Competitive ELISA Research Articles

Abstract 3135: BSI-730, a bispecific antibody targeting HER2 and PD-L1 for the development of a first-in-class bispecific ADC

Abstract Background: Antibody-drug conjugates (ADCs) have become a promising therapeutic option for patients with cancer. Bispecific ADCs targeting two tumor-associated antigens have potential differential anti-tumor advantages. HER2 and PD-L1 are co-expressed in some tumor cell types, such as breast cancer, gastric cancer, lung adenocarcinoma and urothelial carcinoma. A bispecific ADC targeting HER2 and PD-L1 should provide therapeutic benefit, particularly for patients with HER2 low cancer. Dual-target mediated cytotoxicity combined with PD-L1 mediated immunotherapy provides an innovative approach for cancer patients. Experimental procedures: An anti-PD-L1 monoclonal antibody was identified from A/J mice immunized with PD-L1-ECD-Fc and screened by the Biosion proprietary SynTracer® High Throughput Endocytosis Platform. A “knob-in-hole” bispecific antibody, BSI-730, targeting HER2 and PD-L1 was created with trastuzumab and the humanized anti-PD-L1 scFv. Target binding specificity, binding activity, and affinity of BSI-730 were evaluated by protein-based ELISA, cell-based FACS and Biacore-based SPR, and the ligand blocking activity of PD-L1 portion was measured by competitive ELISA and cell-based FACS. The internalization activities on various cancer cell lines with different expression levels of HER2 and PD-L1 were evaluated by using the SynTracer® Platform. Summary: BSI-730, a HER2/PD-L1 bispecific antibody, showed comparable binding affinity to HER2 as compared to trastuzumab, as well as comparable bioactivity to the parental anti-PD-L1 antibody regarding PD-L1 binding and PD-1/PD-L1 blocking. Based on a cell-based reporter assay, BSI-730 was able to reverse PD-L1 mediated T-cell suppression and exhibited comparable potency to the parental anti-PD-L1 antibody. The internalization of BSI-730 on HER2 low cancer cell lines was stronger than that of trastuzumab whereas the internalization of BSI-730 on HER2 high cancer cell lines was comparable to that of trastuzumab. Conclusion: BSI-730 is a novel bispecific antibody for the development of a first-in-class bispecific ADC with the potential advantage of increased potency that can be further investigated to treat HER2 and PD-L1 co-expression tumors, particularly HER2 low tumors. Citation Format: Xiaoyao Hao, Hongyan Li, Xiaodong F. Liu, Jinyu Liu, Wenwen Dai, Ying Pan, Hui-Han Hu, Jun Li, Shukai Xia, Qun Lyu, Hugh M. Davis, Mingjiu Chen, Zeyu Peng. BSI-730, a bispecific antibody targeting HER2 and PD-L1 for the development of a first-in-class bispecific ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3135.

Abstract 7666: Type XVII collagen as potential biomarker of epithelial damage in patients with atopic dermatitis and patients with metastatic colorectal cancer

Abstract Background Type XVII collagen is a membrane-associated collagen with interrupted triple-helices (MACIT) facilitating adhesion between the basement membrane (BS) and epithelial cells. Mutations in COL17A1 lead to junctional epidermolysis bullosa (JEB), while autoimmunity against type collagen XVII ectodomain can cause bullous pemphigoid (BP). In multiple epithelial cancers, both type XVII collagen and its shed ectodomain are associated with cancer development and invasiveness. This study aims to assess the potential of type XVII collagen as an epithelial damage biomarker in patients with metastatic colorectal cancer (mCRC) and in patients with skin diseases like atopic dermatitis (AD) and psoriasis. Methodology Type XVII collagen ectodomain levels were measured in serum using a competitive ELISA (nordicPRO-C17). The study included two cohorts: cohort 1 comprised 212 patients with mCRC and 28 healthy controls and cohort 2 included patients with AD (n=8) or psoriasis (n=3). PRO-C17 levels in mCRC patients were compared to 28 healthy controls using a t-test. The association between PRO-C17 levels and overall survival (OS) was evaluated by categorizing patients into low PRO-C17 levels (tertiles 1+2) and high PRO-C17 levels (tertile 3). Kaplan-Meier curves and the log-rank test were used to assess differences in survival between the two groups. The independent predictive value of PRO-C17 was evaluated using multivariate Cox analysis. PRO-C17 levels in AD and psoriasis patients were compared to 15 healthy controls using one-way ANOVA, followed by multiple comparisons with Dunn's test. Results PRO-C17 levels were elevated (p<0.05) in mCRC patients treated with bevacizumab compared to healthy controls. Patients with high PRO-C17 levels had a shorter overall survival (390 days) compared to those with low levels (539 days) (p=0.007). High levels of the biomarker were associated with an increased risk of mortality and PRO-C17 levels were independent of other risk factors, such as the number of drugs in the treatment regimen, tumor location, synchronous metastatic disease, primary tumor resection, performance status, and the number of metastatic sites (HR=1.43, 95% CI 1.01-2.01, p=0.044). Further, patients with AD had increased PRO-C17 levels (p=0.0057), while patients with psoriasis showed no significant differences in biomarker levels compared to healthy controls (p=0.58). Conclusions This study demonstrated that high PRO-C17 levels were independently associated with shorter OS and an increased risk of mortality in patients with mCRC. Additionally, elevated levels of PRO-C17 were observed in patients with AD. These findings suggest that type XVII collagen may serve as a reflection of epithelial damage in the invasive front during tumor metastasis. The role of this biomarker in other cancer types and skin diseases warrants further exploration. Citation Format: Marina Crespo-Bravo, Neel Nissen, Rasmus Pedersen, Mogens Boisen, Maria Liljefors, Astrid Johansen, Julia Johansen, Morten Karsdal, Nicholas Willumsen. Type XVII collagen as potential biomarker of epithelial damage in patients with atopic dermatitis and patients with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7666.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.

Abstract 1350: KD6001: A promising new anti-CTLA-4 human monoclonal antibody for cancer therapy

Abstract Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a crucial immune checkpoint that negatively regulates T cell activation. Inhibiting this negative regulation through CTLA-4 blockade has emerged as a promising therapeutic strategy against cancer. Herein, we present the preclinical pharmacology study of KD6001, a novel anti-CTLA-4 IgG1 kappa human monoclonal antibody with superior properties compared to ipilimumab, a currently approved anti-CTLA-4 mAb. Surface plasmon resonance (SPR) analysis revealed that KD6001 exhibits a higher affinity for CTLA-4 compared to ipilimumab (KD = 0.57 nM vs 1.20 nM). Competition ELISA experiments demonstrated that KD6001 is significantly more potent than ipilimumab in disrupting CTLA-4 interactions with CD80 (IC50 = 16 ng/mL vs 93 ng/mL) and CD86. Further, amino acid point mutation analysis indicated that two specific residues following the CD80/CD86 binding site (99MYPPPY104) are critical for ipilimumab binding, whereas mutation of the two residues had minimal impact on KD6001's CTLA-4 binding activity. These findings suggest that KD6001 and ipilimumab bind distinct epitopes on CTLA-4. In vitro experiments demonstrated that KD6001 enhances the production of IL-2 and IFNγ in PHA-activated human lymphocytes and exhibits synergistic anti-tumor effects when combined with anti-PD-1/L1 antibodies. Moreover, in vivo studies utilizing syngeneic murine tumor models established in human CTLA-4 gene knock-in mice revealed that KD6001 effectively inhibits or delays tumor growth in colon cancer MC38, melanoma B16, and hepatocellular carcinoma Hepa1-6 models. Additionally, KD6001 demonstrated synergistic anti-tumor effects when combined with a mouse anti-PD-1 antibody or ibuprofen. Toxicity evaluation in cynomolgus monkeys revealed that KD6001 was generally well-tolerated at doses ranging from 1 mg/kg to 10 mg/kg administered intravenously once weekly for four weeks. The primary pathological changes observed were lymphocyte/monocyte infiltration or increased lymphocyte/monocyte infiltration in several organs, consistent with KD6001's mechanism of action. Notably, the similar affinity of KD6001 for human and cynomolgus monkey CTLA-4 suggests that the toxicity profile observed in monkeys accurately reflects the dose-toxicity relationship in human. The promising preclinical data presented herein support the further clinical development of KD6001 as a potential therapeutic agent for cancer treatment. Currently, clinical trials evaluating KD6001 in patients with advanced melanoma are ongoing. Citation Format: Yi Chen, Peng Li, Guobo Fang, Heng Wu, Chi Zhang, Zeling Cai. KD6001: A promising new anti-CTLA-4 human monoclonal antibody for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1350.

Simultaneous identification of three emergent stimulant laxative adulterants in slimming foods using only one antibody.

Stimulant laxatives were recently found to be abused in slimming foods, resulting in harmful effects on consumers. To ensure the safety of relative products, sensitive yet multiplex immunoassays are crucial in rapid screening of stimulant laxatives. However, there are few immunoassays for these substances, and even less for broad-specific recognition. Thus, in this work, four theoretically promising haptens of emerging stimulant laxative bisacodyl were rationally designed using molecular modeling and synthesized to immune animals, whose feasibility was confirmed by the obtained broad-specific antibody. Based on this unique antibody, a highly sensitive multiplex competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with low limits of detection for bisacodyl, sodium picosulfate, and BHPM (0.23, 13.68, and 0.11 ng/mL). In spiked sample recovery test and real sample detection, this ciELISA exhibited acceptable consistency with the validation method, demonstrating high accuracy and applicability of our method. This reliable multiplex ciELISA proceeds the rapid screening of stimulant laxatives in slimming foods.

Generation of a highly specific recombinant full-length antibody for detecting ethirimol in fruit and environmental water

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a KD of 5.71 × 10-10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

On-site rapid detection of perfluorooctanoic acid by visual immunochromatographic strip biosensor in domestic water and real human samples

The International Agency for Research on Cancer (IARC) classifies PFOA as a Class 1 carcinogen. Here, a new naked-eye PFOA immunochromographic strip was developed to recognize PFOA in domestic water and real human samples within 10 min based on a novel custom designed anti-PFOA monoclonal antibody (mAb) 2A3, which was firstly an immune rapid detection method for PFOA has been proposed. Using computer simulation techniques such as quantum computing to assist in designing the structural formula of PFOA semi antigen, which hapten was firstly proposed. The half maximal inhibitory concentration of PFOA monoclonal antibody (mAb) 2A3 was 2.4 μg/mL. Using mAb 2A3, we developed an immunochromatographic strip (ICS) for detecting PFOA in real samples. The developed method generated results in 10 min, with visual detection limits of 20, 20, and 200 μg/mL and limit of detection of 50, 200, and 500 μg/mL for water, blood and urine samples, respectively. The established ICS and indirect competitive enzyme-linked immunosorbent assay were used to analyze the actual samples, and the results were confirmed by LC-MS/MS. Our study findings showed that the ICS and ic-ELISA can quickly detect PFOA in actual samples.

Preparation of limonin monoclonal antibody and establishment of a sensitive icELISA for analyzing limonin in citrus and herbal samples

Limonin is an intensely bitter and highly oxidized tetracyclic triterpenoid secondary metabolite, which is abundant in the Rutaceae and Meliaceae, especially in Citrus. In order to detect limonin content in complex substrates such as citrus and traditional Chinese medicine, monoclonal antibodies specifically recognizing limonin were prepared and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was established. The median inhibition concentration (IC50) was 5.40 ng/mL and the linear range was 1.25–23.84 ng/mL. The average recoveries from citrus peel and pulp samples were 95.9%–118.8% and 77.5%–113.1%, respectively. Moreover, the contents of limonin in 6 citrus samples and 4 herbal samples were analyzed by icELISA and UPLC-MS, and the results of the two methods were consistent. This validation is sufficient to demonstrate that the developed immunoassay is applicable for the detection of limonin in citrus and herbal samples and has the advantage of high efficiency, sensitivity, and convenience.

Establishment of Indirect Competitive Enzyme-linked Immunosorbent Assay (ic-ELISA) for Copper ion (Cu2+) in Raw Meat Products

Adding an appropriate amount of copper to feed can promote the growth and development of livestock; however, a large amount of heavy metal copper can accumulate in livestock through the enrichment effect, which poses a serious threat to human health. Traditional Cu2+ detection relies heavily on complex and expensive instruments, such as inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS); thus, convenient and simple rapid detection technologies are urgently needed. In this paper, synthesized copper antigens were used to immunize mice and highly specific anticopper monoclonal antibodies were obtained, which were verified to exhibit high affinity and specificity. Based on the above antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for the rapid detection of copper content in pork. The standard inhibition curve of the method was obtained by antigen–antibody working concentration screening, in which the half inhibitory concentration (IC50) was 11.888 ng/mL, the limit of detection (LOD) was 0.841 ng/mL and the correlation coefficient R2 of the curve was 0.998. In the additive recovery experiment, the recovery rate ranged from 90% to 110%, and the coefficient of variation (CV) was less than 10%, indicating that the method achieved high accuracy and precision. Finally, the results of ic-ELISA combined with Bland-Altman analysis showed a high correlation with ICP-MS, and the correlation coefficient (R2) reached 0.990 when the copper concentration was less than 200 ng/mL. Thus, the ic-ELISA method exhibits high reliability.

The hapten rigidity improves antibody performances in immunoassay for rifamycins: Immunovalidation and molecular mechanism

The immunogenicity of haptens determines the performance of the resultant antibody for small molecules. Rigidity is one of the basic physicochemical properties of haptens. However, few studies have investigated the effect of hapten rigidity on the strength of an immune response and overall antibody performance. Herein, we introduce three molecular descriptors that quantify hapten rigidity. By using of these descriptors, four rifamycin haptens with varied rigidity were designed. The structural and physicochemical feasibility of the designed haptens was then assessed by computational chemistry. Immunization demonstrated that the strength of induced immune responses, i.e., the titer and affinity of antiserum, was significantly increased with increased rigidity of haptens. Furthermore, molecular dynamic simulations demonstrated conformation constraint of rigid haptens contributed to the initial binding and activation of naïve B cells. Finally, a highly sensitive indirect competitive enzyme-linked immunosorbent assay was developed for detection of rifaximin, with an IC50 of 1.1 μg/L in buffer and a limit of detection of 0.2–11.3 μg/L in raw milk, river water, and soil samples. This work provides new insights into the effect of hapten rigidity on immunogenicity and offers new hapten design strategies for antibody discovery and vaccine development of small molecules.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach, however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, -Kv1.1 and -Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Impact of chronic psychological stress on platelet membrane fatty acid composition in a rat model of type 1 diabetes Mellitus

BackgroundChronic stress and diabetes mellitus are highly associated with oxidative stress and inflammation, resulting in cell membrane disruption and platelet activity. This study aims to evaluate the impact of chronic psychological stress on the composition of the platelet phospholipid membrane and platelet activation in type 1 diabetes mellitus (T1DM).MethodsWe enrolled 35 mature healthy female Wistar rats and randomly divided them into 4 groups, namely the control group (n = 9), stress group (n = 10), T1DM group (n = 8), and T1DM + Stress group (n = 8). The Wistar rats were treated in different experimental conditions for 28 days while being provided free access to feed and water. The concentration of corticosterone in blood serum and hair samples was measured using a competitive enzyme-linked immunosorbent assay. Gas chromatography-mass spectrometry was conducted to identify the methyl esters of fatty acids (FAs) in the platelet phospholipid membrane. A quantitative determination of 11-dehydro-thromboxane B2 in the blood serum was also performed using a competitive enzyme-linked immunosorbent assay.ResultsAfter 28 days, the concentration of corticosterone in blood serum (ng/mL) was observed to be higher in the stress group as compared to the T1DM and T1DM + Stress groups (P = 0.031 and P = 0.008, respectively). The percentage of C 16:0 FA in the platelet membrane was greater in the T1DM + Stress group, but its levels of C 20:1 omega (ω) 9 FA, including C 18:3ω3 FA, C 20:5ω3 FA, and the total sum of ω3 FAs, were lower as compared to the control group (P = 0.016; P = 0.016; P = 0.031; P = 0.016, P = 0.031). The concentration of 11-dehydro-thromboxane B2 in blood serum (pg/mL) was observed to be higher in the stress group than in rats with T1DM (P = 0.063).ConclusionChronic psychological stress is related to higher levels of corticosterone, saturated FAs acids in the platelet membrane, and greater platelet activation. This study proves how a low percentage of unsaturated fatty acids in the DM and stress groups indicates the disturbing impact of the oxidative/inflammatory environment to lipid metabolism and neuroendocrine response.

Sero-Detection of HEV in Cancer Patients and Healthcare Workers, Sudan

Background: Sudan, as a developing country, suffers from most of the risk factors related to the transmission of the hepatitis E virus (HEV). Objective: This study aimed to determine the sero-frequency of HEV in cancer patients and healthcare workers and its associated risk factors. Methods: A descriptive cross-sectional design was followed, recruited populations were 120 cancer patients and 120 healthcare workers. Sociodemographic data and risk factors of the study subject were collected using a questionnaire and patient records. HEV-specific anti-HEV IgM and IgG antibodies screened by a competitive ELISA technique. A Chi-square test was determined for the association between HEV IgG sero-positivity andsubject information. Results: The percentage of cancer patients positive for anti-HEV IgG and IgM antibodies was 50% (60/120) and 6.7% (8/120) while for the healthcare workers group was 25.8% (31/120) and 4.2% (5/120) respectively. The most documented risk factors for HEV among cancer patients were consumption of raw meat 94.2% (113/120), contact with animals 88.3% (106/120), and history of blood transfusion 81.7% (98/120) while raw meat consumption was commonly found in 94.2% (113/120) of healthcare workers group. A significant association was observed between IgG sero-positive healthcare workers and consumption of raw meat (p-value = 0.005). Conclusion: The HEV sero-positivity was found at a high rate in both study groups, which requires further studies on its prevalence, epidemiology, and prevention.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Seroprevalence and associated risk factors of equine herpesvirus type-1/-4 in selected districts of Northwest Amhara, Ethiopia

This study aimed to estimate the prevalence, determine the distribution, and identify the epidemiological risk factors of EHV-1/-4 infections in selected districts of Northwest Amhara Region. 460 serum samples were collected from equines using multistage cluster sampling technique, and a competitive Enzyme-linked immunosorbent assay (cELISA) was performed. Various risk factors for the occurrence of EHV-1/-4 were considered. Statistical analysis was performed using R version 4.3.1. 65.9% (303) equids were tested positive for antibodies against EHV-1/-4. Based on district, the highest prevalence was recorded in Wogera (86.1%), while the lowest was in Debark (47.4%). There was a significant difference (p <0.05; 95% CI: 1.1067993–3.682843) in the prevalence of EHV-1/-4 among species and donkeys are 2.019 times more likely to get an EHV infection than horses. The prevalence of EHV-1/-4 was highest in equids with the age of 3–8 years and lowest in < 3 years, and the difference was statistically significant (p <0.05; 95% CI: 1.9812042–6.771820). Statistically significant variation (p <0.05; 95% CI: 1.1173822–2.684013) was also observed between sex of equids in which females had 1.73 times higher chance to get EHV infection than males. Higher prevalence was found in lactating equids (81.6%), followed by pregnant equids (74.6%), and dry equids (66.4%). Generally, this study indicated a high and wide distribution of EHV-1/-4 infection in the study area, which needs due attention. Devising strategies to prevent and minimize the spread and occurrence of the infection is crucial.

Seroprevalence of peste des petits ruminants disease in Indonesian buffaloes may be an emerging threat to small ruminants

Background and Aim: The peste des petit ruminants (PPR) is a disaster-class virus that causes catastrophic drawbacks to small ruminant industries in affected countries. As PPR disease has been reported in neighboring countries, Indonesia, which has a large population of sheep and goats, has become prone to the emerging threat of infection. Because the virus can also infect other animals with subclinical manifestations, large ruminants, such as buffaloes, may play an important role in spreading the virus in the environment. Therefore, the main objective of this study was to identify PPR seroprevalence in the buffalo population of Indonesia. Materials and Methods: A competitive enzyme-linked immunosorbent assay was performed to identify the specific antibody for PPR viruses in the buffalo population using serum bank collection from the National Research and Innovation Agency, Indonesia. Results: PPR virus seroprevalence was detected in buffalo from Central Java, East Java, and East Nusa Tenggara Province in Indonesia. Although seroprevalence was low in the population, the antibody titer was relatively high in the positive samples. Sex and age were identified as determinant factors in the seroprevalence distribution of the buffalo population. Conclusion: The presence of antibodies against the PPR virus in buffaloes may indicate that PPR virus is circulating in the buffalo population of Indonesia. Keywords: antibody, buffalo, Indonesia, peste des petit ruminants, seroprevalence.

Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile

BackgroundTargeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells.MethodsIn order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059.ResultsGenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile.ConclusionGenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

Adjustment for Renal Function Improves the Prognostic Performance of Urinary Thromboxane Metabolites.

Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. We sought to define optimal TXB2-M cutpoints for aspirin users and nonusers and determine if adjusting TXB2-M for estimated glomerular filtration rate (eGFR) in addition to urinary creatinine improved mortality risk assessment. Urinary TXB2-M were measured by competitive ELISA in 1363 aspirin users and 1681 nonusers participating in the Framingham Heart Study. Cutpoints were determined for TXB2-M and TXB2-M/eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time. Multivariable models were compared using the Akaike Information Criterion (AIC). A cohort of 105 aspirin users with heart failure was used for external validation. Optimized cutpoints of TXB2-M were 1291 and 5609 pg/mg creatinine and of TXB2-M/eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg·min/creatinine·mL·1.73 m2), for aspirin users and nonusers, respectively. TXB2-M/eGFR cutpoints provided more robust all-cause mortality risk discrimination than TXB2-M cutpoints, with a larger unadjusted hazard ratio (2.88 vs 2.16, AIC P < 0.0001) and greater differences in restricted mean survival time between exposure groups (1.46 vs 1.10 years), findings that were confirmed in the external validation cohort of aspirin users. TXB2-M/eGFR cutpoints also provided better cardiovascular/stroke mortality risk discrimination than TXB2-M cutpoints (unadjusted hazard ratio 3.31 vs 2.13, AIC P < 0.0001). Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin use.

Seroprevalence, Serotyping, and Associated Risk Factors of Foot and Mouth Diseases in Bovine in Western Amhara Regional State, North Western Ethiopia

Foot and mouth disease (FMD) is the most contagious disease of animals. The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats, deer, and is characterized by fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats. The study aimed to determine seroprevalence of FMD (Foot and Mouth Disease), to identifay type of serotypes and to know associated risk factors. A total of 389 sera samples were collected from cattle in four districts of the north western Amhara region and subjected to a 3ABC enzyme-linked immune-sorbent assay. The overall seroprevalence of FMDV was 5.66% (22/389); (95%; CI: 3.34% to 7.98%). The 22 positive samples were subjected to solid phase competitive ELISA to identify specific serotypes. The occurrence of FMD was higher in Adet (OR= 12.8), greater in the semi-intensive than extensive production systems (OR=10.4) and highly occurred in the cross breed than local breed cattle (OR=3.56). Serotypes identified in the four districts were type O, SAT2, and A. This study revealed that FMD is a prevalent and endemic disease. Thus, awareness creation to the stakeholders on the control and prevention of a disease is required. Further epidemiological investigation and vaccine trials should be conducted.