Prozone Research Articles

Dual-Fluorescent Quantum Dot Nanobead-Based Lateral Flow Immunoassay for Simultaneous Detection of C-Reactive Protein and Procalcitonin.

Simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) at the point of care is crucial for the management of infections in patients with inflammation and in critical care settings. The challenge of detecting high concentrations of CRP alongside low concentrations of PCT in plasma from inflammatory patients has limited the clinical application of multiplexed immunoassays. Herein, we developed a lateral flow immunoassay (LFIA) that employs quantum dot nanobeads (QDNBs) of varying sizes and colors to enable the simultaneous quantification of PCT and CRP in human plasma. To extend the dynamic range of CRP detection, we combined QDNBs with smaller particle sizes with the CRP detection antibodies, thereby increasing the assay's dynamic range and reducing the hook effect. At the same time, the stronger fluorescence emitted by these larger QDNBs, in conjugation with the PCT detection antibodies, allows for the detection of PCT at the nanogram level, meeting the demand for high sensitivity. The results show that this method can detect CRP concentrations from 0.1 to 3 mg/L and PCT with a detection limit of 0.09 ng/mL, which is on par with clinically used methods. By employing this dual-color and dual-size QDNB labeling strategy, we successfully achieved simultaneous detection of CRP with a broad dynamic range and PCT with high sensitivity in a one-step point-of-care rapid test.

Characterizing the Cooperative Effect of PROTAC Systems with End-Point Binding Free Energy Calculation.

Proteolytic targeting chimeras (PROTACs), as an emerging type of drug, function by proximity-based modalities that narrow the distance between a target protein and the E3 ubiquitin ligase to facilitate the ubiquitination labeling of the target protein for degradation. Although it is evidenced that the cooperativity of the PROTAC ternary interaction is one of the key factors affecting the degradation rate of a target protein, PROTAC design utilizing this indicator is still challenging because of the complicated/flexible interactions in a target-PROTAC-E3 ternary system. Therefore, developing reliable and practicable computational methods is of great interest for PROTAC design. Hence, in this study, we investigate the feasibility of using the end-point binding free energy calculation method, represented by molecular mechanics/Poisson-Boltzmann (generalized-Born) surface area (MM/PB(GB)SA), for characterizing cooperativity (including the stabilization and hook effects) of the PROTAC systems. The result shows that MM/GBSA is a good predictor in characterizing these effects under a relatively long molecular dynamics adjustment (50-100 ns) and low dielectric constant (εin = 1), with the Pearson correlation coefficient (rp) > 0.5 and 0.6 for the stabilization and hook effect, respectively. This study provides a feasible strategy for characterizing the cooperativity of the PROTAC systems, facilitating the rational design of PROTAC molecules.

B-293 Enzyme linked immunosorbent assay development and full validation for the quantification of the antibody conjugated fraction of an antiFAP ADC in human serum

Abstract Background Cancer remains a global public health issue. Despite recent progress in early diagnosis and treatment strategies, development of anti-tumor therapies with reduced systemic toxicity remains a challenge. Antibody-drug conjugates (ADCs), a new, highly-potent drug class are synthesized by linking a small molecule cytotoxic drug to a tumor specific antibody, are an emerging treatment option for many cancers. Despite advances in ADCs, study of the pharmacokinetics (PK) is challenging. A complete PK profile requires at least three analytical methods to determine: conjugated antibody, total antibody, and free drug by Ligand Binding Assays (LBA) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) respectively. Objective To develop and validate an ELISA method to quantify the conjugated antibody fraction of an anti-fibroblast activation protein (FAP) ADC against solid tumors in human serum. Methods Conjugated antibody assay: This indirect sandwich ELISA utilizes human FAP as capture antigen (Abcam) with a proprietary rabbit polyclonal anti-drug IgG as detection antibody. The signal is amplified by the use of a second antibody conjugated to Horse Radish Peroxidase (Abcam). Absorbance is measured at 450nm. Immunoassay method validation was done in compliance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M10 which establishes the limit for precision in CV% and accuracy in Relative Error (RE%) at ± 20% each (± 25% for LLOQ and ULOQ). Additionally, the total error (TE%) for accuracy and precision batches, should be ≤ 30% (≤ 40% at LLOQ and ULOQ). Results The weighted calibration curves were linear over a concentration range 0.5 to 100 µg/mL. A four-parameter regression model with a 1/Y2 weighting factor was adopted to best fit the OD450 values and concentrations. The intra- and inter-assay accuracy and precision for RE% and CV% was <12%, with a TE% < 23% for all concentrations tested. The quantification limit of 0.5 µg/mL complies with ICH criteria with a RE%, CV% and TE% of -10%, 8,5% and 15% respectively. Selectivity and specificity test fall within the criteria indicating that no individual matrix or co-medication affects the samples. The results from the dilution linearity obtained were an RE% of 11, -16 and -6 for 1:50, 1:100 and 1:200 respectively. Furthermore, not hook effect was observed as the dilution QC results gave above the quantification limit. Finally, the analyte was found to be stable at -20° and -80°for 148 days, at room temperature for 24hr and after 5 freeze/thaw cycles with a maximum RE and CV of 12 and 14% respectively in all the stability samples. Conclusions We have developed and optimized an ELISA method for measuring the antibody conjugated fraction of the ADC in serum. This method complies with the criteria of the ICH50 for selectivity, specificity, accuracy and precision, dilution linearity and hook effect in a concentration range between 0.5 - 100 µg/mL. Further, serum specimens are stable for 24h at room temperature, 5 freeze/thaw cycles and 148 days at -20 and -80 °C.

Impact of immunoglobulin preparations on anti-HLA antibody specificity analysis

Abstract Background Donor-specific antibodies (DSAs) targeting human leukocyte antigens (HLAs) substantially reduce the longevity of transplanted organs. Desensitization of DSA-positive renal transplant recipients is achieved through intravenous administration of immunoglobulin (IVIg). However, the presence and detectability of anti-HLA antibodies in IVIg preparations following administration are not fully understood. We aimed to assess whether immunoglobulin preparations contain anti-HLA antibodies that can be detected as passive antibodies when administered into the body. Methods We evaluated 3 immunoglobulin preparations from different pharmaceutical companies, using anti-HLA class I and II antibody specificity tests and immunocomplex capture fluorescence analysis (ICFA). Results Direct testing for anti-HLA antibodies resulted in high background errors, particularly for Venoglobulin. Diluting Venoglobulin to physiological concentrations revealed the presence of anti-HLA class I antibodies; however, no common alleles were found between the specificity identification test and ICFA. For Glovenin and Venilon, anti-HLA class I and II antibodies were detected; however, variability was observed across different test reagent lots. Moreover, dilution of the globulin formulation revealed a prozone phenomenon. Conclusion The administration of IVIg complicates the accurate detection of anti-HLA antibodies, underscoring the need for careful interpretation of test results post-IVIg administration.

A Novel Electrochemiluminescence (ECL) Immunoassay for Detection of Anti-drug Antibodies (ADAs) to a Monoclonal Antibody (mAb) PYX-106 in Human Serum

Background: PYX-106 is a fully human monoclonal antibody (mAb), targeting sialic acid binding immunoglobulin-like lectin-15 (Siglec-15), an immunosuppressive agent with widespread expression across various tumor types. Methods: To facilitate the evaluation of immunogenicity for PYX-106 in human serum, a validated method was established to enable the detection (screening, confirmatory, and titration) of antibodies to PYX-106 in human serum samples. Results: The Screening Cut-Point Factor (SCPF), Confirmatory Cut-Point (CCP), and Titration Cut-Point Factor (TCPF) were found to be 1.51, 26.9%, and 1.86, respectively. Sensitivity was determined to be 1.81 ng/mL in the screening assay and 1.25 ng/mL in the confirmatory assay. Low Positive Control (LPC) was set at 6.00 ng/mL, and High Positive Control (HPC) was set at 1000 ng/mL. The drug tolerance was up to 500 μg/mL at the HPC level, up to 241 μg/mL at the ADA 100 ng/mL level, and up to 38.9 μg/mL at the LPC level. The intra-assay percent coefficient of variation (%CV) was ≤ 2.1% for Positive Controls (PCs) in the screening assay and ≤ 1.0% for PCs in the confirmatory assay. The inter-assay %CV was ≤ 15.3% for PCs in the screening assay and ≤ 2.1% for PCs in the confirmatory assay. No hook effect, hemolysis effect, lipemia effect, or bilirubin interference was found in this ADA method. Anti-PYX-106 antibodies were found stable in human serum for at least 23 hours 51 minutes at room temperature or after six freeze/thaw cycles. Conclusion: Anti-PYX-106 ADA, bioanalytical assay validation, was reported for the first time in any biological matrix. This ADA method has been successfully applied to human sample analysis to support a clinical study.

A plug-and-play monofunctional platform for targeted degradation of extracellular proteins and vesicles

Existing strategies use bifunctional chimaeras to mediate extracellular protein degradation. However, these strategies rely on specific lysosome-trafficking receptors to facilitate lysosomal delivery, which may raise resistance concerns due to intrinsic cell-to-cell variation in receptor expression and mutations or downregulation of the receptors. Another challenge is establishing a universal platform applicable in multiple scenarios. Here, we develop MONOTAB (MOdified NanOparticle with TArgeting Binders), a plug-and-play monofunctional degradation platform that can drag extracellular targets into lysosomes for degradation. MONOTAB harnesses the inherent lysosome-targeting ability of certain nanoparticles to obviate specific receptor dependency and the hook effect. To achieve high modularity and programmable target specificity, we utilize the streptavidin-biotin interaction to immobilize antibodies or other targeting molecules on nanoparticles, through an antibody mounting approach or by direct binding. Our study reveals that MONOTAB can induce efficient degradation of diverse therapeutic targets, including membrane proteins, secreted proteins, and even extracellular vesicles.

Study on the Hook Effect in the Detection of Beta2-Microglobulin with Different Reagents in Fully Automated Biochemical Analyzers

Study on the Hook Effect in the Detection of Beta2-Microglobulin with Different Reagents in Fully Automated Biochemical Analyzers

Diagnostic pitfalls in assessment of ferritin following CAR-T-cell therapy: Understanding the hook effect.

The hook effect is a well-described but clinically underappreciated immunoassay interference, where a falsely lowered result is caused by analyte excess. We describe a situation in which ferritin immunoassay results from a 27-year-old female with immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome were more than 1000 times lower at a reference laboratory than those determined in-house after dilution. This case underscores the importance for clinical care providers to be aware of the impact of the hook effect on ferritin measurements, and to promptly communicate with the laboratory when there are discrepancies between clinical symptoms and test results.

A Novel Electrochemiluminescence (ECL) Immunoassay for the Quantitation of Monoclonal Antibody (mAb) PYX-106 in Human Serum

Background:: PYX-106 is a novel monoclonal antibody (mAb), targeting the sialic acidbinding immunoglobulin-like lectin 15 (Siglec-15) in the Tumor Microenvironment (TME). Precise measurement of PYX-106 is essential for the thorough assessment of PYX-106 pharmacokinetics in clinical investigations. Methods:: A novel Electrochemiluminescence (ECL) immunoassay for the quantitation of PYX- 106 in human serum was developed and validated. Biotinylated anti-PYX-106 antibody Bio-A1A1 was employed as the capture antibody, and ruthenylated anti-PYX-106 antibody Ru-A3G10 was utilized as the detection antibody in the ECL immunoassay on Meso Scale Discovery (MSD) platform. Results:: This assay was fully validated in terms of selectivity, accuracy, precision, hook effect, stability, etc., with a dynamic range from 50.0 to 2,500 ng/mL in human serum under the 2018 U.S. Food and Drug Administration (FDA) guidance and the 2022 U.S. FDA ICH M10 guidance. Conclusion:: PYX-106 bioanalytical assay validation was reported for the first time in a biological matrix, and this assay has been successfully applied to support a clinical trial PYX-106-101.

Nano-Proteolysis Targeting Chimeras (Nano-PROTACs) in Cancer Therapy.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have the capability to induce specific protein degradation. While playing a revolutionary role in effectively degrading the protein of interest (POI), PROTACs encounter certain limitations that impede their clinical translation. These limitations encompass off-target effects, inadequate cell membrane permeability, and the hook effect. The advent of nanotechnology presents a promising avenue to surmount the challenges associated with conventional PROTACs. The utilization of nano-proteolysis targeting chimeras (nano-PROTACs) holds the potential to enhance specific tissue accumulation, augment membrane permeability, and enable controlled release. Consequently, this approach has the capacity to significantly enhance the controllable degradation of target proteins. Additionally, they enable a synergistic effect by combining with other therapeutic strategies. This review comprehensively summarizes the structural basis, advantages, and limitations of PROTACs. Furthermore, it highlights the latest advancements in nanosystems engineered for delivering PROTACs, as well as the development of nano-sized PROTACs employing nanocarriers as linkers. Moreover, it delves into the underlying principles of nanotechnology tailored specifically for PROTACs, alongside the current prospects of clinical research. In conclusion, the integration of nanotechnology into PROTACs harbors vast potential in enhancing the anti-tumor treatment response and expediting clinical translation.

Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood.

Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis. We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood. An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma. Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples. The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.

Combating Prozone Effects and Predicting the Dynamic Range of Naked-Eye Nanoplasmonic Biosensors through Capture Bioentity Optimization.

Accurately quantifying high analyte concentrations poses a challenge due to the common occurrence of the prozone or hook effect within sandwich assays utilized in plasmonic nanoparticle-based lateral flow devices (LFDs). As a result, LFDs are often underestimated compared to other biosensors with concerns surrounding their specificity and sensitivity toward the target analyte. To address this limitation, here we develop an analytical model capable of predicting the prozone effect and subsequently the dynamic range of the biosensor based on the concentration of the capture antibody. To support our model, we conduct a sandwich immunoassay to detect C-reactive protein (CRP) in a phosphate-buffered saline (PBS) buffer using an LFD. Within the experiment, we investigate the relationship between the CRP dynamic range and the prozone effect as a function of the capture antibody concentration, which is increased from 0.1 to 2 mg/mL. The experimental results, while supporting the developed analytical model, show that increasing the capture antibody concentration increases the dynamic range. The developed model therefore holds the potential to expand the measurable range and reduce costs associated with quantifying biomarkers in diverse diagnostic assays. This will ultimately allow LFDs to have better clinical significance before the prozone effect becomes dominant.

Design algorithm for detecting falsely low urinary microalbumin results due to the hook effect

Design algorithm for detecting falsely low urinary microalbumin results due to the hook effect

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

Development and Clinical Validation of a Hook Effect-Based Lateral Flow Immunoassay Sensor for Cerebrospinal Fluid Leak Detection.

Rapid detection of cerebrospinal fluid (CSF) leaks is vital for patient recovery after spinal surgery. However, distinguishing CSF-specific transferrin (TF) from serum TF using lateral flow immunoassays (LFI) is challenging due to their structural similarities. This study aims to develop a novel point-of-care diagnostic assay for precise CSF leak detection by quantifying total TF in both CSF and serum. Capitalizing on the substantial 100-fold difference in TF concentrations between CSF and serum, we designed a diagnostic platform based on the well-known "hook effect" resulting from excessive analyte presence. Clinical samples from 37 patients were meticulously tested using the novel LFI sensor, alongside immunofixation as a reference standard. The hook effect-based LFI sensor exhibited outstanding performance, successfully discriminating positive clinical CSF samples from negative ones with remarkable statistical significance (positive vs negative t -test; P = 1.36E-05). This novel sensor achieved an impressive 100% sensitivity and 100% specificity in CSF leak detection, demonstrating its robust diagnostic capabilities. In conclusion, our study introduces a rapid, highly specific, and sensitive point-of-care test for CSF leak detection, harnessing the distinctive TF concentration profile in CSF compared with serum. This novel hook effect-based LFI sensor holds great promise for improving patient outcomes in the context of spinal surgery and postsurgical recovery. Its ease of use and reliability make it a valuable tool in clinical practice, ensuring timely and accurate CSF leak detection to enhance patient care.

119 A Hook Effect-based Lateral Flow Immunoassay Sensor for Cerebrospinal Leak Detection

INTRODUCTION: A rapid test for the presence of cerebrospinal fluid (CSF) would be ideal in the management of postoperative fluid collections after spinal surgery. In order to ascertain the nature of the fluid the gold standard has been to send the fluid for gel electrophoresis which is time consuming and expensive in order to determine the presence or absence of beta-2 transferrin. METHODS: A cassette containing a lateral flow strip was used with two conjugates: a gold nanoshell-anti-TF antibody conjugate (conjugate 1) and a gold nanoshell-lectin conjugate (conjugate 2).After a sample is applied, a chromatographic sandwhich forms in two detection zones to yield a detectable dark line: Test line 1 (T1) where anti-TF antibodies are embedded form a sandwich reaction with analyte (TF) and with conjugate 1 and test line 2 (T2) where embedded serum glycoproteins react with conjugate 2. A control line with anti-mouse IgG antibody is embedded to form a reaction with conjugate 1. We availed the well-known hook effect as both T1 and T2 detection zone development was inhibited by the saturation of binding sites of the conjugates and embedded material. RESULTS: 18 clinical samples of either CSF or postoperative drain fluid (serum) were tested using a third generation LFI. There were 7 CSF samples and 11 serum samples and one with buffer only. The hook effect-based LFI successfully discriminated positive clinical CSF samples from negative samples in all cases. CONCLUSIONS: This novel hook effect-based LFI sensor offers a fast, highly accurate and easy to use POC test for CSF leak.

Control line failure in Angiostrongylus vasorum point-of-care serology test in dogs with angiostrongylosis due tosuspected hook effect.

Angiostrongylosis is a significant differential for a diverse range of clinical signs in dogs, many of whom present acutely and sometimes with fatal consequences. Point-of-care diagnostic assays include a commercially available Angiostrongylus vasorum qualitative direct lateral flow assay. Case records from one referral centre from dogs with an invalid A. vasorum lateral flow assay, comprising an absent control line alongside a visible test line, were reviewed. As control line failure was hypothesised to be due to antigen excess; where available the A. vasorum lateral flow assay was repeated using dilutions of the original serum. Six dogs had an invalid A. vasorum lateral flow assay result. Five dogs had presented with acute-onset, severe clinical disease consistent with angiostrongylosis, and one dog was a clinically healthy in-contact. Clinical suspicion of angiostrongylosis was confirmed using alternative diagnostic testing and/or response to treatment. Repetition of the A. vasorum lateral flow assay, in four cases, using diluted plasma (10% to 12.5% v/v) resulted in the appearance of a control line alongside the visible test line. A heavy burden of A. vasorum infection resulting in angiostrongylosis should be suspected in dogs with compatible clinical signs and an invalid A. vasorum lateral flow assay result due to control failure alongside a visible test line. Repetition of the test with a diluted serum may be considered to account for the hook effect, also known as the postzone phenomenon, as a possible cause.

Characterization of a multiple-identity Vampire. Matthew Clairmont in A Discovery of Witches by Deborah Harkness

The combination of historical and fantastic literatura featured with romance, conflict and supernatural characters seem to be the ingredients of the new bestseller sagas focused on creatures such as vampires. An example is the All Souls Trilogy by Deborah Harkness. This autor uses different characteristics to report a current shift in traditional style, traits, and behaviour that the vampire figure seems to have experienced. This study has been set out with the objective of examining the character of the vampire Clairmont aimed at exploring the complexity of his multiple identities and demonstrating a shift in his role and traits regarding the traditional vampire. This has been done through a corpus study utilizing a thematic qualitative textual analysis. The study has revealed seven distinct themes related to new vampire characters and their potential to create a hook effect in the aforementioned readers.

Development and Validation of an ADA-Tolerant Assay for Quantification of an Exatecan-Based ADC in Monkey Plasma.

The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody-drug conjugate (ADC) in monkey plasma. The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.

Fuzzy-Based Anti-Swing Control for Variable-Length Cable-Suspended Aerial Transportation Systems Considering the Hook Effect

Fuzzy-Based Anti-Swing Control for Variable-Length Cable-Suspended Aerial Transportation Systems Considering the Hook Effect