Sandwich ELISA Research Articles

P091 PROENKEPHALIN CONCENTRATION AND ITS DETERMINANTS IN PATIENTS WITH END-STAGE KIDNEY DISEASE TREATED WITH HEMODIALYSIS AND PERITONEAL DIALYSIS

Background and Objective. Proenkephalin (PENK) has been recently shown to reflect glomerular dysfunction and predict new-onset acute kidney injury and heart failure. While previous studies investigated PENK utility in individuals with preserved renal function, PENK concentration in patients with end-stage kidney disease (ESKD) remained to be established. The study aimed to assess plasma PENK concentration in patients with end-stage kidney disease (ESKD) treated with hemodialysis (HD) and peritoneal dialysis (PD) and to investigate its correlation with heart failure. Additionally, we aimed to determine whether PENK is removed during a hemodialysis session. Methods. 88 patients with ESKD (41 females) undergoing HD (n=66, 75%) and PD (n=22, 25%) were enrolled in this cross-sectional study. Blood samples for PENK determination were collected before and at the end of the hemodialysis session in HD patients and at a single time point in PD patients. Plasma proenkephalin concentration was assessed using a sandwich ELISA immunoassay. Samples for measurement of creatinine, urea, and NT-proBNP were drawn simultaneously. Results. The median (IQR) plasma PENK concentration in the overall population was 1.492 ng/ml (1.071-4.380). Both median eGFR (5 mL/min/1.73 m2 [4-7] vs. 5 mL/min/1.73 m2 [4-8], p=0,856) and plasma PENK levels (1.368 ng/ml [1.068-4.030] vs. 1.706 ng/ml [1.211-5.858], p=0.305) did not differ significantly between HD and PD patients. In HD patients, median PENK concentration was significantly higher before than after hemodialysis (1.368 vs. 2.061; p=0.003). No correlation was found between PENK level and urea (p=0.192), eGFR (p=0.922), duration of dialysis (p=0.637), and residual urine output (p=0.784). Heart failure (p=0.961), EF (p=0.361), and NT-proBNP (p=0.949) were not associated with increased PENK concentration. Conclusions. Our study proved that PENK is not removed during hemodialysis session. PENK concentration does not reflect renal function and cardiac status in patients with ESKD. The fact that PENK is not removed by hemodialysis calls into question the usefulness of PENK as an everyday use biomarker in acute kidney injury.

Exploring the Association of VEGF-A and ANGPTL2 with the Prognosis of Non-proliferative and Proliferative Diabetic Retinopathy.

Introduction Diabetic retinopathy (DR) is a microvascular ailment that can arise from the long-term effects of diabetes mellitus. It can potentially cause retinal damage that could endanger vision and cause blindness. The worsening of DR is mainly linked to poor glycemic control, uncontrolled hypertension, and dyslipidemia. There is a need for alternative and clinically significant novel molecules involved in the pathogenesis of DR because the diagnostic and prognostic markers have reached a limit. Materials and method This study included sex and age-matched diabetic patients with proliferative stage (N = 70), non-proliferative stage (N = 80), and control (N = 80, without the sign of DR). These patients were recruited from outpatients in the Department of Ophthalmology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India. A random blood sample was collected from each study participant, and the serum was separated after centrifugation and stored at -80 °C for batch analysis. The biomarkers vascular endothelial growth factor (VEGF-A) and angiopoietin-like protein-2 (ANGPTL2) were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) technique, and the laboratory parameters such as fasting blood sugar (FBS), lipid profile, blood urea nitrogen (BUN), creatine, and glycated hemoglobin (HbA1C) were also assessed. Results We observed statistically significant differences in theduration of diabetes, FBS, total cholesterol (TC), triglyceride level (TGL), BUN, and creatine (p<0.05), and the mean age of study participants was 52.95±8.20 years in the control group, 53.85±10.20 years in the proliferative diabetic retinopathy (PDR) group, and 55.02±7.65 in the non-proliferative diabetic retinopathy (NPDR) group. Furthermore, ANGPTL2 levels were statistically significant according to the severity of the disease (p<0.001*), and they were also linked (p<0.05) with established markers such as VEGF-A. Conclusion Thus, our research implies that the up-regulated markers might be linked to the disease's advancement and could serve as a prognostic indicator or therapeutic target for DR.

Unique content of breastmilk: neurotrophic growth factors in breastmilk at 2 years and beyond.

The aim of our study was to show the presence of neurotrophic factors in breast milk that have a significant impact on neurocognitive development of children aged two years and beyond. Mothers expressed at least 5mL of breast milk into sterile containers when their children 18, 24, and ≥ 25months of age, and then specimens were transferred to Eppendorf tubes and stored at -20 °C. One day before the analysis, specimens were kept at +4°C and then thawed at room temperature to prepare them for analysis. Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and S100B neurotrophic growth factor levels were analyzed using the sandwich enzyme-linked immunosorbent assay (ELISA) principles. Sixty-two mothers with children aged 18months were included in the study. The mean age of the mothers was 33.4 (± 0.71) years. Due to the detection limits of the commercial kits, BDNF and S100B analyses could not be conducted. Therefore, only GDNF was analyzed. The presence of GDNF was found in the breast milk samples taken at 18, 24, and ≥ 25months, and the median (min max) values were 315,505ng/mL (193,067 750,718), 316,721ng/mL (161,278l-752,252), and 564,577ng/mL (238,528-781,104) respectively. There were no significant differences between GDNF levels of breast milk samples collected from the same mother at the three different time points (18, 24, and ≥ 25months) (p = 0.278). Conclusion: Our study was the first to show the presence of neurotrophic factors in the breast milk of mothers with healthy children over one year of age. Our results provide evidence-based data on the importance of breastfeeding until children are at least two years of age. What is Known: • Presence of Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and S100B neurotrophic growth factor have been shown in the breast milk of mothers whose infants are the first year of life. What is New: • Glial Cell Line-Derived Neurotrophic factors continue to present in breast milk of mothers with children aged 18, 24, and ≥ 25months, without any significant difference in level between months.

In situ growth of double-hybrid nanoflowers on paper for the visual detection of carcinoembryonic antigen

In this study, the hemoglobin, egg white, copper phosphate organic–inorganic hybrid nanoflowers (H-E-Cu-NFs), and filter paper-based-egg white-Ca3(PO4)2 HNFs (FP-E-Ca-NFs) were synthesized using a mild biomineralization method. Antibodies (Ab) were labeled on the surface of nanoflowers through highly specific interactions between avidin and biotin. The FP-E-Ca-NFs@Biotin N-hydroxysuccinimide ester–Anti-carcinoembryonic antigen primary antibodies (Biotin-NHS-Ab1)/carcinoembryonic antigen (CEA)/H-E-Cu-NFs@Biotin-NHS-Ab2 immune-biosensing system was constructed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to achieve the visual detection of CEA. Among them, H-E-Cu-NFs have abundant enzyme-like catalytic sites of Fe3+/Cu2+, which can effectively improve the efficiency of the colorimetric reaction. In contrast, E-Ca-NFs have no catalytic sites, and the directional design of catalytic performance realizes the specific identification and signal amplification of CEA detection. The as-developed immune-biosensing system showed a detection range of 0.5 ∼ 100 ng/mL, with the lowest detection limit of 0.5 ng/mL and the CEA recovery between 89.5 % ∼ 108.1 % in human serum samples. Compared to traditional ELISA methods, this immune-biosensing system has the advantages of easy operation, low cost, rapid detection, and on-site detection.

Generation of rabbit single-chain variable fragments with different physicochemical and biological properties by complementary determining region-grafting technology

In this study, we have demonstrated a CDR grafting technology for the generation of rabbit scFvs with different antigen recognition and physicochemical properties. The antigen-binding affinity of the CDR-grafted anti-CRP scFv, C1R/B1R (V1), which was generated by the complementary determining region/framework region (CDR/FR) definition based on the traditional numbering rule, was insufficient when compared to that of the original clone, C1R, suggesting that the amino acid residues outside the original CDRs might significantly contribute to antigen recognition in rabbit scFvs. We redefined new CDRs and FRs to maintain antigen-binding affinities through the extension of multiple amino acid residues for CDRH1 and CDRH2, based on the amino acid sequence alignments of rabbit scFvs isolated from phage libraries. The new version successfully maintained the antigen binding affinity. CDR-grafted scFvs possessing a common CDR sequence and different FR sequences were successfully generated based on this new CDR/FR definition, and their physicochemical properties were further investigated. The antigen-binding activities of rabbit scFvs on Maxisorp varied between the tested clones in sandwich ELISA, supporting the idea that the combination of CDR with different FRs might change the physicochemical properties of scFvs on a solid material. The CDR-grafted scFvs possessing a frame sequence of anti-CRP scFv C2R maintained the ability to bind to protein L, and were successfully purified. Expression titers showed improved solubility by diminishing the amount of insoluble scFvs. Thus, the method developed in this study is promising for generating alternatives with strict antigen binding recognition and different physicochemical properties.

Revolutionizing ELISA: A one-step blocking approach using lipid bilayer coatings

Enzyme-linked immunosorbent assay (ELISA) is an essential technique for biomolecule detection in diagnostics and research, but traditional protocols are time-consuming and variable. Conventional blocking agents like bovine serum albumin (BSA) pose ethical issues and potential assay interference. This study presents a one-step lipid-blocking approach to simplify the ELISA procedure. Compared to conventional blockers, the Lipid Universal Coating Assembly (LUCA) antifouling blocker showed comparable sensitivity while significantly reducing processing time and steps. Furthermore, it showed improved co-blocking efficacy and signal intensity in conventional protocols when combined with minimal BSA concentrations. Stability tests under accelerated aging confirmed the robustness of the LUCA antifouling blocker. Additionally, it effectively facilitated antibody detection for COVID-19 antigens in direct ELISA and human IL-6 antigen in Sandwich ELISA, suggesting its versatile applicability. Taken together, our findings reveal that one-step lipid blocking not only streamlines the ELISA process but also maintains high sensitivity and specificity, providing an efficient, user-friendly, and environmentally friendly alternative to traditional ELISA blockers and a robust platform for rapid diagnostics.

Straightforward high-temperature antibody immobilization for rapid and simple microfluidic paper-based ELISA for detection of β’-component (Onc k 5) protein, a major IgE-binding protein in salmon roe

Detection of allergenic proteins in food is required for maintaining the health of persons with food allergies. Enzyme-linked immunosorbent assay (ELISA) is a common technique for the detection of proteins. However, conventional ELISA is expensive, time-consuming, and needs technical expertise. Here, a microfluidic paper-based analytical device (µPAD) combined with a sandwich ELISA technique is developed for rapid, simple, and sensitive colorimetric detection of β’-component protein, a major allergen in salmon roe. The developed µPAD-ELISA employs a capture antibody and enzyme-labeled detection antibody. In addition to the µPAD, a technique for immobilization of the capture antibody has been developed, which completes the immobilization within 5 min by drying the antibody solution on a paper substrate in an oven at 50 °C without any chemical modification. The immobilized antibody exhibited higher binding capability to an antigen and stability against washing processes than those immobilized at room temperature. Since this technique allows simple and rapid preparation of the ELISA in the µPAD, the present µPAD-ELISA completed the analysis within only 15 min, including the immobilization step, compared to a conventional ELISA, which takes about 19 h. This µPAD-ELISA offered a good limit of detection of 1.59 ng mL−1 and reduced the amounts of reagents by a factor of about 17–67, which decreases the analysis cost. This µPAD-ELISA approach employing the present antibody immobilization is expected to be an effective strategy for the detection of allergenic proteins in food.

Major Vault Protein/Lung Resistance-Related Protein: A Novel Biomarker for Inflammation and Acute Infections.

Vault particles are large cytoplasmic ribonucleoprotein particles that participate in inflammation. The aim of this study was to assess the diagnostic and prognostic value of major vault protein (MVP) in patients with inflammation, in order to determine whether MVP could be used as a biomarker for infection or inflammation. We also aimed to compare the diagnostic impact of MVP compared to other conventional measurements, such as CRP or white blood cell (WBC) counts. CRP and MVP levels were measured in 111 sera samples from 85 patients with inflammation admitted to a tertiary-care hospital and 26 healthy individuals during an 18-month period (2019-2020), using nephelometry and a custom MVP sandwich ELISA assay, respectively. In addition, WBC counts were measured using a commercial assay. MVP levels were found to be elevated in patients with inflammation compared to healthy individuals (p < 0.0001). Moreover, MVP levels were higher in patients with inflammation due to an infectious etiology compared to those with non-infectious etiology (p = 0.0006). MVP levels significantly decreased during the first four days of infection in response to antibiotic treatment, while CRP levels showed a less-sensitive decline. An ROC curve analysis demonstrated that MVP and CRP have similarly high diagnostic accuracy, with AUCs of 0.955 and 0.995, respectively, followed by WBCs with an AUC of 0.805. The ROC curves demonstrated that MVP has the potential to serve as a diagnostic biomarker for inflammation and infection. Additionally, MVP levels may reflect the efficacy of antibiotic treatment.

The clinical importance of IFN-γ and human epididymis protein 4 in Egyptian patients with epithelial ovarian cancer combined with HPV infection

BackgroundHigh-grade Epithelial Ovarian Cancer (HGEOC) is an aggressive disease that usually presents at an advanced stage. Thus, detecting the circulating cytokines (IFNγ and TNF-α) may serve as a biomarker to identify malignancy and manage therapeutic decisions. ObjectivesAssessing the clinical importance of inflammatory mediators and tumor markers in EOC Egyptian patients compared with benign cases. Moreover, identifying the distinct inflammatory mediators in EOC patients combined with HPV infection. MethodsThis study was conducted on 61 Egyptian patients, divided into 25 patients with HGEOC, 22 patients with LGEOC, and 14 benign ovarian tumor cases. Measurements of serum HE4, CA125, CEA, and CA19-9 were determined by Roche Elecsys immunoassays. Serum levels of TNF-α and IFN-γ were measured using quantitative sandwich ELISA. Quantitative genotyping of HPV DNA types 16, 18, and 45 was assessed for the HPV DNA-positive samples. ResultsHPV DNA was detected in 25.53 % of malignant cases, HPV 16 was detected in 50 % of HPV-positive cases, and only 1 case of HPV 18 was detected out of 12 positive cases. The Human Epididymis protein 4 (HE4) was statistically different between patients with EOC and benign cases (p-value = 0.007) and between HPV DNA positive and HPV DNA negative cases (p-value = 0.008). The serum levels of IFN- γ were statistically different between HGEOC and LGEOC (p-value < 0.001), while the serum levels of TNF-α didn’t differ statistically between the two groups. ConclusionIFN-γ could be used as a biomarker to discriminate HGEOC and LGEOC. Initial evidence for the possible association between HE4 and the progression of HPV-associated EOC was speculated.

Effects of YM087 and VPA985 on the T237M mutant receptor functionality in nephrogenic diabetes insipidus

Abstract Objectives Mutations detected in the AVPR2 gene (arginine vasopressin type 2 receptor) are known to cause nephrogenic diabetes insipidus (NDI). Several pharmacological chaperones (PCs) target misfolded AVPR2 proteins and rescue them from the quality control system of the cell. In this study, we investigated the effect of YM087 and VPA985, which are PCs, on T273M-AVPR2 mutant that are known to cause NDI. Methods The total and cell surface expressions of T273M in COS-1 cells were measured by sandwich ELISA and flow cytometry after the cells were treated with YM087 and VPA985 separately. In addition, the cAMP accumulation assay was performed. Results It was observed that VPA985 treatment significantly increased the cell surface expression and slightly increased the maximum cAMP response of T273M. Both YM087 and VPA985 decreased the ligand concentrations which were responsible for making half of the maximum response of the T273M mutant receptor. Conclusions PCs have different potential effects on different AVPR2 mutants. Therefore, studying the effectiveness of PCs in rescuing AVPR2 mutants and making them functional again may contribute to the development of new therapeutic strategies.

Development of a novel sandwich immunoassay based on targeting recombinant Francisella outer membrane protein A for the diagnosis of tularemia.

Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.

Unravelling the Significance of NLRP3 and IL-β1 in Oral Squamous Cell Carcinoma and Potentially Malignant Oral Disorders: A Diagnostic and Prognostic Exploration.

Nucleotide-binding domain-like receptor protein 3 (NLRP3), an inflammasome, is reported to be dysregulated or aberrantly expressed in chronic inflammation, leading to a myriad of inflammatory disorders, autoimmune diseases, and cancer. This study aimed to explore the expression and role of NLRP3 protein and the secreted cytokine IL-β1 in oral squamous cell carcinoma (OSCC) and potentially malignant oral disorders (PMOD). Tissue NLRP3 expression was quantified using sandwich ELISA in 30 cases each of OSCC, PMOD, and normal oral mucosa. Serum IL-β1 level was also measured by ELISA to determine their correlation. In surgically treated OSCC cases, pathological parameters such as tumor size, depth of invasion (DOI), pTNM stage, and perineural & lymphovascular invasion were assessed and correlated with NLRP3 & IL-β1 levels to investigate their roles in tumor progression, invasion, and metastasis. Tissue NLRP3 expression was markedly elevated in OSCC, with significant IL-β1 levels observed in the serum of both OSCC and PMOD cases. Both markers showed a pronounced increase with the severity of dysplasia, indicating a strong association (p = 0.003%). The expression levels of tissue NLRP3 and serum IL-β1 were positively correlated with DOI and tumor size. Furthermore, their elevated levels, alongside higher histological grades, indicate roles in the dedifferentiation and progression of tumor cells. The findings indicated that increased expression of NLRP3 and IL-β1 in PMOD correlates with higher transformation rates, along with tumor progression and dedifferentiation in OSCC. Consequently, these markers hold promise as valuable targets for prognostic assessment, diagnostics, and therapeutic strategies in OSCC.

Rapid determination of α-lactalbumin in raw cow's milk and recombined milk using an immunochromatographic assay strip analysis method

α-Lactalbumin (α-La) is a common allergenic protein in dairy products, posing a potential hazard to human health. Therefore, there is an urgent need to develop an efficient and sensitive analytical method to assess the α-La content in food. After screening by cell fusion assay, we obtained a pair of monoclonal antibodies (mAbs) (mAb 1 and 9). The calculated limit of detection of the sandwich enzyme-linked immunosorbent assay (ELISA) was 3.99 ng/mL. By labeling mAbs with colloidal gold, we developed a sensitive and rapid immunochromatographic assay (ICA) strip method for the detection of α-La in dairy products. The established ICA strips showed high sensitivity and specificity for the detection of α-La, with a calculated limit of detection and a visual limit of detection of 10.33 and 50 ng/mL, respectively. The average recoveries for the determination of raw cow's milk and recombined milk using ICA strips ranged from 97.60% to 102.96%. The ICA strips showed good correlation with the high-performance liquid chromatography (HPLC) method when analyzing actual samples. The above results indicated that both methods can be used for the determination of α-La in dairy products.

Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common cause of severe thrombocytopenia in dogs. The pathogenesis of nonassociative, primary ITP (pITP) appears complex, with ill-defined thrombopoietic response. Develop an immunoassay to measure plasma canine thrombopoietin (TPO) concentration and characterize TPO concentrations in dogs with pITP. Forty-one healthy dogs, 8 dogs in an induced ITP model (3 control, 5 ITP), and 58 pITP dogs. Recombinant canine TPO (rcTPO) was purchased and its identity confirmed by mass spectrometry. Monoclonal antibodies were raised to rcTPO and used to configure a sandwich ELISA using streptavidin-biotin detection. Assay performance, coefficients of variability, and healthy dog plasma TPO reference interval (RI) were determined, followed by assay of ITP samples. Assay dynamic range was 15 pg/mL (lower limit of detection) to 1000 pg/mL TPO, with limit of quantitation of 62 pg/mL. Plasma TPO RI was 0 to 158 pg/mL, with plasma TPO <62 pg/mL for 35/41 healthy dogs. All dogs with induced ITP developed marked increases in plasma TPO concentration. Peak values ranged from 515 to >6000 pg/mL. In contrast, only 2/58 pITP dogs had TPO values above RI. Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP. This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans. The TPO assay provides a new tool to study thrombopoiesis in pITP and other thrombocytopenic syndromes in dogs.

Impact of the Hydrophilicity of Poly(sarcosine) on Poly(ethylene glycol) (PEG) for the Suppression of Anti-PEG Antibody Binding.

A method of poly(ethylene glycol) (PEG) conjugation is known as PEGylation, which has been employed to deliver therapeutic drugs, proteins, or nanoparticles by considering the intrinsic non- or very low immunogenic property of PEG. However, PEG has its weaknesses, and one major concern is the potential immunogenicity of PEGylated proteins. Because of its hydrophilicity, poly(sarcosine) (P(Sar)) may be an attractive-and superior-substitute for PEG. In the present study, we designed a double hydrophilic diblock copolymer, methoxy-PEG-b-P(Sar) m (m = 5-55) (mPEG-P(Sar) m ), and synthesized a triblock copolymer with hydrophobic poly(l-isoleucine) (P(Ile)). We validated that double hydrophilic mPEG-P(Sar) block copolymers suppressed the specific binding of three monoclonal anti-PEG antibodies (anti-PEG mAbs) to PEG. The results of our indirect ELISAs indicate that P(Sar) significantly helps to reduce the binding of anti-PEG mAbs to PEG. Importantly, the steady suppression of this binding was made possible, in part, thanks to the maximum number of sarcosine units in the triblock copolymer, as evidenced by sandwich ELISA and biolayer interferometry assay (BLI): the intrinsic hydrophilicity of P(Sar) had a clear supportive effect on PEG. Finally, because we used P(Ile) as a hydrophobic block, PEG-P(Sar) might be an attractive alternative to PEG in the search for protein shields that minimize the immunogenicity of PEGylated proteins.

Detection and Evaluation of Procalcitonin Variants As Diagnostic Tools in Systemic Inflammation

BACKGROUND: Procalcitonin is an indicator of systemic inflammation associated with major surgery or sepsis. Procalcitonin exists in a full-length and truncated variant as a result of dipeptidylpeptidase-4 (DPP4)-cleavage. We recently identified differential biological activity of both variants. Here, we present an immunoassay-based method for the separate detection of procalcitonin variants and correlation to clinical data in patients with severe systemic inflammation. METHODS: Rabbits were immunized with peptides of N-terminal sequences of both human procalcitonin variants and polyclonal antibodies purified from rabbit plasma. Antibodies were used for the detection of procalcitonin variants in an indirect sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available monoclonal anti-procalcitonin antibody as capture. Serum was collected from 19 septic patients exhibiting hyperprocalcitonemia as part of a cross-sectional study; clinical data were analyzed and correlated with procalcitonin variant measurements. DPP4 activity was determined by a DPP4 activity assay. RESULTS: Purified antibodies allowed for the separate detection of both procalcitonin variants in all patients. Levels of truncated procalcitonin (truncPCT) correlated with DPP4-activity (Pearson’s R = 0.85, P &lt; .001) and negatively correlated with patients’ Sequential Organ Failure Score (SOFA) scores (Pearson’s R = –0.56, P = .013). In contrast, the correlation between full-length procalcitonin (fullPCT) and SOFA scores was positive (Pearson’s R = 0.56, P = .013). Separation of the patient collective into groups with higher amounts of fullPCT versus truncPCT revealed higher SOFA scores in patients with fullPCT &gt; truncPCT (mean ± standard error of the mean; 11. 3 ± 0.8 vs 6. 1 ± 1.5, P = .003). Patients with fullPCT &gt; truncPCT showed a tendency towards higher doses of vasopressor (0. 2 ± 0.1 vs 0. 1 ± 0.03 µg/kg/min norepinephrine within the first 24 hours after sepsis diagnosis, P = .062) and exhibited higher creatinine (2. 0 ± 0.2 vs 1. 4 ± 0.3mg/dL, P = .019) and leukocyte levels (31. 0 ± 5.4 vs 12. 8 ± 1.9cells/µL, P = .012). In addition, patients with fullPCT &gt; truncPCT were more often subjected to treatment with hydrocortisone (49.0 vs 0%, P = .018). CONCLUSIONS: Polyclonal antibodies generated using procalcitonin N-terminal variant peptides as immunogens are suitable for procalcitonin variant assessment. The separate detection of procalcitonin variants may offer additional diagnostic value and can be correlated with organ dysfunction and clinical outcomes in patients with systemic inflammation.

Development and Application of Automated Sandwich ELISA for Quantitating Residual dsRNA in mRNA Vaccines.

The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day.

Quantitative Detection of the CP4-EPSPS Protein in Genetically Modified Soybeans Using a Novel Fluorescence Immunoassay Assay.

Protein-based detection methods, enzyme-linked immunosorbent assays (ELISAs) and lateral flow strips, have been widely used for rapid, specific, and sensitive detection of genetically modified organisms (GMOs). However, the traditional ELISA method for the quantitative detection of GMOs has certain limitations. Herein, a quantum dot (QD)-based fluorescence-linked immunosorbent assay was developed using QDs as fluorescent markers for the detection of glyphosate-resistant protein (CP4-EPSPS) in the MON89788 soybean. The end-point fluorescent detection system was carried out using QDs conjugated with a goat anti-rabbit secondary antibody. Compared with the conventional sandwich ELISA method, the newly developed fluorescence-linked immunosorbent assay was highly sensitive and accurate for detecting the CP4-EPSPS protein. The quantified linearity was achieved in the range of 0.05-5% (w/w) for the MON89788 soybean sample. The recovery of protein extracted from mixed MON89788 soybean samples ranged from 87.67% to 116.83%. The limits of detection and limits of quantification were 0.7101 and 2.152 pg/mL, respectively. All of the results indicated that the QD-based fluorescence-linked immunosorbent assay was a highly specific and sensitive method for monitoring the CP4-EPSPS protein in GMOs.

Analysis of management factors influencing Anaplasma marginale transmission

The aim of this study was to identify factors that play a key role in the epidemiology of bovine anaplasmosis by adapting a model primarily developed for cattle babesiosis. A cross-sectional observational study was conducted to study the proportion of calf herds in endemic stability/instability for A. marginale in a semi-arid area of Argentina. The A. marginale inoculation rate (h) was calculated from age-specific seroprevalence using double-antigen sandwich ELISA in 58 herds of 4.5–8.5-month–old calves. Herds were considered to be in endemic instability (EI) at h < 0.005 and, therefore, at risk of anaplasmosis outbreaks. A generalized linear model was performed to explore husbandry practices associated with differences in A. marginale transmission. Additionally, spatial clustering of herds with the same immunological status was analyzed using spatial scan statistics (SatScan, Bernoulli model). Spearman's correlation was used to explore a possible association between A. marginale h and Babesia bovis and B. bigemina h (data obtained in previous works). Almost half (43 %) of the herds were in the EI zone for A. marginale. Calves raised under forage combinations had a greater risk of being in EI (OR = 5.41, CI95 %OR = 1.43–20.41) than those reared exclusively on permanent pastures, where cattle density is higher (P = 0.01). Moreover, calves from herds treated only with pyrethroids to control ticks had more chances of being in EI (OR = 4.16, CI95 %OR = 1.12–15.38) than calves from herds receiving different acaricide combinations (P = 0.03). Calves from herds subjected to more than two treatments against Haematobia irritans had higher odds for EI (OR = 5.69, CI95 %OR = 1.24–26.11) than those from herds using fewer than two treatments (P = 0.02). The spatial analysis revealed no spatial clustering of the immune status of the herds (P = 0.67 and P = 0.74 for low and high incidence rates, respectively). A significant variation between farms was observed in A. marginale h (CV = 90.38 %). The correlation analysis revealed a strong epidemiological link of A. marginale h with B. bovis h (Rho=0.794, P<0.001) and B. bigemina h (Rho=0.839, P<0.001). Given that R. microplus is the only vector of B. bovis and B. bigemina in the region, the results of this work strongly suggest an active and significant role of R. microplus in the transmission of A. marginale.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.