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Immunoassays are bioanalytical methods in which the quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Immunoassays have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug...
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... methods that have been applied in phar- maceutical analysis, based on whether the separation step is or is not required, can be classified into heterogeneous or homogeneous assay, respectively. These methods can be performed in either competitive or non-competitive de- signs. The choice from these designs is based on nature of the analyte, labeling chemistry available and the ana- lytical parameter required from the assay (e.g. sensitivity, dynamic range, and precision). Competitive design of im- munoassays can be carried out in an antigen-capture or antibody-capture format, depending on whether the solid phase is coated with antibody or antigen (analyte), respec- tively. The features of these formats are shown in Figure 1. In the antigen-capture format ( Figure 1A), the competi- tion reaction occurs between the analyte (in sample) and a labelled analyte for the binding to a limited amount of anti-analyte antibody coated onto a solid support. After equilibration and separation, the label activity on the solid phase is measured, and the measured signal is inversely correlated to the concentrations of analyte in the sample (66). In antibody-capture format ( Figure 1B), the analyte (or its protein conjugate) is coated onto a solid support. The competition occurs between the analyte (in sample) and the immobilized analyte for the binding to a limited amount of labelled anti-analyte antibody. After equilibration and sep- aration, the activity of the label bound to the solid support is measured, and the signal is inversely correlated to the concentration of the analyte. The non-competitive design (usually called "two-site" or "sandwich" assay) is used for large analytes possessing more than one recognition epit- opes on the molecule. It requires two antibodies that bind to non-overlapping epitopes on the analyte molecules. One of the two antibodies is bound to the solid phase, and the second one is labelled and used for detection. Figure 2 il- lustrates the features of this assay. The sample analyte is allowed to bind to an immobilized antibody. After wash- ing, the solid support (contains the formed analyte-anti- body complex) is incubated with an excess of the second labelled antibody, which binds to the remaining epitope on the analyte molecule. After washing, the activity of the la- bel bound to the solid support is ...
Citations
... Since no assays were validated for FVU samples, our study compared three distinct total human IgG quantification methods, each with different protocols and readouts. Homogeneous assays (HTRF and AlphaLISA) offered several advantages over the BioRad ELISA-based assay, including reduced hands-on time, shorter run times, and lower variability, even though they have a narrower dynamic range 46 . These assays operate on an energy transfer mechanism between fluorophores or beads, whereas the BioRad assay relies on Luminex technology 44,47 . ...
First-void urine (FVU) samples, containing human papillomavirus (HPV)-specific IgG from female genital tract secretions, provide a non-invasive option for disease monitoring and vaccine impact assessment. This study explores the utility of FVU for IgG quantification, exploring stability and compatibility with DNA preservation methods, alongside various IgG enrichment methods. Healthy female volunteers provided FVU and serum samples. FVU was collected with or without urine conservation medium (UCM) and stored under different conditions before freezing at −80 °C. Four IgG enrichment methods were tested on FVU samples. All samples were analyzed using three total human IgG quantification assays and an in-house HPV16-specific IgG assay. Samples stored with UCM buffer had higher total and HPV16-specific IgG concentrations (p ≤ 0.01) and IgG remained stable for at least 14 days at room temperature. Among IgG enrichment methods, Amicon filtration (AM) and AM combined with Melon Gel purification (AM-MG) provided similar HPV16-IgG concentrations, correlating strongly with serum levels. Protein G magnetic beads methods were incompatible with time-resolved fluorescence-based assays. This study highlights FVU as a reliable and convenient sample for IgG quantification, demonstrating stability for at least 14 days at room temperature and compatibility with UCM DNA preservation. It emphasizes the need to select appropriate IgG enrichment methods and confirms the suitability of both AM and AM-MG methods, with a slightly better performance for AM-MG.
... Table I below summarises a number of immunoassay approaches for determining vancomycin concentrations in human fluids that have been substantially investigated and developed. The RIA method uses radioactive marker compounds that are very harmful to the environment and health, and such method has proved to experience cross-reactivity with degradation products and needs more time and costs than other types of immunoassay (Darwish, 2006). Not only RIA but also FPIA are subject to cross-reactivity, which leads to biased results in patients with renal impairment. ...
Background: Administration of vancomycin in treating infections caused by Methicillin-resistant Staphylococcus aureus (MRSA) requires therapeutic drug monitoring (TDM). The immunoassay method and high-performance liquid chromatography (HPLC) are the two methods of choice for examining vancomycin levels, with their respective advantages. Objective: This study aims to review the validity of immunoassay and HPLC methods, as well as consider which method is appropriate, effective, and efficient for TDM in the clinical setting. Method: Related articles were searched for using the keywords "immunoassay", "vancomycin", "HPLC", "bioanalysis", and "human" in the PubMed, Google Scholar, and Science Direct databases. Result: A total of 20 publications examined immunoassays, whereas 23 articles covered HPLC. Both the immunoassay and HPLC methods provided acceptable bioanalytical validation values. Conclusion: The immunoassay method is an option for routine sample analysis that requires fast results, but this method is not recommended for patients with high immunoglobulin levels. The HPLC method is a choice because it offers better selectivity and sensitivity.
... Based on molecular recognition, numerous proteins, aptamers, and molecular imprints have been developed for various analytical methods, such as assays or sensors, and have provided powerful tools for diagnostics. 1,2 In the development of usable biomolecular recognition elements, the goal is usually to obtain a molecule with both a high affinity and specificity to the target ligand. Steroid hormones characterize a wide range of physiological and pathological states. ...
In the development of proteins, aptamers, and molecular imprints for diagnostic purposes, a major goal is to obtain a molecule with both a high binding affinity and specificity for the target ligand. Cushing syndrome or Addison’s disease can be diagnosed by cortisol level tests. We have previously characterized and solved the crystal structure of an anti-cortisol (17) Fab fragment having a high affinity to cortisol but also significant cross-reactivity to other glucocorticoids, especially the glucocorticoid drug prednisolone. We used native mass spectrometry (MS) to determine the binding affinities of nine steroid hormones to anti-cortisol (17) Fab, including steroidogenic precursors of cortisol. Based on the results, the number of hydroxyl groups in the structure of a steroid ligand plays a key role in the antigen recognition by the Fab fragment as the ligands with three hydroxyl groups, cortisol and prednisolone, had the highest affinities. The antibody affinity toward steroid hormones often decreases with a decrease in the number of hydroxyl groups in the structure. The presence of the hydroxyl group at position C11 increased the affinity more than did the other hydroxyl groups at positions C17 or C21. The binding affinities obtained by native MS were compared to the values determined by surface plasmon resonance (SPR), and the affinities were found to correlate well between these two techniques. Our study demonstrates that native MS with a large dynamic range and high sensitivity is a versatile tool for ligand binding studies of proteins.
... Cortisol, commonly known as the body's stress hormone, enables the body to remain active and alert in stressful situations [3]. Traditional methods of measuring depression included the use of questionnaires and scales such as the Hamilton Depression Rating Scale [4], as well as the measurement of cortisol levels in blood, urine, or saliva through immunoassays (which are bioanalytical methods where the quantitation of the analyte depends on the reaction of an antigen and an antibody [5]). However, these methods are unreliable. ...
... Immunoassays are biosensors in which an analyte reacts with an antibody (that is usually bound to a surface), and with the help of a label (which can be an enzyme, fluorescent molecule, or a radioisotope), the amount of analyte is determined [13]. Known for their high specificity and sensitivity, numerous immunoassays have been tried for the testing of botulinum toxins. ...
The Botulinum neurotoxin (BoNT) has the title of the most potent poison known to man. It causes a serious condition known as botulism. Guidelines and regulations for the industrial processing of food have made foodborne botulism rarer, but the use of the toxin in pharmaceutical industries and its potential as a bioweapon make detection methods of the toxin a necessary thing. Currently, the Mouse Lethality Bioassay (MLB) is still considered the golden standard of botulinum toxin detection even though it has several limitations. There had subsequently been the development of several alternatives to the MLB, including immunoassays, endopeptidase-based assays, and more. This paper gives an overview of the mechanisms behind botulinum toxins, current methods of detection, and the methods advantages and disadvantages.
... Our research team has developed 3D-printed immunoarrays for clinical diagnostics for the detection mainly of proteins in biological fluids as biomarkers for cancers [21][22][23][24][25] and COVID-19 [26]. Microfluidic systems have been designed to detect analytes, including hormones, enzymes, drugs, and disease-specific biomarkers [27,28]. The significance of 3D-printed immunoarrays in clinical diagnostics lies in their sensitivity, specificity, and versatility. ...
... They can be designed to detect low concentrations of analytes with high precision and accuracy. In addition, assay designs can facilitate fast and easy protocols, requiring only small amounts of sample and minimal equipment [19][20][21][22][23][24][25][26][27][28][29][30]. ...
Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay’s accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses.
... Immunoassay methods have been widely used in many important areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetic and bioequivalence studies in drug discovery and pharmaceutical industries" (Darwish, 2006). In hospital settings, their application is mainly confined to disease diagnosis and therapeutic drug monitoring. ...
Introduction: Immunoassays are expensive laboratory tests performed to diagnose diseases. It was emphasized to optimize the spending on care services at government hospitals to sustain free healthcare services in Sri Lanka. Optimization of services cannot be achieved without understanding the present through detailed cost analysis. The NHSL has no mechanism to measure the cost incurred for immunoassays. Objectives: The study's objective was to estimate the pertest cost of immunoassays at the NHSL and compare that with the private sector tariffs. Method: It was a single-setting bottom-up micro-costing study combined with a top-down approach conducted at NHSL. Reagent costs were considered direct costs and the rest of the expenses were indirect costs. Results: The pertest direct costs for immunoassays were in the range of SLR.772 to SLR. 6250. The indirect costs per test were estimated to be SLR.114. Indirect costs incurred by; the remuneration of human resources (62%), the opportunity cost of the building (15%), consumables (11%), depreciation cost of equipment (9%), and electricity charges (4%). Conclusion: The pertest costs of immunoassays were estimated to be in the range of SLR.886 to SLR.6364. The tariffs of the leading private hospital network were found to be much higher (158% to 1260%) than the pertest costs at NHSL. Recommendation: Considering the overwhelming contribution of reagent costs to the total pertest costs of immunoassays (87% to 98%), the efficient and effective sourcing of reagents could be the amicable way to contain the expenses for immunoassays.
... Predictive and mechanistic models of these binding associations [4] have provided physicians with a basis to scientifically define physiological and pathophysiological states to aid in the diagnosis, prognosis, and treatment of disease in their patients [2]. These models have also provided researchers biotechnological tools to further advance their understanding in subject areas such as molecular mechanism elucidation [5], drug discovery [6], and the evaluation of food and environment safety [7]. ...
... What distinguishes immunoassays from other assays is their employment of an immune complex, relying on the reaction between an antibody and an antigen for the measurement of an unknown concentration of analyte in a sample [9]. Immunoassays can be broadly defined as quantitative and qualitative techniques of measuring analytes using antibody-antigen interactions [6], coupled with a detection system that generates a signal response from a label (e.g., radioisotopic, enzymatic, fluorescent, or chemiluminescent [10]). Once the immune complex is formed, the label activity is measured and interpolated using a standard curve [6] that represents the measured signal as a function of the concentration [11]. ...
... Immunoassays can be broadly defined as quantitative and qualitative techniques of measuring analytes using antibody-antigen interactions [6], coupled with a detection system that generates a signal response from a label (e.g., radioisotopic, enzymatic, fluorescent, or chemiluminescent [10]). Once the immune complex is formed, the label activity is measured and interpolated using a standard curve [6] that represents the measured signal as a function of the concentration [11]. ...
An immunoassay is an analytical test method in which analyte quantitation is based on signal responses generated as a consequence of an antibody–antigen interaction. They are the method of choice for the measurement of a large panel of diagnostic markers. Not only are they fully automated, allowing for a short turnaround time and high throughput, but offer high sensitivity and specificity with low limits of detection for a wide range of analytes. Many immunoassay manufacturers exploit the extremely high affinity of biotin for streptavidin in their assay design architectures as a means to immobilize and detect analytes of interest. The biotin–(strept)avidin system is, however, vulnerable to interference with high levels of supplemental biotin that may cause elevated or suppressed test results. Since this system is heavily applied in clinical diagnostics, biotin interference has become a serious concern, prompting the FDA to issue a safety report alerting healthcare workers and the public about the potential harm of ingesting high levels of supplemental biotin contributing toward erroneous diagnostic test results. This review includes a general background and historical prospective of immunoassays with a focus on the biotin–streptavidin system, interferences within the system, and what mitigations are applied to minimize false diagnostic results.
... The remaining studies were conducted using enzyme-multiplied immunoassay technique (EMIT), non-stability indicating liquid chromatographymass spectrometry (LC-MS), spectrophotometric method and bioassays. Historically, EMIT and other immunoassays were commonly used in hospital laboratories and pharmaceutical stability studies [108]. Despite its selectivity, the immunoassay analytical technique may not be specific to the intact drug compared to the stability-indicating assays. ...
Background:
This systematic review summarises the stability of less commonly prescribed antibiotics in different peritoneal dialysis solutions that could be used for culture-directed therapy of peritonitis, which would be especially useful in regions with a high prevalence of multidrug antibiotic-resistant strains.
Methods:
A literature search of Medline, Scopus, Embase and Google Scholar for articles published from inception to 25 January, 2023 was conducted. Only antibiotic stability studies conducted in vitro and not recently reviewed by So et al. were included. The main outcomes were chemical, physical, antimicrobial and microbial stability. This protocol was registered in PROSPERO (registration number CRD42023393366).
Results:
We screened 1254 abstracts, and 28 articles were included in the study. In addition to those discussed in a recent systematic review (So et al., Clin Kidney J 15(6):1071-1078, 2022), we identified 18 antimicrobial agents. Of these, 9 have intraperitoneal dosing recommendations in the recent International Society for Peritoneal Dialysis (ISPD) peritonitis guidelines, and 7 of the 9 had stability data applicable to clinical practice. They were cefotaxime, ceftriaxone, daptomycin, ofloxacin, and teicoplanin in glucose-based solutions, tobramycin in Extraneal solution only and fosfomycin in Extraneal, Nutrineal, Physioneal 1.36% and 2.27% glucose solutions.
Conclusions:
Physicochemical stability has not been demonstrated for all antibiotics with intraperitoneal dosing recommendations in the ISPD peritonitis guidelines. Further studies are required to determine the stability of antibiotics, especially in icodextrin-based and low-glucose degradation products, pH-neutral solutions.
... Successful monitoring, disease prevention and overall care for public health rely on the effectiveness of biomarkers regarding food and clinical samples. IAs are based on the quantitation of an analyte through the reaction of an antigen (Ag) and an antibody (Ab) [2,3]. They are selective and sensitive; however, labeling agents are commonly used to increase sensitivity and catalyze the reaction [4]. ...
Nanotechnology offers new possibilities in molecular diagnostics, with nanoparticles gaining attention as biosensor upgrades. This study evaluates gold-coated silver nanoplates coated with PEG for enhanced protection, aiming to detect Spike protein with higher sensitivity, and emphasizes the importance of considering complex environments and appropriate controls for specific binding and accurate analysis. The sensitivity of antibody-coated PEGAuTSNPs as tools for immunoassays is demonstrated through fibronectin (Fn)– anti-fibronectin binding within an isolated extracellular matrix as a complex and native environment of Fn. Moreover, the optimal functionalization volume of Spike protein was determined (4 µg/mL of PEGAuTSNP). Anti-Spike was added to confirm binding, while the TJP1 protein was used as a negative control. The same experiment was used in the presence of horse serum to simulate a complex environment. According to Localized Surface Plasmon Resonance analysis and Dynamic Light Scattering size measurements, anti-Spike exhibited a stronger affinity for the nanoplates, causing TJP1 to be replaced by the antibody on the nanoplates’ surface. Future research will involve exploring alternative complex environments, filtering larger molecules, and the optimization of immunoassay performance.
