Antigen-antibody Reaction Research Articles

Plasmonic biochips with enhanced stability in harsh environments for the sensitive detection of prostate-specific antigen.

Hollow and porous plasmonic nanomaterials have been demonstrated for highly sensitive biosensing applications due to their distinctive optical properties. Immunosensors, which rely on antibody-antigen interactions, are essential constituents of diverse biosensing platforms owing to their exceptional binding affinity and selectivity. The majority of immunosensors and conventional bioassays needs special storage conditions and cold chain systems for transportation. Prostate-specific antigen (PSA), a serine protease, is widely employed in the diagnosis of prostate cancer. In this study, we present the successful utilization of a biopolymer-preserved plasmonic biosensor with improved environmental stability for the sensitive detection of PSA. The preserved plasmonic biosensors exhibited sustained sensitivity in the detection of PSA, achieving a limit of detection of 10 pg mL-1. Furthermore, these biosensors exhibited remarkable stability at elevated temperatures for one week.

A Stereological Approach to Quantify Immunohistochemical Staining in Zebrafish Larvae.

Immunohistochemistry (IHC) is a powerful research tool to localize specific antigens in whole-mount or tissue sections of embryos with labeled antibodies based on antigen-antibody interactions. Stereological methods are nowadays an essential tool to quantify cells or other types of structures in an unbiased and reproducible manner. In this chapter, a general protocol for a stereological estimation of the relative volume density of each structural component (Vv), which can be applied to any organ/structural component, will be described.

Whole-Mount Immunohistochemical and Immunofluorescence Assays in Zebrafish Embryos.

Zebrafish embryos are an important organism used as an in vivo model in a wide variety of disciplines from the past to the present. Immunohistochemistry analyses are an important method used to determine the localization of specific antigens in tissue sections with labeled antibodies depending on antigen-antibody interactions in zebrafish embryos. Immunofluorescence assays are an immunohistochemistry method that uses fluorophores to determine diverse cellular antigens. Zebrafish embryos and larvae, with their small size, are the most ideal model organisms for whole-mount immunohistochemical and immunofluorescent methods today. The small size of these organisms allows simultaneous evaluation of different tissues and organs, and results are obtained in a shorter time. In this section, whole-mount immunohistochemical and immunofluorescent analysis methods in zebrafish embryos, and larvae are summarized in detail, taking into account different studies and recent advances.

Synergistic acceleration of machine learning and molecular docking for prostate-specific antigen ligand design.

Prostate-specific antigen (PSA) serves as a critical biomarker for the early detection and continuous monitoring of prostate cancer. However, commercial PSA detection methods primarily rely on antigen-antibody interactions, leading to issues such as high costs, stringent storage requirements, and potential cross-reactivity due to PSA variant sequence homology. This study is dedicated to the precise design and synthesis of molecular entities tailored for binding with PSA. By employing a million-level virtual screening to obtain potential PSA compounds and effectively guiding the synthesis using machine learning methods, the resulting lead compounds exhibit significantly improved binding affinity compared to those developed before by researchers using high-throughput screening for PSA, substantially reducing screening and development costs. Unlike antibody detection, the design of these small molecules offers promising avenues for advancing prostate cancer diagnostics. Furthermore, this study establishes a systematic framework for the rapid development of customized ligands that precisely target specific protein entities.

Highly sensitive detection of a neurodegenerative protein biomarker by using the pyro-electrohydrodynamic jet

A set of protein biomarkers are largely recognized as responsible of neurodegeneration mechanisms and hence as potential targets to be detected in low abundant concentrations in body fluids for performing early diagnosis. As an example, the Tau protein experiences a transition phase from a native disorder conformation into a preaggregation state, which leads to fibrillization processes. Here we show the possibility to detect Tau in urine samples at sub-picogram level, through the concentration effect of the pyro-electrohydrodynamic (p-jet) technique. An immunofluorescence protocol is applied to concentrated p-jet spots able to reduce drastically the diffusion effects in the antibody-antigen reaction. A set of diluted samples were prepared, and the fluorescence signal was detected by a confocal scanner. We achieved an excellent linear response with a significant signal-to-noise ratio down to 0.25 pg/mL. In perspective, the technique could be integrated into a compact device to be used for monitoring the early stage associated to neurodegenerative syndromes in different scenarios such as for example in long-term human space exploration missions.

Recent advancements in the specific determination of carcinoembryonic antigens using MOF-based immunosensors.

Carcinoembryonic antigens (CEAs) are prominent cancer biomarkers that enable the early detection of numerous cancers. For effective CEA screening, rapid, portable, efficient, and sensitive diagnosis approaches should be devised. Metal-organic frameworks (MOFs) are porous crystalline materials that have received major attention for application in high-efficiency signal probes owing to their advantages such as large specific surface area, superior chemical stability and tunability, high porosity, easy surface functional modification, and adjustable size and morphology. Immunoassay strategies using antigen-antibody specific interaction are one of the imperative means for rapid and accurate measurement of target molecules in biochemical fields. The emerging MOFs and their nanocomposites are synthesized with excellent features, providing promising potential for immunoassays. This article outlines the recent breakthroughs in the synthesis approaches of MOFs and overall functionalization mechanisms of MOFs with antigen/antibody and their uses in the CEA immunoassays, which operate according to electrochemical, electrochemiluminescent and colorimetric techniques. The prospects and limitations of the preparation and immunoassay applications of MOF-derived hybrid nanocomposites are also discussed at the end.

Apremilast in Erythema Nodosum Leprosum: A Case Series

Erythema nodosum leprosum (ENL) is an immune-mediated inflammatory reaction (antigen-antibody reaction involving complement) of leprosy. Available standard treatment in the form of steroids or thalidomide does not work in all ENL patients. Apremilast is an oral phosphodiesterase-4 inhibitor with potent immunomodulatory action. At present, apremilast is approved for chronic plaque psoriasis and psoriatic arthritis, however, not in ENL. The aim of this study was to assess the effectiveness of apremilast in chronic recalcitrant ENL. It was a case series, in which apremilast was started in 10 consecutive patients with recalcitrant ENL with 10 mg morning dose with a daily increment of 10 mg until day 6, when the recommended dose of 30 mg twice daily for adults is reached. Follow-up was done every 1 month till 6 months. Three patients showed good responses. The drug was well tolerated with few side effects. The most common adverse effect reported was headache (n = 5), followed by nausea (n = 4) and diarrhea (n = 4). Apremilast has lower cost and better safety profile compared to thalidomide and may be an option with limited effectiveness for ENL.

Cu-MOFs@AuPtNPs nanozyme-based immunosorbent assay for colorimetric detection of alpha-fetoprotein.

Accurate detection of tumor biomarkers in blood is crucial for diagnosing and treating tumor disease. In this study, a metal enzyme-linked immunosorbent assay (MeLISA) was fabricated for the ultrasensitive and naked-eye detection of tumor biomarker alpha-fetoprotein (AFP) in clinical serum samples. Herein, novel copper metal-organic frameworks and gold platinum nanoparticle composites (Cu-MOFs@AuPtNPs) were synthesized for the first time by an in situ method, which showed an enormous specific surface area and excellent peroxidase (POx) mimicking properties. Cu-MOFs@AuPtNPs linked with antibodies targeting AFP served as a signal nanoprobe to amplify the detection signal. Additionally, the specificity of MeLISA was significantly enhanced through a conventional antigen-antibody reaction and efficient blocking of non-specific sites with BSA. Under optimal conditions, the sandwich-type MeLISA exhibited a wide range from 0.001 to 1000 ng mL-1 (R2 = 0.997) and a low detection limit of 0.86 pg mL-1 (S/N = 3) with acceptable stability, selectivity, and reproducibility. It is noteworthy that the suggested MeLISA performed exceptionally well in detecting clinical serum samples, which were visible to the naked eye and did not require complex platforms. To sum up, the innovative MeLISA based on Cu-MOFs@AuPtNPs provides an alternative method for early cancer diagnosis, particularly in economically backward areas where simple diagnostic apparatus is extremely desirable.

Structural Characterization of Peptide Antibodies.

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery. To better understand the underlying mechanisms of antibody-antigen interaction, here, we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class.

Visualizing the cellular internalization of therapeutic antibodies via pH-sensitive release of AIEgen.

Among the fastest-growing bio-pharmaceuticals, therapeutic antibodies have achieved unprecedented success in treating various diseases. Though powerful, issues such as inefficacy or acquired resistance are waiting to be addressed to benefit more patients with improved therapeutic outcomes. In addition to in vivo distribution, the cellular spatiotemporal information including the antibody-antigen interaction and subsequent internalization is found to be important for the therapeutic effects. To better understand the cellular fate of therapeutic antibodies, especially the cellular internalization process, we employed a pH-sensitive linker to attach a red-emissive AIEgen onto the antibody. The resulting antibody conjugate will undergo AIEgen release to liberate brilliant fluorescence inside acidic endo/lysosomes, allowing wash-free visualization of the internalization process and facilitating the evaluation of antibody-drug efficacy.

Immunohistochemistry of Brain Tissues.

Immunohistochemistry (IHC) is the basis of histological or pathological analysis and is widely used to enable the detection and characterization of proteins in various organ tissues, including brain tissues. IHC is commonly performed on formalin-fixed paraffin-embedded (FFPE) tissues because of their easy storage and versatility. IHC is a key method for providing more accurate analysis of localization and function of neurons, neuroendocrine cells, and neural stem cells in the brain and other nervous systems. The related cells such as glial cells and neurovascular units have also been analyzed by IHC. Visualization of antibody-antigen interactions can be performed primarily in one of the following ways: chromogenically stained IHC and fluorescently stained IHC. In chromogenically stained IHC, an antibody is chemically conjugated to an enzyme, such as peroxidase, that can be reacted with a suitable substrate to give a colored product. In fluorescently stained IHC, the antibodies are finally tagged with fluorescent chemicals such as fluorescein isothiocyanate (FITC) or rhodamine. Here, we describe the standard methods of IHC applied to brain slice sections. Furthermore, an automated immunostainer is presented as another option for standardized immunohistochemistry.

Label-free detection of Aβ-42: a liquid crystal droplet approach for Alzheimer's disease diagnosis.

This study introduces a biosensor based on liquid crystals (LC) designed to detect the Aβ-42 biomarker, commonly associated with Alzheimer's disease. The sensor utilizes LC droplets created using a PEI/Tween-20 surfactant mixture, arranged radially in an aqueous solution. These droplets are coated with the Aβ1-16 antibody, enabling the detection of the Aβ1-42 biomarker. The key advantage of this biosensor lies in its ability to directly translate the antigen-antibody interaction into a change in the molecular orientation of the LC droplets, simplifying the detection process by removing additional procedural steps. Specifically, this immunoassay induces a transformation in the nematic droplets orientation from radial to bipolar upon successful antigen binding. When only the Aβ1-16 antibody coated the LC droplets, no change in orientation was detected, confirming the reaction's specificity. The orientation shift in the LC droplets indicates the formation of an immunocomplex between the Aβ1-16 antibody and the Aβ1-42 antigen. The LC droplet immunoassay effectively detected Aβ1-42 antigen concentrations ranging from 45 to 112.5 μM, with the Aβ1-16 antibody immobilized on the droplets at a concentration of 1 μg mL-1. These findings suggest that the LC microdroplets' orientational behavior can be harnessed to develop a biosensor for the in vivo detection of various proteins or pathogens in a PBS aqueous medium. Owing to its label-free nature and distinct optical signaling, this LC droplet-based immunoassay holds promise for further development into a cost-effective, portable diagnostic tool.

Epizootic Hemorrhagic Disease Agar Gel Immunodiffusion Assay.

Agar gel immunodiffusion assay (AGID) is a laboratory test which detects specific antigen-antibody interactions by the development of visible precipitation lines in a semisolid matrix. Here we describe the preparation of agar gel plates, the method to test serum samples by AGID for the presence of EHDV antibodies, and the interpretation of test results. This test has known cross-reactivity to bluetongue antibodies; therefore positive samples by this assay require additional confirmatory testing; generally, its use should be limited to healthy animal attestations where required.

Isolation of Peroxidase enzyme from various vegetables and coupling to IgG

Coupling of IgG with peroxidase enzyme finds various applications in determining the antigen-antibody interactions, in diagnostics or for research purpose. In the present study, Peroxidase enzyme was isolated from various vegetables like tomato (Solanum lycopersicum), cabbage (Brassica oleracea) and Radish (Raphanus sativus) and partial purification of enzymes was done with ammonium sulphate fractionation to 80% saturation. The active peroxidase enzyme was coupled to Goat anti-human IgG and the efficiency of coupling procedure was tested against Protein A sepharose purified human IgG. The coupling procedure was done by using sodium periodate and coupling was confirmed by dot blot and ELISA procedures. The present study provides an indigenous method to isolate an active peroxidase enzyme and couple to various IgG and use in immuno detection methods at very cost effective manner.

A liquid crystal-based biosensor for sensitive detection of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-α) is a cytokine secreted by the macrophages and Th1 cells of the immune system in response to inflammation. Given its significance as a biomarker with elevated levels in physiological fluids in various conditions, there is an increasing demand for a simple and accurate TNF-α detection strategy. In this article, we present a liquid crystal (LC)-based biosensor developed for sensitive TNF-α detection. The biosensor operates as follows: TNF-α and detection antibodies (DAbs) form complexes during preincubation. These complexes then bind with the surface-immobilized capture antibodies (CAbs), facilitating the antigen-antibody reaction between the CAbs and the TNF-α/DAb complexes. This target recognition interaction alters the surface topography, disrupting the vertical orientation of LCs produced by dimethyloctadecyl[3-(trimethoxysilyl)-propyl]ammonium chloride. The orientational change in the LCs can be easily visualized with a polarized optical microscope, resulting in brighter images as TNF-α levels rise. Our results demonstrated a linear range of 5.00-500pg/mL, with a limit of detection and limit of quantification being 1.08 and 3.56pg/mL, respectively. Recovery experiments on diluted saliva samples produced reasonable results, with TNF-α recoveries ranging from 97.1% ± 2.58% to 107% ± 5.95%.

Evaluation of AlphaFold antibody-antigen modeling with implications for improving predictive accuracy.

High resolution antibody-antigen structures provide critical insights into immune recognition and can inform therapeutic design. The challenges of experimental structural determination and the diversity of the immune repertoire underscore the necessity of accurate computational tools for modeling antibody-antigen complexes. Initial benchmarking showed that despite overall success in modeling protein-protein complexes, AlphaFold and AlphaFold-Multimer have limited success in modeling antibody-antigen interactions. In this study, we performed a thorough analysis of AlphaFold's antibody-antigen modeling performance on 427 nonredundant antibody-antigen complex structures, identifying useful confidence metrics for predicting model quality, and features of complexes associated with improved modeling success. Notably, we found that the latest version of AlphaFold improves near-native modeling success to over 30%, versus approximately 20% for a previous version, while increased AlphaFold sampling gives approximately 50% success. With this improved success, AlphaFold can generate accurate antibody-antigen models in many cases, while additional training or other optimization may further improve performance.

The multiple arrays of a PD1-derived peptide on chromatin specifically bind to PD-L1 and induce doxorubicin resistance in cancer cell lines

Programmed cell death-1 (PD-1) and its ligand 1 (PD-L1) have been extensively studied to block the activation of the PD-1 axis immune checkpoint pathway on T cells. However, recent evidence suggests that PD-1-independent PD-L1 pathways in cancer cells and macrophages are important in cellular proliferation and survival of those cell types but the underlying mechanism is little understood. To recapitulate the binding of multiple PD-1 to the high levels of PD-L1 expression on cancer cell membranes, recombinant histones carrying a PD-1 receptor-derived peptide (PDR) were prepared and used to assemble a PDR-displayed nucleosome array. PDR-displayed chromatin showed physiologically spaced nucleosomes, unaffected by N-terminal histone tails and biotinylated DNA modifications. PDR-displayed chromatin exhibited selective binding to the breast cancer cell line with high PD-L1 expression, MDA-MB-231, where saturated binding occurred within 3 h, comparable to well-known antigen-antibody reactions. Notably, PDR-displayed chromatin enhanced resistance to doxorubicin in PD-L1-overexpressed cancer cells, revealing a PD-L1 level-dependent effect on cell survival upon chemotherapy. Our study sheds light on the potential of PDR-displayed chromatin as a tool to induce chemoresistance in cancer cells without employing anti-PD-L1 antibodies or soluble PD-1 receptors. Further investigation into the underlying signaling pathways and therapeutic applications is warranted, positioning PDR-displayed chromatin as a valuable tool for understanding PD-L1-mediated intracellular signaling pathways in cancer cells with high PD-L1 expression.

Bioengineered cellulosic paper micro-device for serum albumin detection in clinical range

Chronic Kidney Disease (CKD) is becoming one of the major causes of morbidity and mortalities in 21st century. We have developed a bioengineered cellulosic paper device for the quantification of albumin (ALB) in physiological samples. The paper surface was activated and antibodies specific to target biomarker was immobilized on engineered paper surface. Every step after modification was characterized by FTIR, XPS, SPM and optical analysis. Further, the device model was designed using CAD file, and a 3-D cascade device was fabricated with in-built constant light source to provide proper and controlled environment for in-situ image analysis. After adding the sample on the bioengineered paper, the antigen-antibody reaction takes place, after that addition of dye results in change of color from yellow to blueish-green within 40 s. An optical method was employed for the analysis of the images by recognizing the specific area and the color intensity. Additionally, the immunosensor specificity was evaluated on number of molecules that are usually found in the serum sample. The linear dynamic range of the developed immunosensor has been reported to be 1–60 mg/mL, covering the normal as well as clinical range of ALB in physiological samples with a detection limit of 0.049(±0.002) mg/mL. With good precision and recovery, the device was able to successfully determine the ALB concentrations in serum sample. The developed device has simple and user-friendly interface and it may also help diagnosing CKD in personalized settings.

Research progress of colloidal gold technology

The colloidal gold technique is a technique that combines antigen-antibody immune reaction with colloidal gold labeling for antigen and antibody detection. This paper introduces the background, principle, application, advantages and disadvantages of this technique. The colloidal gold technique uses colloidal gold particles as markers to bind with antigens or antibodies and show red bands on the test strip. It has three methods: the double antibody sandwich method, competition method and indirect method. It can be used for things like environmental monitoring, food safety, and clinical diagnostics. It has the advantages of rapid, simple, low cost, and good stability, but also has the disadvantages of low sensitivity, poor specificity, difficult quantification, etc. In this paper, the background, principle, preparation method, application, advantages and disadvantages of colloidal gold are reviewed to provide readers with a comprehensive and systematic understanding. Although colloidal gold technology has made many achievements, there are still some challenges and difficulties in realizing the industrial application of colloidal gold, such as how to improve the stability, sensitivity, specificity and repeatability of colloidal gold.

An Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Circulating Pi*Z Alpha1-Antitrypsin Polymers.

Enzyme-linked immunosorbent assay (ELISA) is a sensitive immunoassay based on specific antigen-antibody reaction that is used for quantitative/qualitative analysis of various analytes in serum, plasma, saliva, cell and tissue lysates, and urine. ELISAs are typically performed in multi-well plates and depending on the design require coating antibody/antigen, analyte, detection antibodies, buffer, wash solution, and substrate/chromogen. Here we describe highly specific monoclonal antibody-based ELISA that detects circulating polymers formed by Pi*Z variant of human alpha-1-antitrypsin (Z-AAT). The circulating Z-AAT polymers are present in all individuals with inherited Pi*Z AAT deficiency. Thus, our assay provides a useful tool to examine the clinical significance and utility of Z-AAT polymers.