic cells when compared to antibodies, due to their rapid attachment to nanocarriers and equal targeting specificity. This would replace antibodies as the current "gold standard" of targeting moieties.

Rapid Nuclease-Assisted Selection of High-Affinity Small-Molecule Aptamers.

Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.

Sensitivity and specificity of rapid antibody diagnostic test for diagnosis COVID 19 in pediatric patients

Objective: Aim of this observational is to evaluate the sensitivity and specificity a rapid diagnostic test for COVID-19 for screening COVID-19 pediatric patients. Study Design: 179 Patients under 18 years old with a history of COVID-19 symptoms, and who underwent PCR and/or reference antibody testing for COVID-19. Results: The sampel of this study consisted of 179 patient who 100 sunject with reactive in rapid antibody diagnostic test COVID 19 which consisting of 40 subject positive SARS-CoV2 PCR swab examination and 60 subject negative SARS-CoV2 PCR swab examination. The patient with nonreactive result in rapid antibody diagnostic test COVID 19 which consisting of 19 subject positive SARS-CoV2 PCR swab examination and 60 subject negative SARS-CoV2 PCR swab examination. The sensitivity of the test was 67%. Specificity was 50%. There was substantial agreement between SARS-CoV2 PCR results and a rapid antibody diagnostic test COVID 19. Conclusion: The current evaluation of antibody-based system shows low sensitivity and low specificity result.

Lateral Flow Glyco-Assays for the Rapid and Low-Cost Detection of Lectins-Polymeric Linkers and Particle Engineering Are Essential for Selectivity and Performance.

Lateral flow immuno-assays, such as the home pregnancy test, are rapid point-of-care diagnostics that use antibody-coated nanoparticles to bind antigens/analytes (e.g., viruses, toxins or hormones). Ease of use, no need for centralized infrastructure and low-cost, makes these devices appealing for rapid disease identification, especially in low-resource environments. Here glycosylated polymer-coated nanoparticles are demonstrated for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems introduced here use glycans, not antibodies, to provide recognition: a “lateral flow glyco-assay,” providing unique biosensing opportunities. Glycans are installed onto polymer termini and immobilized onto gold nanoparticles, providing colloidal stability but crucially also introducing assay tunability and selectivity. Using soybean agglutinin and Ricinus communis agglutinin I (RCA120) as model analytes, the impact of polymer chain length and nanoparticle core size are evaluated, with chain length found to have a significant effect on signal generation—highlighting the need to control the macromolecular architecture to tune response. With optimized systems, lectins are detectable at subnanomolar concentrations, comparable to antibody-based systems. Complete lateral flow devices are also assembled to show how these devices can be deployed in the “real world.” This work shows that glycan-binding can be a valuable tool in rapid diagnostics.

Glycan-Based Flow-Through Device for the Detection of SARS-COV-2.

The COVID-19 pandemic, and future pandemics, require diagnostic tools to track disease spread and guide the isolation of (a)symptomatic individuals. Lateral-flow diagnostics (LFDs) are rapid and of lower cost than molecular (genetic) tests, with current LFDs using antibodies as their recognition units. Herein, we develop a prototype flow-through device (related, but distinct to LFDs), utilizing N-acetyl neuraminic acid-functionalized, polymer-coated, gold nanoparticles as the detection/capture unit for SARS-COV-2, by targeting the sialic acid-binding site of the spike protein. The prototype device can give rapid results, with higher viral loads being faster than lower viral loads. The prototype’s effectiveness is demonstrated using spike protein, lentiviral models, and a panel of heat-inactivated primary patient nasal swabs. The device was also shown to retain detection capability toward recombinant spike proteins from several variants (mutants) of concern. This study provides the proof of principle that glyco-lateral-flow devices could be developed to be used in the tracking monitoring of infectious agents, to complement, or as alternatives to antibody-based systems.

Theranostic Applications of Antibody-Based Systems in Human Diseases.

Since antibodies are one of the most successful classes of biopharmaceuticals for cancer treatment, it is very important to discuss the functionality and biological activity of endogenous antibodies in the diagnosis and treatment of the diseases. Antibodies act on tumor cells in diverse ways, including engaging in antibody-dependent effector mechanisms, internalizing to deliver toxins, and displaying direct effects on cells. In this review, we outline three kinds of antibody-based therapeutic systems, namely Antibody-based Drug Conjugates (Ab-DCs), Antibody-based Recruiting Small Molecules (Ab-RSMs) and Carbohydrate Epitopes Recruiting Natural Antibodies (CERNA) for human disease treatment and imaging. These antibody-based therapeutic systems are able to strengthen the binding ability of antibodies with disease-relevant cells or viruses, leading to their immuno-mediated therapeutic responses and clearance. Research in these fields shows exciting potential at the junction of organic chemistry and immune-biology. Since the applications of antibodies are of great importance, it is necessary to functionalize antibodies or develop other antibody-based synthetic strategies to modulate the human immune system, and ultimately achieve the treatment of diseases.

TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation.

Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.

An integrated microfluidic system for the isolation and detection of ovarian circulating tumor cells using cell selection and enrichment methods.

Gynecological cancer is difficult to be diagnosed at early stages. The relatively high mortality rate has been a serious issue accordingly. We herein reported a diagnosis method by using circulating tumor cells (CTCs) which have been extensively explored as a potential tool for diagnostics and prognostics of ovarian cancers. Nonetheless, the detection of CTCs still remains a challenge because of the difficulty in isolating them from whole blood samples since they are shed into the vasculature from primary tumors and circulate irregularly in the bloodstream in extremely low concentrations. In this work, we reported a new, integrated microfluidic system capable of (1) red blood cells lysis, (2) white blood cell (WBC) depletion via a negative selection process, and (3) capture of target cancer cells from whole blood samples using aptamer-binding technology. Furthermore, this is the first time that an aptamer was used to capture ovarian cancer cells owing to its high affinity. The new microfluidic chip could efficiently perform the entire process in one hour without human intervention at a high recovery rate and a low false positive detection rate when compared with antibody-based systems. A high recovery rate for the isolation of CTCs within a short period of time has been reported when compared to the traditional negative or positive selection approach by using traditional antibody biomarkers. More importantly, "false positive" results from WBCs could be significantly alleviated due to the high specificity of the cancer cell-specific aptamers. The developed integrated microfluidic system could be promising for the isolation and detection of CTCs, which could be used for early diagnosis and prognosis of cancers.

Industry Update: the Latest Developments in Therapeutic Delivery

The present industry update covers the period 16 February–15 March 2012, which included the announcement of two large deals in the drug conjugation space – Mersana partnered its Fleximer technology with Endo for application to antibody drug conjugates, and Angiochem partnered with GlaxoSmithKline for application of its LRP-1 receptor-targeting technology to lysosomal storage diseases. Two Boston, MA, USA-area start-ups reached early funding milestones: 4s3 Bioscience, developing an antibody-based system for enhanced intracellular delivery of proteins, closed a US$20 million Series A round, while Kala Pharmaceuticals, developing a mucus-penetrating particle technology, completed a US$11.2 million seed round. Organogenesis’ cell-based mucogingival therapy and Discovery Labs’ synthetic peptide-based pulmonary surfactant received product approvals. Endocyte announced plans to submit marketing applications for its folate-receptor targeting-based diagnostic and therapy for ovarian cancer.

Evolution of the immune system at geological and local scales

This review will contest the idea that the adaptive immune system of mammals represents an evolutionary advance which superseded the less evolved, innate immune system of invertebrates. General conclusions about the evolution of immune systems will be applied to current infectious diseases of humans common in conditions of poverty. Many invertebrates have mechanisms for diversifying the repertoire of receptors specific for pathogen-associated molecules. The lamprey uses true genetic rearrangement to produce a repertoire comparable to that of our antibody-based system. Both adaptive immune systems may have evolved independently from an efficient, pre-existing, lymphocyte-based, innate immune system, the importance of which has only recently been described in mammals.Genetic variation in human populations is associated with susceptibility and resistance to infectious diseases. Specifically, human leucocyte antigen and natural killer receptor alleles are associated with susceptibility or resistance to HIV. However, the extent to which these will exert long-term selective pressure is unclear. Human pathogens do to exert selective pressures on the immune system, but these are unlikely to do more than change allele frequencies in the short term. In the long term, pathogen-driven evolution seems to promote expansion of recognition repertoire in adaptive and innate immune systems.

Allergen determination in food: reference materials and traceability of results

Food allergens are more or less denatured mixtures of non-defined proteins in complex matrices. The most common technique to measure these proteins is an antibody-based system as, e.g., a commercial ELISA system which consists of an extraction procedure, suitable antibodies and a calibrator. Until now, only a few attempts to standardize these different ELISA systems were performed and the most promising way seems to be a standardization of the calibrator. It is shown that we have currently no methods to fully characterize these calibrators, which are always complex protein mixtures. This also means that the production of reference materials containing these calibrators is not possible. It is proposed to choose a different way because we will never have an unbroken traceability chain. As described in ISO 17511:2003 for the clinical area, one possibility could be the selection of a commercial preparation (e.g. skim milk powder) to which all test kit manufacturers will relate their results on and in a second step to select a reference method.

Standardization in allergen determination

An overview is given of sensitization and allergic reaction on food, along with minimal eliciting doses for selected allergens (milk, egg, peanut), followed by the legislative situation in the European Community, the USA, Canada, Australia/New Zealand and Japan. The main focus of the paper discusses methods for food allergen determination (chromatography, DNA-based, antibody-based) and their limitations. Special emphasis is given on antibody-based systems, which are at the moment the most advanced and validated methods available. Examples are given of the current status of standardization for egg, milk, nuts, prolamins, glutelins and for clinical validated reference samples from the EU project EuroPrevall. Recommendations for ongoing activities are also included.

Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Molecular quantification of slug density in the soil using monoclonal antibodies

Molecular ecology techniques are increasingly used to study invertebrate foodwebs and trophic interactions in the field. However, the study of subterranean foodwebs is currently constrained by the difficult, laborious and often expensive methods that need to be employed to simply measure invertebrate population densities accurately. Here we describe and field-test a novel monoclonal antibody-based system for tracking slug populations. Proteins were extracted from soil blocks using sodium chloride and a new slug-specific monoclonal antibody was developed, capable of detecting the proteins liberated by the salt. Detection sensitivity and limits, using enzyme-linked immunosorbent assays (ELISA), were measured for a range of soils and the system proved to be effective, whether the soil was heavy clay or sandy. The sensitivity of the assay varied between soils and needed to be calibrated by ELISA. There was a linear relationship between slug biomass in any given soil and slug proteins detected by ELISA. A field experiment was performed comparing ELISA with the most accurate conventional approach. The latter involved taking blocks of soil from the field and flooding them gradually over 10 days to drive slugs to the surface, where they were collected and weighed. Parallel blocks of soil were taken 1 m away and subjected to the salt extraction/ELISA approach. Results using the two systems proved to be very similar, but ELISA produced results more rapidly. The many advantages of using ELISA to measure slug density are discussed.

Integrating molecular detection and response to create self-signalling antibodies

Monoclonal antibodies are reproducible, specific, and cost-effective molecular probes; use outside the laboratory is, however, restricted by technical limitations. Addressing these constraints, the first self-signalling antibodies are now described, where specific antigen binding causes release of bound reporter from bispecific antibodies (BsAb) to generate a detectable signal. The report examines the concept that two different antibody binding sites in close proximity can promote interaction between molecules recognised by these sites, generating a signal by molecular crowding. Signal strength is found to increase with increasing homogeneity for a BsAb reactive with multimeric surfactant antigen; signal response is linear for a BsAb reactive with univalent small analyte deoxypyridinoline. Self-signalling is consistent with intramolecular steric hindrance. This is the first report detailing integration of two different functions, molecular detection and signal response, into BsAbs and with detection of large and small analytes, has generic application to antibody-based systems.

Inducible Clustering of Membrane-Targeted SH3 Domains of the Adaptor Protein Nck Triggers Localized Actin Polymerization

Background: SH2/SH3 adaptor proteins play a critical role in tyrosine kinase signaling pathways, regulating essential cell functions by increasing the local concentration or altering the subcellular localization of downstream effectors. The SH2 domain of the Nck adaptor can bind tyrosine-phosphorylated proteins, while its SH3 domains can modulate actin polymerization by interacting with effectors such as WASp/Scar family proteins. Although several studies have implicated Nck in regulating actin polymerization, its role in living cells is not well understood.Results: We used an antibody-based system to experimentally modulate the local concentration of Nck SH3 domains on the plasma membrane of living cells. Clustering of fusion proteins containing all three Nck SH3 domains induced localized polymerization of actin, including the formation of actin tails and spots, accompanied by general cytoskeletal rearrangements. All three Nck SH3 domains were required, as clustering of individual SH3 domains or a combination of the two N-terminal Nck SH3 domains failed to promote significant local polymerization of actin in vivo. Changes in actin dynamics induced by Nck SH3 domain clustering required the recruitment of N-WASp, but not WAVE1, and were unaffected by downregulation of Cdc42.Conclusions: We show that high local concentrations of Nck SH3 domains are sufficient to stimulate localized, Cdc42-independent actin polymerization in living cells. This study provides strong evidence of a pivotal role for Nck in directly coupling ligand-induced tyrosine phosphorylation at the plasma membrane to localized changes in organization of the actin cytoskeleton through a signaling pathway that requires N-WASp.

Production and characterization of a human recombinant monoclonal Fab fragment specific for influenza A viruses.

A human recombinant monoclonal Fab fragment that specifically recognizes all the influenza A virus strains tested was produced in transformed Escherichia coli using the phage display technique. No strain of influenza B virus reacted with it. It was purified after four cycles of panning and by a single passage through an immunoaffinity column. About 1 mg of pure monoclonal antibody was obtained from 1 liter of culture medium in 3 working days. The Fab fragment reacted with a viral 27-kDa protein, which could reasonably be a matrix protein. Indirect immunofluorescence tests performed on virus-infected MDCK cells showed that this Fab fragment was at least equally efficient as other commercial monoclonal antibody-based systems in detecting influenza A viral infections. The potential advantages of human recombinant Fabs on murine monoclonal antibodies are discussed.

Cryptosporidiosis.

Cryptosporidiosis is a self-limited diarrheal disease that occurs in the community setting but can be chronic and potentially serious in immunocompromised patients. Community outbreaks are often associated with water-borne transmission. Cryptosporidium research has increased dramatically since the human disease was first recognized in 1976. The present review summarizes recent work in three of the several areas of active Cryptosporidium investigation. Molecular techniques have revealed that current taxonomic designations need re-evaluation and that humans are host to several Cryptosporidium spp. that were once believed to be limited to the veterinary realm. These findings have important public health implications for water quality standards in the USA and other developed countries. Second, techniques for detecting the parasite in infected individuals (or environmental samples) have progressed from acid-fast staining of fecal smears to the currently used antibody-based systems (enzyme immunoassays and immunofluorescent assays). New molecular methodologies, based on polymerase chain reaction amplification of gene loci, are being developed to improve the sensitivity and specificity for diagnostic and epidemiologic purposes. Third, curative therapy is attained only by an effective immune response or the reconstitution of a failing immune system in the compromised host. However, several drugs are in development, and compounds such as nitazoxanide appear to hold some promise. Cryptosporidiosis continues to be a serious problem in immunocompromised patients and on a worldwide scale in undernourished infants and children. The lack of an effective treatment, and the propensity of the parasite to survive in and be transmitted through source waters make this an important public health threat.

Antibody-based systems for the detection of bacillus anthracis in environmental samples

There is a necessity for rapid immunodiagnostic techniques for the detection and identification of Bacillus anthracis in environmental specimens. The technology available for accomplishing this ranges in complexity from a simple dipstick type assay to complex biosensors. We have developed antigen capture dipstick assays for a series of infectious agents including an assay for B. anthracis protective antigen and one for B. anthracis spores. These immunochromatographic assays use colloidal gold to visualize the reaction and take approximately 15 min to perform. We will also describe our current effort in the development of two antigen detection biosensors and discuss the sensitivity and specificity of the assays in environmental specimens.

Antibody-Directed Enzyme Prodrug Therapy

A 2- or 3-stage system can overcome some of the limitations of antibody-based attempts to restrict the action of cytotoxic agents to tumour sites. These systems use the antibody to direct an enzyme, which is unique to extracellular locations in humans, to tumour sites. The tumour-located enzyme is used to activate a subsequently administered prodrug. As with any antibody-based system, the main limitation lies in the distribution of the antibody or antibody conjugate. However, in contrast to drugs or radioisotopes, an enzyme can be inactivated in nontumour tissues or subjected to rapid clearance without toxic effects. The conjugation of an enzyme to an antibody increases its immunogenicity and may require the administration of immunosuppressive agents or the development of nonimmunogenic fusion proteins. A small scale pilot clinical trial has shown the general feasibility of this approach.