Mixture Of Antibodies Research Articles

A multispecific antibody against SARS-CoV-2 prevents immune escape in vitro and confers prophylactic protection in vivo.

Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Establishment of a Raman nanosphere based immunochromatographic system for the combined detection of influenza A and B viruses’ antigens on a single T-line

A simple and rapid system based on Raman nanosphere (R-Sphere) immunochromatography was developed in this study for the simultaneous detection of Influenza A, B virus antigens on a single test line (T-line). Two types of R-Sphere with different characteristic Raman spectrum were used as the signal source, which were labeled with monoclonal antibodies against FluA, FluB (tracer antibodies), respectively. A mixture of antibodies containing anti-FluA monoclonal antibody and anti-FluB monoclonal antibody (capture antibody) was sprayed on a single T-line and goat anti-chicken IgY antibody was coated as a C-line, and the antigen solution with known concentration was detected by the strip of lateral flow immunochromatography based on surface-enhanced Raman spectroscopy (SERS). The T-line was scanned with a Raman spectrometer and SERS signals were collected. Simultaneous specific recognition and detection of FluA and FluB were achieved on a single T-line by analyzing the SERS signals. The findings indicated that the test system could identify FluA and FluB in a qualitative manner in just 15 minutes, with a minimum detection threshold of 0.25 ng ml-1, excellent consistency, and specificity. There was no interference with the other four respiratory pathogens, and it exhibited 8 times greater sensitivity compared to the colloidal gold test strip method. The assay system is rapid, sensitive, and does not require repetitive sample pretreatment steps and two viruses can be detected simultaneously on a single T-line by titrating one sample, which improves detection efficiency, and provide a reference for developing multiplexed detection techniques for other respiratory viruses.

Single-Batch Expression of an Experimental Recombinant Snakebite Antivenom Based on an Oligoclonal Mixture of Human Monoclonal Antibodies.

Oligoclonal antibodies, which are carefully defined mixtures of monoclonal antibodies, are valuable for the treatment of complex diseases, such as infectionss and cancer. In addition to these areas of medicine, they could be utilized for the treatment of snakebite envenoming, where recombinantly produced monoclonal human antibodies could overcome many of the drawbacks accompanying traditional antivenoms. However, producing multiple individual batches of monoclonal antibodies in an industrial setting is associated with significant costs. Therefore, it is attractive to produce oligoclonal antibodies by mixing multiple antibody-producing cell lines in a single batch to have only one upstream and downstream process. In this study, we selected four antibodies that target different toxins found in the venoms of various elapid snake species, such as mambas and cobras, and generated stable antibody-producing cell lines. Upon co-cultivation, we found the cell line ratios to be stable over 7 days. The purified oligoclonal antibody cocktail contained the anticipated antibody concentrations and bound to the target toxins as expected. These results thus provide a proof of concept for the strategy of mixing multiple cell lines in a single batch to manufacture tailored antivenoms recombinantly, which could be utilized for the treatment of snakebite envenoming and in other fields where oligoclonal antibody mixtures could find utility.

Modelling the mechanisms of antibody mixtures in viral infections: the cases of sequential homologous and heterologous dengue infections.

Antibodies play an essential role in the immune response to viral infections, vaccination or antibody therapy. Nevertheless, they can be either protective or harmful during the immune response. Moreover, competition or cooperation between mixed antibodies can enhance or reduce this protective or harmful effect. Using the laws of chemical reactions, we propose a new approach to modelling the antigen-antibody complex activity. The resulting expression covers not only purely competitive or purely independent binding but also synergistic binding which, depending on the antibodies, can promote either neutralization or enhancement of viral activity. We then integrate this expression of viral activity in a within-host model and investigate the existence of steady-states and their asymptotic stability. We complete our study with numerical simulations to illustrate different scenarios: firstly, where both antibodies are neutralizing and secondly, where one antibody is neutralizing and the other enhancing. The results indicate that efficient viral neutralization is associated with purely independent antibody binding, whereas strong viral activity enhancement is expected in the case of purely competitive antibody binding. Finally, data collected during a secondary dengue infection were used to validate the model. The dataset includes sequential measurements of virus and antibody titres during viremia in patients. Data fitting shows that the two antibodies are in strong competition, as the synergistic binding is low. This contributes to the high levels of virus titres and may explain the antibody-dependent enhancement phenomenon. Besides, the mortality of infected cells is almost twice as high as that of susceptible cells, and the heterogeneity of viral kinetics in patients is associated with variability in antibody responses between individuals. Other applications of the model may be considered, such as the efficacy of vaccines and antibody-based therapies.

Molecular imaging supports the development of multispecific cancer antibodies.

Multispecific antibodies are engineered antibody derivatives that can bind to two or more distinct epitopes or antigens. Unlike mixtures of monospecific antibodies, the binding properties of multispecific antibodies enable two specific molecules to be physically linked, a characteristic with important applications in cancer therapy. The field of multispecific antibodies is highly dynamic and expanding rapidly; to date, 15 multispecific antibodies have been approved for clinical use, of which 11 were approved for oncological indications, and more than 100 new antibodies are currently in clinical development. Nevertheless, substantial challenges limit the applications of multispecific antibodies in cancer therapy, particularly inefficient targeting of solid tumours and substantial adverse effects. Both PET and single photon emission CT imaging can reveal the biodistribution and complex pharmacology of radiolabelled multispecific antibodies. This Review summarizes the insights obtained from preclinical and clinical molecular imaging studies of multispecific antibodies, focusing on their structural properties, such as molecular weight, shape, target specificity, affinity and avidity. The opportunities associated with use of molecular imaging studies to support the clinical development of multispecific antibody therapies are also highlighted.

An Electrochemical Biosensor for the Detection of Pulmonary Embolism and Myocardial Infarction.

Due to the clinical similarities between pulmonary embolism (PE) and myocardial infarction (MI), physicians often encounter challenges in promptly distinguishing between them, potentially missing the critical window for the correct emergency response. This paper presents a biosensor, termed the PEMI biosensor, which is designed for the identification and quantitative detection of pulmonary embolism or myocardial infarction. The surface of the working electrode of the PEMI biosensor was modified with graphene oxide and silk fibroin to immobilize the mixture of antibodies. Linear sweep voltammetry was employed to measure the current-to-potential mapping of analytes, with the calculated curvature serving as a judgment index. Experimental results showed that the curvature exhibited a linear correlation with the concentration of antigen FVIII, and a linear inverse correlation with the concentration of antigen cTnI. Given that FVIII and cTnI coexist in humans, the upper and lower limits were determined from the curvatures of a set of normal concentrations of FVIII and cTnI. An analyte with a curvature exceeding the upper limit can be identified as pulmonary embolism, while a curvature falling below the lower limit indicates myocardial infarction. Additionally, the further the curvature deviates from the upper or lower limits, the more severe the condition. The PEMI biosensor can serve as an effective detection platform for physicians.

Hazelnut proteins detection in cookies using an immersible label-free photonic chip sensor

Rapid and sensitive methods to detect allergenic proteins in foods at the point-of-need could help to protect allergic consumers from life-threatening accidental exposure. In this context, the development of a label-free optical immunosensor for detecting hazelnut proteins in cookies is presented. The sensor is based on silicon photonic chips containing two integrated Mach-Zehnder interferometers (MZI) with light input and output on one side of the chip, and sensing window openings on the other side. Coupling with a white-light LED and a spectrometer for signal recording is achieved via a bifurcated fiber. The sensing window of one of the MZIs is modified with a mouse monoclonal antibody against hazelnut proteins, while the other sensing window is modified with bovine serum albumin to serve as blank. The assay follows a two-step sandwich immunoassay format, involving a 10-min reaction with the hazelnut proteins calibrator or cookie sample, followed by a 2-min reaction with a mixture of monoclonal antibodies. All reactions were performed by immersing the chip side where the MZIs windows are located into the solutions. The recorded spectrum is processed in real-time to transform the spectrum shifts due to immunoreactions taking place on the sensing windows of the two MZIs into phase shifts. The developed assay detects hazelnut proteins at concentrations as low as 25 ng/mL in calibrators prepared in buffer or in aqueous extract of hazelnut-free cookies. Additionally, cookies containing hazelnuts were analyzed demonstrating the sensor ability for fast and sensitive detection of the hazelnut proteins in commercial products.

Oligo cyc-DEP: On-chip cyclic immunofluorescence profiling of cell-derived nanoparticles.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98±3% (n=12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies.

Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death

Type I interferons (IFN-I) are implicated in exacerbation of tuberculosis (TB), but the mechanisms are unclear. Mouse macrophages infected with Mycobacterium tuberculosis (Mtb) produce IFN-I, which contributes to their death. Here we investigate whether the same is true for human monocyte-derived macrophages (MDM). MDM prepared by a conventional method markedly upregulate interferon-stimulated genes (ISGs) upon Mtb infection, while MDM prepared to better restrict Mtb do so much less. A mixture of antibodies inhibiting IFN-I signaling prevents ISG induction. Surprisingly, secreted IFN-I are undetectable until nearly two days after ISG induction. These same antibodies do not diminish Mtb-infected MDM death. MDM induce ISGs in response to picogram/mL levels of exogenous IFN-I while depleting similar quantities from the medium. Exogenous IFN-I increase the proportion of dead MDM. We speculate that Mtb-infected MDM produce and respond to minute levels of IFN-I, and that only some of the resultant signaling is susceptible to neutralizing antibodies. Many types of cells may secrete IFN-I in patients with TB, where IFN-I is likely to promote the death of infected macrophages.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Abstract 5792: Rapid and efficient generation of panels of T cell engaging (TCE) multispecific antibodies via complementary technologies

Abstract A goal of immunotherapy is to induce an anti-tumor response that is potent and specific. However, there is increasing understanding that currently approved monospecific checkpoint inhibitors are not effective for all patients and/or indications. Thus, investigators are increasingly pursuing multispecific antibody treatments in an attempt to enhance clinical outcomes. These treatments may either take the form of mixtures (administered simultaneously or in series) of multiple monospecific antibodies, or as single entity, multispecific antibodies (‘multispecifics’). In the case of multispecifics, the large number of possible topologies and complexity of manufacture necessitate the development and application of a robust set of complementary technologies. We have developed an exemplary set of such technologies. Herein, we demonstrate the ability to direct desired antibody chain pairing (HC-HC and HC-LC), isolate and engineer single-domain antibodies, as well as generate large (>100) panels of multispecific antibodies with diverse topologies from a limited number of input molecules. These technologies can be leveraged in combination with our affinity and developability-optimized TCE αCD3 and αCD28 antibody panels to achieve desired potency without the need for additional discovery or engineering. Citation Format: Cory Ahonen, Nathan Sharkey, Melissa Durkin, Andrew Avery, Michael Battles, Beth Sharkey, Kyle Barlow, Arvind Sivasubramanian, Caitlin Stein, Bradley Lunde, Kevin Schutz, Bianka Prinz, Rob Pejchal, Paul Widboom, Eric Krauland. Rapid and efficient generation of panels of T cell engaging (TCE) multispecific antibodies via complementary technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5792.

Multiplex Cyclic Fluorescent Immunohistochemistry.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

New approach to generating of human monoclonal antibodies specific to the proteolytic domain of botulinum neurotoxin A.

Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure.

Novel Pharmacological Treatment Options of Steroid-Refractory Graft-versus-Host Disease

Background. Graft-versus-host disease (GVHD) is a potentially fatal complication of allogeneic hematopoietic stem cell transplant. The mainstay of treatment is corticosteroids, which are ineffective in 30–50% of cases. Steroid-refractory GVHD (SR-GVHD) confers a poor prognosis, with high mortality rates despite appropriate therapy. While there is no reliable treatment for SR-GVHD, a variety of novel therapeutic options are slowly emerging and have yet to be examined simultaneously. Objectives. This review evaluates the potential of novel therapeutic options, as well as their efficacy and safety, for the treatment of SR-GVHD. Study Design. The literature search was conducted in PubMed, Cochrane, and Embase, employing MeSH terms and keywords. The studies had to be prospective phases 1, 2, or 3. We excluded retrospective and nonoriginal studies. Results. While the only approved drug for acute GVHD is ruxolitinib with an impressive overall response rate of 73.2% and a complete response of 56.3%, several monoclonal antibodies and other agents are currently under investigation, offering promising results. These include anti-CD2, anti-CD147, IL-2 antagonist, a mixture of anti-CD3 and anti-CD7 antibodies, anti-CD25, monoclonal antibody to a4b7 on T-cells, anti-CD26, pentostatin, sirolimus, denileukin diftitox, infliximab, itacitinib, and alpha-1 antitripsin. However, the toxicities associated with these novel drugs need further investigation. For chronic GVHD, approved options include ruxolitinib with an ORR of up to 62%, ibrutinib with an ORR of up to 77%, and belumosudil with an ORR of up to 77%. Meanwhile, emerging treatments include tyrosine kinase inhibitors such as nilotinib, rituximab, and low-dose IL-2, as well as axatilimab and pomalidomide. Conclusion. While their efficacy needs to be better evaluated through large-scale, multicenter, randomized clinical trials, these novel agents show potential and could provide a better alternative for SR-GVHD treatment in the future.

B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture

Abstract Introduction Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS). Methods In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer. Preliminary data The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture. Novel aspect De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.

Development of a surface plasmon resonance based immunosensor for diclofenac quantification in water

A Surface Plasmon Resonance (SPR) biosensor based on an inhibition immunoassay was developed for the detection of diclofenac (DCF) in aqueous solution. Due to the small size of DCF, an hapten-protein conjugate was produced by coupling DCF to bovine serum albumin (BSA). DCF-BSA conjugate formation was confirmed via MALDI-TOF mass spectrometry. The resulting conjugate was immobilized onto the surface of a sensor fabricated via e-beam deposition of a 2 nm chromium adhesion layer followed by a 50 nm gold layer onto precleaned BK7 glass slides. Immobilization onto the nano thin gold surface was accomplished by covalent amide linkage through a self-assembled monolayer. Samples were composed of a mixture of antibody at a fixed concentration and DCF at different known concentrations in deionized water, causing the inhibition of anti-DCF on the sensor. The DCF-BSA was obtained with a ratio of 3 DCF molecules per BSA. A calibration curve was performed using concentrations between 2 and 32 μg L−1. The curve was fitted using the Boltzmann equation, reaching a limit of detection (LOD) of 3.15 μg L−1 and limit of quantification (LOQ) of 10.52 μg L−1, the inter-day precision was calculated and an RSD value of 1.96% was obtained; and analysis time of 10 min. The developed biosensor is a preliminary approach to the detection of DCF in environmental water samples, and the first SPR biosensor developed for DCF detection using a hapten-protein conjugate.

Abstract 2296: Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma

Abstract Background: Circulating tumor cells (CTCs) are promising real-time, noninvasive biomarkers to assess therapy response and to monitor disease. However clinical applications of CTCs in urothelial carcinomas (UC) remain limited. There is a need for sensitive and disease-specific CTC assay for urothelial cancer. As CTCs are very rare in blood compared to blood cells and may vary due to tumor heterogeneity, detection and isolation of them solely based on one biomarker (e.g., EpCAM) would be insufficient. To achieve better results and increase clinical relevance, we designed a CTC capture device that was functionalized with multiple antibodies specific to UC. Methods: We engineered a novel UC-CTC capture device using nanotechnology and biomimetic cell rolling. To capture UC cells, we examined 4 biomarkers: EpCAM, Nectin-4, Trop-2, and FGFR3, that are highly expressed in UC. We engineered CTC assays using single and multi-biomarker capture surfaces. The capture sensitivity and selectivity of the device were investigated using four bladder cancer cell lines as well as a leukocyte model, HL-60, as negative control. CTCs are defined as CK+/DAPI+/CD45- cells. The capture sensitivity was compared between the different surfaces with each of the different antibodies and the final UC-CTC chip with optimized antibody mix was chosen to detect CTCs from UC patients. Results: We examined the CTC capture using 4 UC cell lines: UM-UC-1, T-24, HT1376, RT4. The devices that utilized single antibodies were much less sensitive (capture efficiencies less than 82%). In comparison, combination antibody capture improved sensitivity to >90%. Negative control using the HL-60 cells showed minimal non-specific binding, <0.3%. We were also able to validate the CTC assay using patient samples and demonstrated high sensitivity. For example, we captured ~302 CTCs/mL in a patient who had early-stage T2 muscle invasive UC. Conclusion: We developed a novel CTC capture device using four UC-specific capture antibodies for the detection and isolation of CTCs in UC. Compared to the configuration of EpCAM alone, the mixture of multiple antibodies improved the capture efficiency up to 1.4-fold for in vitro bladder cancer cell spiking assays. High specificity was also achieved, with low levels of non-specific leukocyte detection. High sensitivity and specificity suggest that this platform has potential impact to detect CTCs from patients with UC. Citation Format: Qinhan Zhou, Michael Poellmann, Nikhila Reddy Sultanpuram, Damla Gunenc, Krista English, Luke Chen, Runze Zhao, Seungpyo Hong, Andrew Wang, Tian Zhang. Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2296.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (−0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

EXPERIENCE OF USING THE IMMUNE-COMPLEX VACCINE PULVAK MAGNIPLEX IN THE PREVENTION OF INFECTIOUS BURSA DISEASE OF BROILERS

The article presents the results of serological control of the effectiveness of vaccination of broilers against infectious bursal disease (IBD) when the immune-complex vaccine Pulvak MagniPlex is used in the incubator.
 Immune-complex vaccines are vaccines of a new generation, the use of which completely eliminates post-vaccination complications in birds and interference phenomena. The vaccine includes a mixture of specific antibodies and the vaccine strain of the IBH virus. The main property of these vaccines is that when administered subcutaneously in day-old chicks or 18-day-old chicken embryos by the in ovo method, immunity is quickly created even in the presence of high maternal antibodies (MAB). Vaccination in the incubator using immune-complex vaccines is more accurate and provides protection to the whole bird, unlike live vaccines, the main method of which is administration with drinking water. Immuno-complex vaccines are effective even in the presence of high MAT in chickens. Due to this, they provide a much higher level of protection due to the high homogeneity of protective antibodies compared to live attenuated vaccines.
 We present the data of serological monitoring on the average titer of antibodies to the IBH virus in blood sera from 15 batches of broilers aged 45-50 days, which were immunized on the first day in the incubator with the immune-complex vaccine Pulvak MagniPlex. It was established that during a single vaccination in the incubator (15 batches), the Pulvak MagniPlex vaccine stimulates the formation of an active immune response to the IBH virus for the entire period of fattening of broilers. The average titers in the blood sera of broilers aged 45-50 days to the IBH virus ranged from 6,995 to 12,388 after a single vaccination in the incubator (15 batches). The percentage of protective antibodies to the IBH virus in 15 batches of broilers varied from 77.3 to 100, which indicates the intensity of immunity to IBH. A single application of the immune-complex vaccine Pulvak MagniPlex in the incubator stimulates the formation of an active immune response to the IBD virus for the entire period of broiler fattening and provides 100 % protection of 12 batches of broilers and 77.3 % of broilers of 3 batches to IBD. The use of Pulvak MagniPlex allows: to create stable immunity against IBD; to form immunity in each individual broiler, regardless of the level of maternal antibodies; to reduce the impact of stress (compared to mass vaccination in the poultry house by the drinking method. Constant control of the effectiveness of the Pulvak MagniPlex immune-complex vaccine by means of serological monitoring of blood sera in ELISA will allow, depending on the epizootic situation, to timely adjust the vaccination scheme of poultry against IBD.