Specific Monoclonal Antibodies Research Articles

Present Scenario and Future Landscape of Payloads for ADCs: Focus on DNA-Interacting Agents

ADCs have emerged as a promising class of therapeutics, combining the targeting specificity of monoclonal antibodies with the cytotoxic potency of small-molecule drugs. Although the majority of approved ADCs are still based on microtubule binder payloads, the recent success of topoisomerase I inhibitors has revitalized interest in the identification of novel agents overcoming present limitations in the field including narrow therapeutic window and chemoresistance. The success of DNA binders as payload for ADCs has been very limited, up to now, due, among other factors, to high hydrophobicity and planar chemical structures resulting in most cases in ADCs with a strong tendency to aggregate, poor plasma stability, and limited therapeutic index. Some of these molecules, however, continue to be of interest due to their favorable properties in terms of cytotoxic potency even in chemoresistant settings, bystander and immunogenic cell death effects, and known combinability with approved drugs. We critically evaluated several clinically tested ADCs containing DNA binders, focusing on payload physicochemical properties, cytotoxic potency, and obtained clinical results. Our analysis suggests that further exploration of certain chemical classes, specifically anthracyclines and duocarmycins, based on the optimization of physicochemical parameters, reduction of cytotoxic potency, and careful design of targeting molecules is warranted. This approach will possibly result in a novel generation of payloads overcoming the limitations of clinically validated ADCs.

Cardiac toxicity of HER-2 targeting antibody-drug conjugates: overview and clinical implications.

Antibody-drug conjugates (ADCs) have recently emerged as a promising therapeutic option that combine the specificity of monoclonal antibodies and the cytotoxic effect of chemotherapy. With numerous ADCs approved and on the market, a particular concern of ADCs that target HER-2 has been their cardiac side effects, in view of the crucial role of HER-2 in cardiac development and physiology. While rarely toxic and generally safe, numerous publications have outlined the consistent association of trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) with the development of cardiac toxicity. Despite not being clinically relevant in most cases, cardiac baseline evaluation, monitoring and early detection of cardiac adverse events remain pivotal with HER-2 targeting ADCs. This review aims to summarize and better characterize the complete cardiac toxicity profile of HER-2 ADCs, with the goal of improving clinical understanding of this adverse event, leading to better recognition, monitoring and management.

Comparison of Survivin Determination by Surface-Enhanced Fluorescence and Raman Spectroscopy on Nanostructured Silver Substrates

Survivin belongs to a family of proteins that promote cellular proliferation and inhibit cellular apoptosis. Its overexpression in various cancer types has led to its recognition as an important marker for cancer diagnosis and treatment. In this work, we compare two approaches for the immunochemical detection of survivin through surface-enhanced fluorescence or Raman spectroscopy using surfaces with nanowires decorated with silver nanoparticles in the form of dendrites or aggregates as immunoassays substrates. In both substrates, a two-step non-competitive immunoassay was developed using a pair of specific monoclonal antibodies, one for detection and the other for capture. The detection antibody was biotinylated and combined with streptavidin labeled with rhodamine for the detection of surface-enhanced fluorescence, while, for the detection via Raman spectroscopy, streptavidin labeled with peroxidase was used and the signal was obtained after the application of 3,3′,5,5′-tetramethylbenzidine (TMB) precipitating substrate. It was found that the substrate with the silver dendrites provided higher fluorescence signal intensity compared to the substrate with the silver aggregates, while the opposite was observed for the Raman signal. Thus, the best substrate was used for each detection method. A detection limit of 12.5 pg/mL was achieved with both detection approaches along with a linear dynamic range up to 500 pg/mL, enabling survivin determination in human serum samples from both healthy and ovarian cancer patients for cancer diagnosis and monitoring purposes.

B-036 Systematic Evaluation of Antibody-Antigen Interactions for Suitability in Immunoassays Using Parallel Microarray ELISA

Abstract Background A critical step in immunoassay development is the selection of the appropriate capture and detection antibodies. The use of suboptimal antibodies can drastically affect the quality of the final assay. Traditional selection methods involve a direct testing of potential antibodies in the final assay setting; a time-consuming and material-intensive exercise that is often too complex to test all possibilities. Alternatives, such as abstract K-on/K-off determinations via SPR or BLI can be costly and often show limited translatability to the ultimate assay format. Here, we describe a novel systematic approach to rapidly screen antibody-antigen interactions for key assay specifications and test for translatability of the findings. Methods Nanoliter volumes of potential capture antibodies were spotted onto the bottom of ELISA wells, generating a microarray of antibodies in each well (>11,000 spots per plate). For the analysis of capture antibody-antigen interactions, a titration series of biotinylated analyte in negative sample matrix was applied. Binding was visualized using strep-polyHRP, precipitating TMB and high resolution scanning of each ELISA well. Antibody pair performance was investigated using unlabeled analyte and biotinylated detection antibody options. Sensitivity, specificity, dynamic range, binding speed, binding strength and matrix compatibility were investigated using time courses, concentration ranges, dilutions, interferent spikes and pH/salt/detergent challenges. Signals were quantified to calculate and statistically compare each possible interaction. Results Antibody-antigen interactions were investigated via parallel microarray ELISA for two example immunoassays: the HBsAg and cTnI test. For both analytes, a panel of antibodies was screened revealing antibodies with diverse characteristics. Striking differences were observed depending on the sample matrix composition. Without optimization, antibodies could be identified showing dynamic assay ranges of 3-4 log orders. Time course experiments revealed high-affinity antibodies suitable for rapid diagnostic tests. For HBsAg, levels down to 0.01 IU/mL (∼40 pg/mL) could be reproducibly detected, which is well below the minimal requirement of 0.13 IU/mL. Reactivity against multiple virus serotypes could be confirmed. Interestingly, for the cTnI test, a strong synergistic effect was observed when specific monoclonal antibodies were combined in the capture mix, resulting in a 6-fold sensitivity enhancement compared to their separate performances. Finally, we show that the results obtained with the parallel microarray ELISA approach are highly translatable to the final assay format, ranging from bead-based dry-chemistry assays to chemiluminescent immunoassays. Conclusions The parallel microarray ELISA evaluation presents a robust and reliable method for identifying the best antibodies for an immunoassay. It allows for a systematic and quantitative screen of numerous antibody options within 1-2 weeks under real immunoassay conditions with minimal antibody amounts. This method has shown to be highly translatable and is customizable to accommodate required assay specifications.

A-068 Development of GIP ELISAs: The Next Level of DiabetesManagement

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total and intact GIP in human plasma, and other biological fluids. Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone consisting of 42 amino acids (1-42aa). This undergoes processing by a dipeptidyl peptidase-4 (DPP-4) and produces GIP (3-42aa) and GIP (3-30aa) fragments. The processed forms have higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. It has also been observed that the GIP response is dependent not only on meal size but also on meal composition. GIP has been found to have physiological and pharmacological relevance in the development of obesity and the pathogenesis of cardiovascular disease besides its involvement in type 2 diabetic pathophysiology. GIP acts in the entero-insular axis as an anabolic hormone that increases insulin levels, which in return increases glycogen and fatty acid synthesis and inhibits the breakdown of fat. GIP also has extra-pancreatic functions as well as roles in the stomach to reduce acid secretion by the parietal cells. On the bone, GIP has a dual effect as it causes the proliferation of osteoblasts as well as inhibits osteoclastic bone resorption. The widespread expression of GIP-R in the brain suggests that GIP might play an essential function in neuro-signaling mechanisms. Methods Highly specific and reproducible total GIP (AL-1013) and Intact GIP (AL-1022) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and intact GIP concentrations in plasma in the respective immunoassays. Results Total and intact GIP ELISAs have been validated using well-characterized sample set and the peptide preparations. The total GIP ELISA detects 1-42aa, 3-42aa, 1-30aa, and 3-30aa in the sample. The intact GIP assay detects GIP (1-42aa) and the most abundant 3-42aa fragment equally in the human plasma. The intact GIP assay does not detect truncated C-terminal fragments(1-30aa). These assays did not cross-react to GRPP, Glucagon, Oxyntomodulin, GLP-1, and GLP-2. Method comparison between intact and total GIP assays using 388 samples yielded a slope of 0.8154 (r=0.958, p < 0.0001). Both the assays are highly reproducible with the total coefficient of variation less than 10%. GIP concentrations were measured in both assays in lean and obese subjects on fat-rich diet at 30-minute intervals. Average concentrations in intact GIP assay at 0 minutes (baseline) in lean and obese subjects (n=10) were 115.6 and 88.5 pg/mL, respectively. Average concentrations in total GIP assay at 0 minutes (baseline) in lean and obese subjects (n=10) were 151.5 and 125.9 pg/mL, respectively. Both total and intact GIP had steep increase in concentrations up to 90 minutes and then flattened up to 180 minutes. The GIP results have been also compared to the mean plasma concentrations of Glucagon, GLP-1, C-Peptide between lean and obese subjects on fat-rich meal. Conclusions Highly sensitive and specific total and intact GIP ELISAs have been developed to reliably quantify these important endocrine and local regulators in physiological and pathophysiological studies for metabolic disorders.

A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.

A-347 A Comprehensive Exdia TRF-LFIA for Simultaneous Quantification of GFAP and NT-proBNP in Distinguishing Ischemic and Hemorrhagic Stroke

Abstract Background The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. Methods We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing three test lines for NT-proBNP, GFAP, and Control. The fluorescence intensity of these test lines on the nitrocellulose membrane was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 hours). A small amount of clinical specimen (serum) was used with a well-assembled cartridge for the measurement of GFAP and NT-proBNP. Results To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Conclusions Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Immunofluorescent detection of protein CoAlation in mammalian cells under oxidative stress.

Previously, we reported the generation and characterisation of highly specific anti-CoA monoclonal antibodies capable of recognizing CoA in various immunological assays. Utilizing these antibodies in conjunction with mass spectrometry, we identified a wide array of cellular proteins modified by CoA in bacteria and mammalian cells. Furthermore, our findings demonstrated that such modifications could be induced by oxidative or metabolic stress. This study advances the utility of anti-CoA monoclonal antibodies in analysing protein CoAlation, highlighting their effectiveness in immunofluorescent assay. Our data corroborates a significant increase in cellular protein CoAlation induced by oxidative agents. Additionally, we observed that hydrogen-peroxide induced protein CoAlation is predominantly associated with mitochondrial proteins.

RELIABILITY OF AMYLOID DETECTION IMMUNOASSAY KITS FOR ALZHEIMER’S DISEASE SCREENING: A PRELIMINARY STUDY

Alzheimer’s Disease is a progressive neurodegenerative disease that damages the brain and is part of dementia. Alzheimer’s dementia is caused by various factors that until now the proper therapy is uncertain. Kit production as a diagnostic tool to support the development of screening for Alzheimer’s disease is a strategic effort. Screening based on detection with Enzyme-Linked Immunosorbent Assay (ELISA) can work effectively and accurately to optimize a specific antibody material or monoclonal antibody to the Aβ42 peptide. This study aimed to research the reliability of the amyloid detection kit which was developed as an alternative screening test for Alzheimer’s disease compared to commercially available kits. Based on the results and interpretation of inter and intra-assay coefficients, the in-house ELISA kit has comparable Aβ42 detection to the commercially available ELISA kit

Prussian-Blue-Nanozyme-Enhanced Simultaneous Immunochromatographic Control of Two Relevant Bacterial Pathogens in Milk

Salmonella typhimurium and Listeria monocytogenes are relevant foodborne bacterial pathogens which may cause serious intoxications and infectious diseases in humans. In this study, a sensitive immunochromatographic analysis (ICA) for the simultaneous detection of these two pathogens was developed. For this, test strips containing two test zones with specific monoclonal antibodies (MAb) against lipopolysaccharides of S. typhimurium and L. monocytogenes and one control zone with secondary antibodies were designed, and the double-assay conditions were optimized to ensure high analytical parameters. Prussian blue nanoparticles (PBNPs) were used as nanozyme labels and were conjugated with specific MAbs to perform a sandwich format of the ICA. Peroxidase-mimic properties of PBNPs allowed for the catalytic amplification of the colorimetric signal on test strips, enhancing the assay sensitivity. The limits of detection (LODs) of Salmonella and Listeria cells were 2 × 102 and 7 × 103 cells/mL, respectively. LODs were 100-fold less than those achieved due to the ICA based on the traditional gold label. The developed double ICA was approbated for the detection of bacteria in cow milk samples, which were processed by simple dilution by buffer before the assay. For S. typhimurium and L. monocytogenes, the recoveries from milk were 86.3 ± 9.8 and 118.2 ± 10.5% and correlated well with those estimated by the enzyme-linked immunosorbent assay as a reference method. The proposed approach was characterized by high specificity: no cross-reactivity with other bacteria strains was observed. The assay satisfies the requirements for rapid tests: a full cycle from sample acquisition to result assessment in less than half an hour. The developed ICA has a high application potential for the multiplex detection of other foodborne pathogens.

The neurotrophic factor artemin and its receptor GFRα3 mediate migraine-like pain via the ion channel TRPM8.

Migraine has a strong genetic foundation, including both monogenic and polygenic types. The former are rare, with most migraine considered polygenic, supported by genome-wide association studies (GWAS) identifying numerous genetic variants associated with migraine risk. Surprisingly, some of the most common mutations are associated with TRPM8, a non-selective cation channel that is the primary sensor of cold temperatures in primary afferent neurons of the somatosensory system. However, it is unlikely that the temperature sensitivity of TRPM8 underlies its role in migraine pathogenesis. To define the basis of the channel's involvement, we reasoned that cellular processes that increase cold sensitivity in the skin, such as the neurotrophic factor artemin, via its receptor GFRα3, also mediate TRPM8-associated migraine-like pain in the meninges. To investigate the role of artemin and GFRα3 in preclinical rodent migraine models, we infused nitroglycerin acutely and chronically, and measured changes in periorbital and hind paw mechanical sensitivity in male and female mice lacking GFRα3, after neutralization of free artemin with specific monoclonal antibodies, or by systemic treatment with a TRPM8-specific antagonist. Further, in wildtypes and mice lacking either GFRα3 or TRPM8, we tested the effects of supradural infusions of a mix of inflammatory mediators, artemin, and a TRPM8-specific agonist on migraine-related pain in mice. We find that mechanical allodynia induced by systemic nitroglycerin, or supradural infusion of inflammatory mediators, involves GFRα3. In addition, neutralization of circulating artemin reduces the nitroglycerin phenotype, demonstrating the importance of this neurotrophic pathway. Further, we show TRPM8 expression in the meninges and that direct supradural infusion of either a TRPM8-specific agonist or artemin itself produces mechanical allodynia, the latter dependent on TRPM8 and ameliorated by concurrent treatment with sumatriptan. These results indicate that neuroinflammatory events in the meninges can produce migraine-like pain in mice via artemin and GFRα3, likely acting upstream of TRPM8, providing a novel pathway that may contribute to migraine pathogenesis.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice.

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibodies to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.IMPORTANCEMost humans are exposed to influenza viruses in childhood and then subsequently exposed to antigenically drifted influenza variants later in life. It is unclear if antibodies elicited by earlier influenza virus exposures impact immunity against new influenza virus strains. Here, we used a mouse model to investigate how an anti-hemagglutinin (HA) monoclonal antibody (mAb) affects de novo B cell and antibody responses to the protein targeted by the monoclonal antibody (HA) and a second protein not targeted by the monoclonal antibody [neuraminidase (NA)]. Collectively, our studies suggest that circulating anti-influenza virus antibodies can potently modulate the magnitude and specificity of antibody responses elicited by secondary influenza virus exposures.

A novel assay of excess plasma kallikrein-kinin system activation in hereditary angioedema

BackgroundCleaved high-molecular-weight kininogen (HKa) is a disease state biomarker of kallikrein-kinin system (KKS) activation in patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH), the endogenous inhibitor of plasma kallikrein (PKa).ObjectiveDevelop an HKa-specific enzyme-linked immunosorbent assay (ELISA) to monitor KKS activation in the plasma of HAE-C1INH patients.MethodsA novel HKa-specific antibody was discovered by antibody phage display and used as a capture reagent to develop an HKa-specific ELISA.ResultsSpecific HKa detection following KKS activation was observed in plasma from healthy controls but not in prekallikrein-, high-molecular-weight kininogen-, or coagulation factor XII (FXII)-deficient plasma. HKa levels in plasma collected from HAE-C1INH patients in a disease quiescent state were higher than in plasma from healthy controls and increased further in HAE-C1INH plasma collected during an angioedema attack. The specificity of the assay for PKa-mediated HKa generation in minimally diluted plasma activated with exogenous FXIIa was demonstrated using a specific monoclonal antibody inhibitor (lanadelumab, IC50 = 0.044 µM).ConclusionsAn ELISA was developed for the specific and quantitative detection of HKa in human plasma to support HAE-C1INH drug development. Improved quantification of the HKa biomarker may facilitate further pathophysiologic insight into HAE-C1INH and other diseases mediated by a dysregulated KKS and may enable the design of highly potent inhibitors targeting this pathway.

Development of a double-antibody sandwich ELISA for detection of SARS-CoV-2 variants based on nucleocapsid protein-specific antibodies.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy-to-use detection methods for SARS-CoV-2 variants are critical for effective epidemic prevention and control. The N protein of SARS-CoV-2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS-CoV-2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2-C11, 5E2-E8, 6C3-D8, 7C8, 9F2-E9, 12H5-D11, 13G2-C10, 14E9-F6, and 15H3-E10. Using these antibodies, we established a sandwich ELISA with 6C3-D8 as the capture antibody and 5F2-C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross-reactivity with MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E. Furthermore, this method successfully detected both wild-type SARS-CoV-2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad-spectrum applicability, providing a robust tool for detecting SARS-CoV-2 variants.

Development of a double-antibody sandwich ELISA for quantification of mutated EPSPS gene expression in rice

Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16–256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %–7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44–445.80 μg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.

Expression of goat poxvirus P32 protein and monoclonal antibody preparation.

P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method.

Епідеміологічні дані, етіологія, патогенез, фактори ризику, клінічні прояви, сучасні методи діагностики і лікування, прогнозування перебігу Неходжкінських лімфом (огляд літератури)

Non-Hodgkin lymphoma (NHL) is a group of malignant diseases characterized by clonal proliferation of lymphoid cells corresponding to different stages of differentiation of normal B- and T- lymphocytes or natural killer (NK) cells. NHL is histopathologically and clinically diverse and divided into two groups: B-cell lymphomas and T-cell lymphomas, and subtypes: indolent (slow) and aggressive, classic and extranodal. The etiology of NHL is environmental, infectious, immunological, iatrogenic and genetic factors. The pathogenesis of NHL consists in: 1) translocations of oncogenes; 2) cytogenetic aberrations and mutations of tumor suppressor genes. Clinical manifestations of NHL: 1) lymphadenopathy; 2) symptoms of the presence of an extranodal tumor (abdominal pain; jaundice; shortness of breath and/or pleural effusion; bleeding, symptoms of gastrointestinal obstruction; symptoms associated with infiltration by lymphoma cells of the skin, thyroid gland, salivary glands, etc.; neurological symptoms of central and/or peripheral origin); 3) symptoms of bone marrow infiltration; 4) general symptoms (fever, night sweats, weight loss, fatigue, loss of appetite). Diagnosis of NHL involves: 1) auxiliary research (histological, immunohistochemical, cytogenetic and molecular, use of more specific monoclonal antibodies, immunophenotypic diagnosis, gene sequencing); 2) diagnostic tactics (estimation of stage and determination of prognostic factors according to IPI); 3) differential diagnosis (covers other causes of symptoms). The choice of treatment program for NHL depends on the type of lymphoma and the patient's condition. The main methods of treatment: wait and watch; monotherapy with alkylating drugs; RT; combined РCT; combined РCT with the use of purine analogs; therapy with monoclonal antibodies; PCT+monoclonal antibodies; HDCT+THSC. Survival prognosis for NHL depends on the origin of the tumor and its stage.

Comparison of polyclonal and monoclonal antibody assays for serum amyloid A in cats: a study based on an automated turbidimetric immunoassay in a primary care veterinary hospital.

Comparing the utility of the anti-human serum amyloid A (SAA)-specific monoclonal and polyclonal antibodies assays (LZ-SAA) with the pure monoclonal anti-human antibody assays (VET-SAA) during clinical practice in primary care hospital populations by measuring SAA measurement in healthy and diseased domestic cats. 52 healthy and 185 diseased client-owned cats. SAA concentration was measured using different LZ-SAA and VET-SAA measurements for healthy and various diseased cats. Sensitivity, specificity, and accuracy were calculated for each disease. VET-SAA has higher sensitivity than LZ-SAA for the most common diseases presenting to primary care veterinary hospitals, including chronic kidney disease, tumors, and gingivostomatitis. Our results reveal the capability of detecting low SAA concentrations in healthy and diseased cats using VET-SAA in contrast to LZ-SAA, which found elevations of SAA concentrations only in diseased cats. Our findings indicate that switching to the new VET-SAA instead of the conventional LZ-SAA will likely enhance the diagnostic performance in primary care veterinary hospitals.

Updated clinical evidence and molecular features of antibody-drug conjugates for advanced urothelial carcinoma

Platinum-based chemotherapy and immunotherapy are the primary systemic treatments for patients with advanced urothelial carcinoma (UC). However, the efficacy of these systemic therapies has yet to be optimized. Antibody-drug conjugates (ADCs), which combine the specificity of monoclonal antibodies with the cytotoxic potency of small-molecule drugs, have emerged as a promising new class of targeted therapies for UC. Currently, the Food and Drug Administration has approved 2 ADCs, namely Padcev and Trodelvy, for the treatment of advanced UC. This review provides an overview of the clinical evidence supporting the use of ADCs in patients with UC and summarizes the molecular features underlying these ADCs, which could be essential for understanding resistance mechanisms and minimizing treatment-related adverse events.