Healthy Human Plasma Research Articles

Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples

BackgroundThe lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes.ResultsEVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates.ConclusionsOur findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

Lutein and Zeaxanthin Enhance, Whereas Oxidation, Fructosylation, and Low pH Damage High-Density Lipoprotein Biological Functionality.

High-density lipoproteins (HDLs) are key regulators of cellular cholesterol homeostasis but are functionally altered in many chronic diseases. The factors that cause HDL functional loss in chronic disease are not fully understood. It is also unknown what roles antioxidant carotenoids play in protecting HDL against functional loss. The aim of this study was to measure how various disease-associated chemical factors including exposure to (1) Cu2+ ions, (2) hypochlorous acid (HOCL), (3) hydrogen peroxide (H2O2), (4) sialidase, (5) glycosidase, (6) high glucose, (7) high fructose, and (8) acidic pH, and the carotenoid antioxidants (9) lutein and (10) zeaxanthin affect HDL functionality. We hypothesized that some of the modifications would have stronger impacts on HDL particle structure and function than others and that lutein and zeaxanthin would improve HDL function. HDL samples were isolated from generally healthy human plasma and incubated with the corresponding treatments listed above. Cholesterol efflux capacity (CEC), lecithin-cholesterol acyl transferase (LCAT) activity, and paraoxonase-1 (PON1) activity were measured in order to determine changes in HDL functionality. Median HDL particle diameter was increased by acidic pH treatment and reduced by HOCl, high glucose, high fructose, N-glycosidase, and lutein treatments. Acidic pH, oxidation, and fructosylation all reduced HDL CEC, whereas lutein, zeaxanthin, and sialidase treatment improved HDL CEC. LCAT activity was reduced by acidic pH, oxidation, high fructose treatments, and lutein. PON1 activity was reduced by sialidase, glycosidase, H2O2, and fructose and improved by zeaxanthin and lutein treatment. These results show that exposure to oxidizing agents, high fructose, and low pH directly impairs HDL functionality related to cholesterol efflux and particle maturation, whereas deglycosylation impairs HDL antioxidant capacity. On the other hand, the antioxidants lutein and zeaxanthin improve or preserve both HDL cholesterol efflux and antioxidant activity but have no effect on particle maturation.

EXPLORING THE HAEMOSTATIC POTENTIAL OF PANCHAVALKALA KASHAYA AND PANCHAVALKALA AQUEOUS EXTRACT: AN IN VITRO STUDY

Acharya Susrutha under the context of Rakthasthambhanopaya (methods of haemostasis) has explained about the different methods to attain Rakthasthambhana or haemostasis. which include Skandana (clotting of blood), Santhana (uniting), Pachana (ripening or digestive measures) and Dahana (thermal cauterisation). Acharya Susrutha while explaining about agantuja vrana chikitsa (treatment of wound) has explained that ‘Pittavat sheeta kriya’ should be done prior to santhana to mitigate the ‘Kshatoshmana’. So, from this it is evident the primary method of management should be done with sheeta (cold) followed by Kashaya rasa dravya (astringent taste drugs) for Santhana. Panchavalkala is a group drug with sheeta veerya (cold potency) and Kashaya rasa (astringent taste) mentioned in Dalhana commentary for Skandana. This study investigates the haemostatic effect of Panchavalkala Kashaya and Panchavalkala aqueous extract by in vitro method. Preliminary phytochemical screening of both trial drugs was done. A total of 10 samples of healthy human platelet poor plasma were mixed in trial drugs, Prothrombin and Activated Partial Thromboplastin time were determined. Data was analysed using Kruskal Wallis test. Phytochemical analysis confirmed that the Panchavalkala kashaya contain alkaloids, tannins, glycosides and saponins. Panchavalkala aqueous extract sample consist of flavonoids, alkaloids, glycosides, tannins and saponins. LCMS Q-TOF analysis shows the presence of compounds Gallic acid, Epicatechin, Leucocynidin and Procyanidin B1. And the HPLC study confirmed that Gallic acid is an active phytoconstituent present in the Panchavalkala aqueous extract. In the present study the trial group Panchavalkala aqueous extract showed more desirable haemostatic effect than Panchavalkala Kashaya on comparison.

Analysis of the Development and Stability of the Plasma Supplier Team in Single-collection Plasma Stations

With the improvement of people s living standard and the progress of medical science, the application of blood products in clinical treatment and disease prevention and control has become more and more extensive and in greater demand. The most important raw material for the production of blood products is healthy human plasma, and the plasma must be collected by the single plasma collection station set up by blood product manufacturers to recruit healthy people (called plasma donors) to voluntarily donate plasma, due to the completely voluntary nature of plasma donors plasma donations, their individual behaviors are susceptible to the influence of a variety of factors, and the need for plasma donors team development and stabilization has posed a serious challenge to single plasma collection stations. This paper briefly dis- cusses the development and stability of the plasma donor team in the single plasma station, hoping to play a guiding role in the development of the blood source of the single plasma industry.

Pharmacokinetic study of five components of Fuzheng Huayu tablets in healthy human plasma.

Natural medicines play a crucial role in clinical drug applications, serving as a primary traditional Chinese medicine for the clinical treatment of liver fibrosis. Understanding the in vivo metabolic process of the Fuzheng Huayu (FZHY) formula is essential for delving into its material basis and mechanism. In recent years, there has been a growing body of research focused on the mechanisms and component analysis of FZHY. This study aimed to examine the pharmacokinetics of FZHY in healthy volunteers following oral administration. Blood samples were collected at designated time intervals after the oral intake of 9.6-g FZHY tablets. A UHPLC-Q/Exactive method was developed to assess the plasma concentrations of five components post-FZHY ingestion. The peak time for all components occurred within 10 min. The peak concentration (Cmax ) values for amygdalin, schisandrin, and schisandrin A ranged from 3.47 to 28.80 ng/mL, with corresponding AUC(0-t) values ranging from 10.63 to 103.20 ng h/mL. For schisandrin B and prunasin, Cmax values were in the range of 86.52 to 229.10 ng/mL, and their AUC(0-t) values ranged from 375.26 to 1875.54 ng h/mL, indicating significant exposure within the body. These findings demonstrate that the developed method enables rapid and accurate detection and quantification of the five components in FZHY, offering a valuable reference for its clinical study.

Plasma microrna quantification protocol.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Orally administered low-molecular weight agaro-oligosaccharides are absorbed into the plasma of healthy humans.

Agaro-oligosaccharides (AOSs) are known to have biological activities, such as anti-inflammatory, anti-tumor, and anti-obesity effects. Although existing evidence suggests the presence of AOSs in peripheral tissues after oral administration, whether AOSs permeate into the blood circulation remains unknown. Thus, we hypothesized that AOSs with low-molecular weight can permeate the human gastrointestinal tract. To test this hypothesis, the time course of absorption was examined by analyzing plasma samples before and 1, 2, and 4 h after ingestion. Analysis was performed using liquid chromatography/mass spectrometry after labeling with p-aminobenzoic ethyl ester. Our results showed that the plasma concentration of agarobiose (Abi) was higher than that of agarotetraose (Ate); however, agarohexaose was not detected. Additionally, plasma levels of Abi and Ate were proportional to the dose. These results suggest that permeation efficiency is dependent on the molecular weight and that the systemic absorption of Abi via the gastrointestinal tract is better than that of Ate. These findings will contribute to a better understanding of the bioactivity of orally administered AOSs in peripheral tissues.

Exosomes from young healthy human plasma promote functional recovery from intracerebral hemorrhage via counteracting ferroptotic injury

Intracerebral hemorrhage (ICH), as a type of life-threatening and highly disabled disease, has limited therapeutic approaches. Here, we show that exosomes derived from young healthy human plasma exhibiting typical exosomes features could facilitate functional recovery of ICH mice. When these exosomes are intraventricularly delivered into the brain after ICH, they mainly distribute around the hematoma and could be internalized by neuronal cells. Strikingly, exosomes administration markedly enhanced the behavioral recovery of ICH mice through reducing brain injury and cell ferroptosis. MiRNA sequencing revealed that microRNA-25-3p (miR-25-3p) was differentially expressed miRNA in the exosomes from young healthy human plasma, compared with exosomes from the old control. Importantly, miR-25-3p mimicked the treatment effect of exosomes on behavioral improvement, and mediated the neuroprotective effect of exosomes against ferroptosis in ICH. Furthermore, luciferase assay and western blotting data illustrated that P53 as assumed the role of a downstream effector of miR-25-3p, thereby regulating SLC7A11/GPX4 pathway to counteract ferroptosis. Taken together, these findings firstly reveal that exosomes from young healthy human plasma improve functional recovery through counteracting ferroptotic injury by regulating P53/SLC7A11/GPX4 axis after ICH. Given the easy availability of plasma exosomes, our study provides a potent therapeutic strategy for ICH patients with quick clinical translation in the near future.

Identification of Neutralizing Anti-HMGB1 IgM Autoantibody and Its B Cell Producers

Abstract Extracellular High Mobility Group Box 1 (HMGB1) is a prototypic damage-associated molecular pattern (DAMP). While a homeostatic level of extracellular HMGB1 may be beneficial for immune defense, tissue repair and tissue regeneration, excessive HMGB1 is linked to chronic inflammation and inflammatory diseases. This prompts an intriguing question: How does a healthy body control the level of extracellular HMGB1? Here we have identified, in the plasma of both healthy humans and healthy mice, an anti-HMGB1 IgM autoantibody that neutralizes extracellular HMGB1 via binding specifically to a 100% conserved epitope, namely HMW4 (HMGB1 98–112). In mice, this anti-HMGB1 IgM (i.e., anti-HMW4 IgM) is produced by peritoneal B-1 cells, and concomitant triggering of their B cell receptor and toll-like receptor 4 by extracellular HMGB1 stimulates the production of anti-HMW4 IgM. The ability of extracellular HMGB1 to induce its own neutralizing antibody suggests a feedback loop limiting the level of this DAMP in a healthy body. Currently, our studies are ongoing to identify and characterize the human counterpart B cells that produce the anti-HMGB1 IgM, by first amplifying HMW4-reactive B cells from human peripheral blood mononuclear cells (PBMCs) in NSG recipient mice, in the presence of B cell-stimulating adjuvants (such as B cell mitogens). These studies serve as the prelude to exploring human anti-HMGB1 IgM down the road for treating patients with inflammatory diseases and provide insight pertaining to the role and regulation of other DAMP molecules.

Autoantibody-Abzymes with Catalase Activity in Experimental Autoimmune Encephalomyelitis Mice.

The exact mechanisms of the evolution of multiple sclerosis are still unknown. At the same time, the development in C57BL/6 mice of experimental autoimmune encephalomyelitis (EAE, simulating human multiple sclerosis) happens as a result of the violation of bone marrow hematopoietic stem cell differentiation profiles integrated with the production of toxic auto-antibodies splitting the basic myelin protein, myelin oligodendrocyte glycoprotein (MOG), histones, and DNA. It has been shown that IgGs from the plasma of healthy humans and autoimmune patients oxidize many different compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities. Here, we first analyzed the changes in the relative catalase activity of IgGs from C57BL/6 mice blood plasma over time at different stages of the EAE development (onset, acute, and remission phases). It was shown that the catalase activity of IgGs of 3-month-old mice is, on average, relatively low (kcat = 40.7 min-1), but it increases during 60 days of spontaneous development of EAE 57.4-fold (kcat = 2.3 × 103 min-1). The catalase activity of antibodies increases by a factor of 57.4 by 20 days after the immunization of mice with MOG (kcat = 2.3 × 103 min-1), corresponding to the acute phase of EAE development, and 52.7-fold by 60 days after the treatment of mice with a DNA-histone complex (kcat = 2.1 × 103 min-1). It is the acceleration of the EAE development after the treatment of mice with MOG that leads to the increased production of lymphocytes synthesizing antibodies with catalase activity. All data show that the IgGs' catalase activity can play an essential role in reducing the H2O2 concentration and protecting mice from oxidative stress.

АНТИОКСИДАНТНЫЙ ПОТЕНЦИАЛ ПЛАЗМЫ КРОВИ БОЛЬНЫХ ОСТРЫМ ПАНКРЕАТИТОМ

Oxidative stress is the most important pathogenetic mechanism of acute pancreatitis (AP). In this study, components of the antioxidant system (AOS), such as ceruloplasmin (CP), albumin (ALB) and uric acid (UA), were studied in the plasma of healthy humans and patients with AP of varying severity. Regardless of the severity of AP, antioxidant protection is reduced. With a mild degree of AP the level of UA is significantly reduced, but compensatory increased level of CP. In medium and high degree of AP the decrease in antioxidant protection activity is associated with a decrease in ALB levels as the prevailing plasma antioxidant, as well as an increase in UA levels above the physiological level.

A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction.

Ubiquinone (UQ) is considered one of the important biologically active molecules in the human body. Ubiquinone determination in human plasma is important for the investigation of its bioavailability, and also its plasma level is considered an indicator of many illnesses. We have previously developed sensitive and selective chemiluminescence (CL) method for the determination of UQ in human plasma based on its redox cycle with dithiothreitol (DTT) and luminol. However, this method requires an additional pump to deliver DTT as a post-column reagent and has the problems of high DTT consumption and broadening of the UQ peak due to online mixing with DTT. Herein, an HPLC (high-performance liquid chromatography) system equipped with two types of online reduction systems (electrolytic flow cell or platinum catalyst-packed reduction column) that play the role of DTT was constructed to reduce reagent consumption and simplify the system. The newly proposed two methods were carefully optimized and validated, and the analytical performance for UQ determination was compared with that of the conventional DTT method. Among the tested systems, the electrolytic reduction system showed ten times higher sensitivity than the DTT method, with a limit of detection of 3.1 nM. In addition, it showed a better chromatographic performance and the best peak shape with a number of theoretical plates exceeding 6500. Consequently, it was applied to the determination of UQ in healthy human plasma, and it showed good recovery (≥97.9%) and reliable precision (≤6.8%) without any interference from plasma components.

Study the effect of isolated human epidermal growth factor receptor (HER2) from plasma on histopathological aspects in some organs of adult female albino rats

This study included an attempt to isolate and purify Human Epidermal Growth Factor Receptor (EGFR) from healthy human plasma using different biochemical technique. Two peaks had been isolated by gel filtration chromatography from the precipitate produced by using ammonium sulfate. The first peak (peak A) had a high level of EGFR. Furthermore, the purity of isolated EGFR peak A had been identified by the high-performance liquid chromatography and by gel electrophoresis technique. The results obtained from high performance liquid chromatography showed that there was a good identity in retention time between the standard and the isolated EGFR peak A. The approximate molecular weight of partially purified EGFR peak A was 75318±100 and 73029±100 dalton using gel filtration chromatography and gel electrophoresis technique respectively. The effect of the isolated EGFR peak A on some histopathological aspects in adult female albino rats had been studied. The result showed that the treatment with three different concentrations of peak A causes alteration in the normal architecture of the liver and kidney characterized by infiltration of lymphocytes, coagulative necrosis, hemorrhage, increase in collagen fibers deposition, hyperplasia, pre-neoplastic lesion with bizarre nucleus, and adenoma. These irreversible pathological changes which occurred in these organs may be converted to malignance if take longer time (more than one month). According to the findings of this study, high levels of HER2 cause irreversible pathological changes in the liver and kidney, which can progress to cancer over time.

Highly efficient TiO2-based one-step strategy for micro volume plasma-derived extracellular vesicles isolation and multiomics sample preparation

Extracellular vesicles (EVs) released from tumor cells and tissues mediate intercellular communication and regulate various important biological processes. The characterization of EVs can provide potential diagnostic biomarkers and therapeutic targets. However, current EV enrichment and sample preparation methods for omics analysis are time-consuming and costly, and a relatively large amount of samples is often required. Here, we report a robust TiO2-mediated multiomics EV preparation method (TiMMEP) for isolating EVs from a small volume of plasma and profiling proteins and metabolites simultaneously. Plasma EVs were initially captured by TiO2, and EVs proteins and metabolites were released by adding 70% acetonitrile. Subsequently, the proteins were precipitated and adsorbed on the surface of TiO2 and the metabolites were collected in the supernatant. More than 700 protein groups and 491 tentatively annotated metabolites were identified in the EVs isolated from 1 μL of plasma. Furthermore, the TiMMEP method was applied for multi-omics EV analysis of ten healthy human plasma samples to demonstrate its feasibility and stability. Thus, the TiMMEP strategy enables a highly sensitive, operationally simple, and high-throughput multiomics analysis of EVs from low volume of plasma, potentially improving disease diagnosis and biomarker discovery.

Simple and fast determination of tetrodotoxin in human plasma based on hydrophilic-interaction/ion-exchange mixed-mode solid phase extraction combined with liquid chromatography-tandem mass spectroscopy

In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the measurement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant matrix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic interaction and ion exchange. 100μL plasma sample was precipitated rapidly with acetonitrile containing 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed with 95% acetonitrile, TTX was eluted with 200μL of 50% acetonitrile containing 1% trichloroacetic acid. 2μL of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide column was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and timesaving (<30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using m/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transition for TTX) and 179.2→63.0 (transition for IS), respectively. The method was linear in the range of 0.1–20 ng/mL for TTX with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra- and inter-assay accuracies were in the range of 98.5%-99.8% (relative standard deviations, RSDs ≤ 5.92%) and 98.8–99.5% (RSDs ≤ 6.23%), respectively. Biases of spiking analysis were ranged from -7.00% to 7.43% for healthy human plasma samples (RSDs ≤ 8.83%) and from -5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high bilirubin plasma samples (RSDs ≤ 6.40%), which proved the good anti-interference property of the method. The results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.

Determination of Blood NOTCH3 Extracellular Domain and Jagged-1 Levels in Healthy Subjects.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common genetic disorder among those responsible for hereditary strokes, and it is caused by a mutation in the NOTCH3 gene on chromosome 19. Blood biomarkers related to the Notch signaling pathway have not been investigated extensively in CADASIL. In this study, we measured the serum and plasma levels of NOTCH3 extracellular domain (N3ECD) and its ligand, Jagged-1, in 279 healthy subjects. The levels of N3ECD and Jagged-1 showed significant correlations in both serum (p < 0.0001, r = 0.2681) and plasma (p < 0.0001, r = 0.4065). The N3ECD levels were significantly higher in the serum than in plasma and tend to increase with age. In contrast, there was no significant difference between the serum and plasma levels of Jagged-1 levels. To summarize, we were able to measure N3ECD and Jagged-1 protein levels in healthy human serum and plasma. Taken together, our findings provide the basis for further studies investigating the clinical use of blood N3ECD and Jagged-1 levels for CADASIL and other Notch signaling-related diseases.

MicroRNA and metabolomics signatures for adrenomyeloneuropathy disease severity.

Adrenomyeloneuropathy (AMN), the slow progressive phenotype of adrenoleukodystrophy (ALD), has no clinical plasma biomarker for disease progression. This feasibility study aimed to determine whether metabolomics and micro-RNA in blood plasma provide a potential source of biomarkers for AMN disease severity. Metabolomics and RNA-seq were performed on AMN and healthy human blood plasma. Biomarker discovery and pathway analyses were performed using clustering, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and regression against patient's clinical Expanded Disability Status Score (EDSS). Fourteen AMN and six healthy control samples were analyzed. AMN showed strong disease-severity-specific metabolic and miRNA clustering signatures. Strong, significant clinical correlations were shown for 7-alpha-hydroxy-3-oxo-4-cholestenoate (7-HOCA) (r 2=0.83, p < 0.00001), dehydroepiandrosterone sulfate (DHEA-S; r 2=0.82, p < 0.00001), hypoxanthine (r 2=0.82, p < 0.00001), as well as miRNA-432-5p (r 2=0.68, p < 0.00001). KEGG pathway comparison of mild versus severe disease identified affected downstream systems: GAREM, IGF-1, CALCRL, SMAD2&3, glutathione peroxidase, LDH, and NOS. This feasibility study demonstrates that miRNA and metabolomics are a source of potential plasma biomarkers for disease severity in AMN, providing both a disease signature and individual markers with strong clinical correlations. Network analyses of affected systems implicate differentially altered vascular, inflammatory, and oxidative stress pathways, suggesting disease-severity-specific mechanisms as a function of disease severity.

Ultrasensitive multiplexed chemiluminescent enzyme-linked immunosorbent assays in 384-well plates

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 μL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags (“customized” assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

A comparative analysis of extracellular vesicles (EVs) from human and feline plasma

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

Collagen Remodeling Proteins, Inflammatory Biomarkers and FABP Regulation in Understanding the Pathogenesis of Atrial Fibrillation

IntroductionAtrial Fibrillation (AF) is the most common sustained form of cardiac arrythmia across the world with around 3 million active cases. Atrial fibrosis is the process by which collagen is deposited within the atria. Atrial fibrosis is correlated with AF. PINP and PICP, the two collagen remodeling proteins focused on in this study, are procollagen type I remodeling proteins and effective biomarkers of collagen deposition and bone formation. PINP exhibits diurnal variation with higher values occurring at night resulting in variance within the data set for PINP in AF patients. Long chain fatty acids (LCFAs), in excess, can lead to lipotoxicity which increases the risk of a potential arrythmia. Fatty Acid Binding Proteins (FABPs) bind to LCFAs to create a lower level of these fatty acid chains in the cytoplasm. Increased LCFAs has a direct correlation with increased FABPs which can ultimately increase the risk of arrythmia.PurposeThe purpose of this study was to analyze levels of collagen remodeling proteins, specifically PINP and PICP, and L‐FABP in an AF cohort to determine whether they can serve as viable biomarkers in the management and diagnosis of AF.HypothesisIt is expected that there will be an upregulation of collagen remodeling proteins, inflammatory biomarkers and FABP in AF patients compared to the NHP.Materials and MethodsThe study was conducted with a sample of 50 normal healthy human plasma and were tested for PINP, PICP, and FABP levels. These normal samples were purchased from George King Biomedical. The blood of the AF patients was collected following IRB protocol, centrifuged, and aliquoted at –80 degrees Celsius. AF plasma samples were analyzed using sandwich ELISA Kits. Results were statistically analyzed by using the IBM SPSS software and PRISM Graphpad.ResultsThe results were collected as mean ± SEM with a percent change of mean (%) to demonstrate upregulation. All three biomarkers PINP, PICP, and FABP demonstrated an upregulation, respectively, of (422.32 ± 43.78, 164.47%), (1.395902 ± 0.178727, 104.86%), and (13186.7517 ± 4256.96265, 157.75% with the outlier and 77.69% without outlier). All three biomarkers were statistically significant with p‐values of &lt;0.0001, &lt;0.01, and &lt;0.0001 respectively for PINP, PICP, and FABP. There was a significant outlier in the FABP data at 295.778.86 pg/mL. This value created a skewness statistic of 7.502 which changed to 3.795 after the outlier was removed. There was a strong, positive correlation between the two collagen remodeling proteins, PICP and PINP, with an r‐value (correlation coefficient) value of 0.62.ConclusionThe results prove that increased structural remodeling, proven by higher levels of collagen remodeling proteins, can indicate AF. FABP had an evident upregulation in the AF patient, but the data was severely skewed right due to an outlier. Once the outlier was removed, the data still demonstrated an upregulation of 77.69%. Elevated levels of these biomarkers suggest that they can serve as surrogate endpoints in the management and diagnosis of AF.