Serum Components Research Articles

Mutual effects of protein corona formation on CdTe quantum dots

Future biomedical applications of nanoparticles will encounter these particles with patients’ serum which might affect the properties and stability of quantum dots and serum proteins at the desired site of action. Therefore, it is essential to clarify the patient-specific serum components, serve as major interaction partners, the spatial distribution of these, and consequently the time-dependent effects of nanoparticle–protein interaction. Here, a biochemical and structural study was performed on the protein corona formation and the corresponding interaction of different sizes of CdTe QDs with human serum proteins to determine if the mutual effects on optical properties by using electrophoresis, chemiluminescence, and fluorescence spectroscopy. The results revealed that interaction with human serum significantly enhanced the stability and photoluminescence of quantum dots. Structural studies of HSA-coated CdTe QDs also showed that corona formation has no adverse effects on protein structure, and the reduction in fluorescence emissions of HSA is due to the direct quenching of aromatics residues by the quantum dot. Improving nanoparticle properties, as well as the lack of structural changes in HSA, can be very useful in biomedical applications and in vivo studies where stability is important.

Immunological fingerprint of 4CMenB recombinant antigens via protein microarray reveals key immunosignatures correlating with bactericidal activity

Serogroup B meningococcus (MenB) is a leading cause of meningitis and sepsis across the world and vaccination is the most effective way to protect against this disease. 4CMenB is a multi-component vaccine against MenB, which is now licensed for use in subjects >2 months of age in several countries. In this study, we describe the development and use of an ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed.

EFFECT OF SOME HERBAL FEED ADDITIVES ON THE PERFORMANCE OF BROILER CHICKENS

The present experiment was conducted in order to investigate the effect of Moringa Oleifera Leaves powder on the performance, some blood serum components, some histomorphological measurements and economic efficiency of broiler chickens. A total of 225 Cobb unsexed one - day old broiler chicks, were distributed at random into 5 treatments each in 3 replicates, 15 chicks each. Moringa Oleifera leaves powder (MOL) was added at the levels of 0, 0.25, 0.50 and 0.75% to low crud protein diet (LCP, 20 and 17%, negative control) compared to the normal crud protein (23 and 20%, positive control), respectively at the starter (1- 21 d) and at the finisher (22- 35d) period. Results indicated that, chicks fed the low crude protein diet supplemented with Moringa Oliefera leaves powder (MOL) had significantly (P ≤ 0.05) higher body weight, body weight gain, daily feed intake and performance index. Feed conversion ratio, European efficiency index and relative economic efficiency were also improved with the supplementation. Chicks fed low crude protein diet supplemented with 0.25 or 0.50% MOL have the highest dressing weight (1753 and 1738g) with no significant differences between them compared and increased serum total protein and glucose levels, while significantly decreased serum total cholesterol and aspartic transaminase (AST) enzyme compared to the other treatments with increasing the levels of MOL up to 0.50%. Morphological sections of small intestine revealed that villi height, width and height: width ratios were increased by the supplementation of different levels of MOLM (0.25 and 0.50%) but not significant except in duodenum villi width and in the ration between height and width. In general, the obtained results indicated that supplementation of 0.25% Moringa Oliefera Leaves, MOL can be used in broiler diets with low protein level to get the heaviest weight, best feed conversion, performance index, due to an increase in small intestinal absorption and activity. This, in turn, resulted in an increase in European efficiency index and economic efficiency under the experimental conditions.

Tau Protein and Its Role in Blood-Brain Barrier Dysfunction.

The blood–brain barrier (BBB) plays a crucial role in maintaining the specialized microenvironment of the central nervous system (CNS). In aging, the stability of the BBB declines and the permeability increases. The list of CNS pathologies involving BBB dysfunction is growing. The opening of the BBB and subsequent infiltration of serum components to the brain can lead to a host of processes resulting in progressive synaptic, neuronal dysfunction, and detrimental neuroinflammatory changes. Such processes have been implicated in different diseases, including vascular dementia, stroke, Alzheimer’s disease (AD), Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, hypoxia, ischemia, and diabetes mellitus. The BBB damage is also observed in tauopathies that lack amyloid-β overproduction, suggesting a role for tau in BBB damage. Tauopathies represent a heterogeneous group of around 20 different neurodegenerative diseases characterized by abnormal deposition of the MAPT in cells of the nervous system. Neuropathology of tauopathies is defined as intracellular accumulation of neurofibrillary tangles (NFTs) consisting of aggregated hyper- and abnormal phosphorylation of tau protein and neuroinflammation. Disruption of the BBB found in tauopathies is driven by chronic neuroinflammation. Production of pro-inflammatory signaling molecules such as cytokines, chemokines, and adhesion molecules by glial cells, neurons, and endothelial cells determine the integrity of the BBB and migration of immune cells into the brain. The inflammatory processes promote structural changes in capillaries such as fragmentation, thickening, atrophy of pericytes, accumulation of laminin in the basement membrane, and increased permeability of blood vessels to plasma proteins. Here, we summarize the knowledge about the role of tau protein in BBB structural and functional changes.

Physiological Response of Pelophylax nigromaculatus Adults to Salinity Exposure.

Simple SummaryThis study explored physiological resilience to and recovery from saline exposure in Pelophylax nigromaculatus, a semi-aquatic frog that is widely distributed in East Asia. Analysis of 11 serum components revealed the physiological response of frogs to either severe saline exposure for six days or moderate saline exposure for forty days, followed by a twenty day recovery period. During exposure to both severe and moderate saline conditions, serum electrolytes increased, protein concentrations in serum decreased, and creatinine, an indicator of renal function, sharply increased. However, renal tissue sampled after the study did not show renal dysfunction. Moreover, serum components that changed during exposure to salinity returned to their initial values during the recovery period. Adult anurans seem capable of resilience, to some extent, to saline conditions.Many freshwater ecosystems are becoming more saline, and amphibians, which have permeable skin, are sensitive to this change. We studied the physiological responses to high salinity and recovery from saline exposure in adult frogs (Pelophylax nigromaculatus). Frogs that experienced severe salinity were exposed to saline conditions for 6 days, while those in the moderate group were exposed to saline conditions for 40 days, followed by a recovery period in freshwater for 20 days. Our data showed that during exposure to saline conditions of severe and moderate groups, serum electrolytes increased, protein concentrations decreased, and creatinine, an indicator of renal function, sharply increased. However, renal tissue sampled after exposure did not show renal dysfunction. In addition, serum components that changed during exposure to salinity returned to their initial values during the recovery period. Thus, adult anurans can be resilient, to some extent, to saline conditions in habitats that experience either rapid or slow salinity changes.

In vitro and In Vivo Immunomodulatory Activity of Physalis angulata Concentrated Ethanolic Extract.

The need for new immunomodulatory drugs is due to the side effects associated with the prolonged use of the currently used immunomodulatory drugs. In this context, the present work aimed to investigate the immunomodulatory effect of an ethanolic concentrated extract from Physalis angulata. The cytotoxicity of samples was determined using peritoneal macrophages though the Alamar Blue assay. The immunomodulatory activity of the ethanolic extract from P. angulata on activated macrophages was determined by measurement of nitrite and cytokine production. The immunosuppressive effects of the ethanolic extract from P. angulata was evaluated on lymphocyte proliferation and cytokine production. The effects of the extract on cell cycle progression and cell death on lymphocytes were evaluated by flow cytometry. Lastly, the ethanolic extract from P. angulata was tested in vivo in toxicological tests and in models of peritonitis and delayed-type hypersensitivity response. The ethanolic extract from P. angulata decreased nitrite, interleukin-6, interleukin-12, and TNF-α production by activated macrophages without affecting the cell viability. In addition, the ethanolic extract from P. angulata inhibited lymphoproliferation and the secretion of interleukin-2, interleukin-6, and IFN-γ, and increased interleukin-4 secretion by activated splenocytes. Flow cytometry analysis in lymphocyte cultures showed that treatment with the ethanolic extract from P. angulata induces cell cycle arrest in the G1 phase followed by cell death by apoptosis. Moreover, mice treated with the extract from P. angulata at 100 or 200 mg/kg did not show signs of toxicity or alterations in serum components. Finally, the ethanolic extract from P. angulata significantly reduced neutrophil migration and reduced paw edema in bovine serum albumin-induced the delayed-type hypersensitivity response model. Our results demonstrate the potential of the ethanolic extract of P. angulata as an alternative for the treatment of immune-inflammatory diseases.

The effect of charge on the uptake and resistance to reduction of platinum(IV) complexes in human serum and whole blood models.

cis- and trans-Platinum(iv) complexes with diaminetetracarboxylate coordination spheres possess the highly desirable property of exhibiting unusual resistance to reduction by blood serum components and endogenous reductants such as ascorbate. At the same time they are rapidly reduced in the intracellular environment of cancer cells. Consequently, they can potentially be tuned to remain intact in vivo until arrival at the tumour target where they are rapidly reduced to yield the active platinum(ii) species. However, in order to achieve this, uptake must be largely restricted to tumour cells and therefore uptake by healthy cells including red blood cells must be prevented. In this proof of concept study, we report on the effect of net charge as a means of controlling the uptake by red blood cells. Using 1H NMR spectroscopy we found that modifying the net charge of the complex does not influence the rate of reduction of the complexes by an excess of ascorbate. Using XANES spectroscopy we found that modifying the net charge of the platinum(iv) complexes decreased the extent of reduction in whole blood, although probably not to the degree needed for the optimal delivery to tumours. Therefore, it is likely to be necessary to adopt higher charges and/or additional strategies to keep platinum(iv) prodrugs out of blood cells.

PERFORMANCE AND HISTOMORPHOLOGICAL PARAMETERS OF BROILER CHICKS FED LOW CRUDE PROTEIN DIET SUPPLEMENTED WITH MORINGA OLEIFERA LEAVES POWDER

The present experiment was conducted in order to investigate the effect of Moringa Oleifera Leaves powder on the performance, some blood serum components, some histomorphological measurements and economic efficiency of broiler chickens. A total of 225 Cobb unsexed one - day old broiler chicks, were distributed at random into 5 treatments each in 3 replicates, 15 chicks each. Moringa Oleifera leaves powder (MOL) was added at the levels of 0, 0.25, 0.50 and 0.75% to low crud protein diet (LCP, 20 and 17%, negative control) compared to the normal crud protein (23 and 20%, positive control), respectively at the starter (1- 21 d) and at the finisher (22- 35d) period. Results indicated that, chicks fed the low crude protein diet supplemented with Moringa Oliefera leaves powder (MOL) had significantly (P ≤ 0.05) higher body weight, body weight gain, daily feed intake and performance index. Feed conversion ratio, European efficiency index and relative economic efficiency were also improved with the supplementation. Chicks fed low crude protein diet supplemented with 0.25 or 0.50% MOL have the highest dressing weight (1753 and 1738g) with no significant differences between them compared and increased serum total protein and glucose levels, while significantly decreased serum total cholesterol and aspartic transaminase (AST) enzyme compared to the other treatments with increasing the levels of MOL up to 0.50%. Morphological sections of small intestine revealed that villi height, width and height: width ratios were increased by the supplementation of different levels of MOLM (0.25 and 0.50%) but not significant except in duodenum villi width and in the ration between height and width. In general, the obtained results indicated that supplementation of 0.25% Moringa Oliefera Leaves, MOL can be used in broiler diets with low protein level to get the heaviest weight, best feed conversion, performance index, due to an increase in small intestinal absorption and activity. This, in turn, resulted in an increase in European efficiency index and economic efficiency under the experimental conditions.

Attribution and identification of absorbed components by HPLC-DAD-ESI-MS after oral administration of Erhuang decoction

To realize the attribution and identification of absorbed components in rat serum after oral administration of Erhuang decoction prepared by semi-bionic enzyme extraction method, the fingerprints of serum samples were established using a HPLC-DAD-ESI-MS method. Thirty-two peaks in Erhuang decoction and 24 peaks in rat serum after oral administration of Erhuang decoction were detected. Among the 24 peaks detected in rat serum, 25 compounds were identified by comparing the retention time and mass spectrometry data with that of reference compounds, or by mass spectrometry analysis and retrieving the reference literatures. Among the identified 25 compounds in vivo, 24 were the original form of compounds absorbed from the detected compounds in vitro, and one was the metabolite compounds of licorice. By analyzing the mass spectrometry or ultraviolet absorption characteristics, other unidentified compounds in vivo were deduced to be the endogenous metabolites in serum or the original form and metabolites of the compounds existed in vivo. Results indicated that HPLC-DAD-ESI-MS is suitable for identifying the bioactive constituents in serum after oral administration of Erhuang decoction, and the findings would be beneficial to further research and development of the pharmacodynamic substance base of Erhuang decoction.

The screening of albumin as a key serum component in preventing release of neutrophil extracellular traps by selectively inhibiting mitochondrial ROS generation.

Neutrophil extracellular traps (NETs) are extracellular DNA webs released from neutrophils to mediate the host antimicrobial defense. As NETs could also induce thrombosis and cause organ injury, their release should be strictly controlled; however, the intrinsic mechanisms that prevent unfavorable NETs are not well understood. Herein, an accidental finding of NET release from human peripheral neutrophils was first described in a serum-free culture, which was later determined to be a conserved NET prevention effect of serum. In contrast to canonical NETs induced by phorbol-12-myristate-13-acetate (PMA), NET formation by serum-free culture was rapid and without prevalent NETosis. Next, albumin was screened out as a key serum component that mediated the suppression of NETs. Moreover, NETs induced upon serum or albumin deficiency were independent of the canonical pathway that involves NADPH oxidase 2 (NOX2) activation and cytosol reactive oxygen species (ROS) production. Instead, the generation of mitochondrial ROS (mtROS) was upregulated to promote NET release. Albumin exhibited mtROS scavenging activity and thus inhibited NETs. Serum-free culture also induced the release of NET-bound oxidized mtDNA, which stimulated interferon-β (IFN-β) production. Overall, our research provides new evidence that characterizes the NET production in serum-free culture and determines the mechanisms by which serum albumin inhibits NETs.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Opportunities of complex hepatosis therapy in cattle

The paper discusses the possibility of using a comprehensive treatment regimen for hepatosis in postpartum cows with the help of preparations with various mechanisms of hepatoprotective and anti-toxic effects. It was proved that enteral administration of Thionontrit-S against the background of intramuscular administration of Livazen to cows with clinical signs of hepatosis has a positive effect on the course of the disease and the recovery timing of animals. The use of a complex treatment regimen contributes to the manifestation of positive dynamics of the main hepatosis syndromes - hepatocellular insufficiency and impaired hepatocyte integrity, which is manifested by an increase in the level of total protein by 8.1%, albumin - by 20.9%, glucose - by 31.6%, triglycerides - by 21.4% and cholesterol - by 27.5% in comparison with the basic treatment. The use of Thionontrit-S as part of the complex treatment of bovine hepatosis made it possible to reduce the activity of the inflammatory process in the liver, which was manifested by a decrease in enzyme activity: AST - by 47.5%, ALT - by 19.9%, alkaline phosphatase - by 1.59 times, and correction of the mineral component of blood serum.

Dendritic cells' characteristics in patients with treated systemic lupus erythematosus.

The systemic lupus erythematosus (SLE) is a chronic autoimmune disease related to a loss of immune tolerance against autoantigens that leads to tissue inflammation and organ dysfunction. Constant stimulation of dendritic cells (DC) with autoantigens is hypothesized to increase the B cells' activity which are involved in production of autoantibodies that play an essential role in the SLE development. We focused our study on detecting alterations in DCs at the cellular and molecular levels in patients with treated SLE, depending on the disease activity and treatment. In order to phenotype subpopulations of DCs, multicolor flow cytometry was used. Transcriptional changes were identified with quantitative PCR, while soluble cytokine receptors were assessed with the Luminex technology. We show that SLE patients display a higher percentage of activated myeloid DCs (mDCs) when compared to healthy people. Both, the mDCs and plasmacytoid DCs (pDCs) of SLE patients were characterized by changes in expression of genes associated with their maturation, functioning and signalling, which was especially reflected by low expression of regulatory factor ID2 and increased expression of IRF5. pDCs of SLE patients also showed increased expression of IRF1. There were also significant changes in the expression of APRIL, MBD2, and E2-2 in mDCs that significantly correlated with some serum components, i.e. anti-dsDNA antibodies or complement components. However, we did not find any significant differences depending on the disease activity. While the majority of available studies focuses mainly on the role of pDCs in the disease development, our results show significant disturbances in the functioning of mDCs in SLE patients, thus confirming mDCs' importance in SLE pathogenesis.

Inhibitory effects of type 2 diabetes serum components in P450 inhibition assays can potential diagnose asymptomatic diabetic mice

Human cytochrome P450 (or CYP) inhibition rates were investigated in sera from high fat diet (HFD)-induced type 2 diabetes (T2D), T2D recovered, and asymptomatic mice models to verify whether P450 inhibition assays could be used for the detection of disease, evaluation of therapeutic effect, and early diagnosis of T2D. In T2D mice, the blood glucose levels markedly increased; while blood glucose levels of recovered mice exceeded 200 mg dL−1, these eventually returned to the levels seen in control mice. In asymptomatic mice fed with short term HFD (stHFD), no changes in blood glucose levels were observed. The inhibition rates of CYP1A2, CYP2A13, and CYP2C18 in T2D mice significantly increased. Whereas in recovered mice, these changes returned to the same levels noted in the control mice. Changes in the inhibition rates of CYP2A13 and CYP2C18 in stHFD mice were similar to those in T2D mice. A receiver operating characteristic (ROC) curve analysis showed high area under the ROC curve (AUC) values (0.879–1.000) of CYP2A13 and CYP2C18 in T2D and stHFD mice, indicating their high diagnostic accuracy. Collectively, this study validates the P450 inhibition assay as a method for the therapeutic evaluation and early diagnosis of T2D mouse models.

Sequential Self-Assembly Using Tannic Acid and Phenylboronic Acid-Modified Copolymers for Potential Protein Delivery.

Tannic acid (TA) can form stable complexes with proteins, attracting significant attention as protein delivery systems. However, its systemic application has been limited due to nonspecific interaction. Here, we report a simple technique to prepare systemically applicable protein delivery systems using sequential self-assembly of a protein, TA, and phenylboronic acid-conjugated PEG-poly(amino acid) block copolymers in aqueous solution. Mixing the protein and TA in aqueous solution led to covering of the protein with TA, and subsequent addition of the copolymer resulted in the formation of boronate esters between TA and copolymers, constructing the core-shell-type ternary complex. The ternary complex covered with PEG exhibited a small hydrodynamic diameter of ∼10-20 nm and prevented an unfavorable interaction with serum components, thereby accomplishing significantly prolonged blood circulation and enhanced tumor accumulation in a subcutaneous tumor model. The technique utilizing supramolecular self-assembly may serve as a novel approach for designing protein delivery systems.

HPLC-DAD-Q-TOF/MS-Based Screening and Analysis of the Multiple Absorbed Bioactive Components in Rat Serum after Oral Administration of Xiexin Tang

Background: Xiexin Tang (XXT) is a classic Traditional Chinese Medicine (TCM) formula that has been used in herbal clinics for more than 1800 years. Recently, many studies have investigated the pharmacological effects and chemical composition of XXT. However, there is little information about systematic studies on the material basis of its efficacy. In the present study, the serum pharmacochemistry technique and HPLC-DAD-Q-TOF/MS were performed to screen and analyze the multiple absorbed bioactive components and metabolites of orally dosed XXT in rat serum. Methods: Bio-samples and herbal extracts were analyzed and detected by HPLC-DAD-Q-TOF/MS. Upon comparison of the chromatograms of the single-constituent decoctions with that of the XXT formulation, the peak quantity and peak intensity of the formulated decoction showed some variation from those of the single-constituent decoctions. Results: Twenty-one serum-adsorbed constituents were identified after intragastric administration of herbal extracts, of which 8 originated from Rhei Radix et Rhizoma (RRR), 5 from Coptidis Rhizoma (CR), and 8 from Scutellariae Radix (SR). The results showed that the main adsorbed constituents in the serum were anthraquinones, anthrones, chromones, and butyrophenones, alkaloids, and flavonoids. Conclusion: The results demonstrate that an effective and reliable analytical method is set up for screening the bioactive components of Chinese herbal medicine, which provided a meaningful basis for further pharmacology and active mechanism research of XXT.

Plasmodium Impairs Antibacterial Innate Immunity to Systemic Infections in Part Through Hemozoin-Bound Bioactive Molecules.

One complication of malaria is increased susceptibility to invasive bacterial infections. Plasmodium infections impair host immunity to non-Typhoid Salmonella (NTS) through heme-oxygenase I (HO-I)-induced release of immature granulocytes and myeloid cell-derived IL-10. Yet, it is not known if these mechanisms are specific to NTS. We show here, that Plasmodium yoelii 17XNL (Py) infected mice had impaired clearance of systemic Listeria monocytogenes (Lm) during both acute parasitemia and up to 2 months after clearance of Py infected red blood cells that was independent of HO-I and IL-10. Py-infected mice were also susceptible to Streptococcus pneumoniae (Sp) bacteremia, a common malaria-bacteria co-infection, with higher blood and spleen bacterial burdens and decreased survival compared to naïve mice. Mechanistically, impaired immunity to Sp was independent of HO-I, but was dependent on Py-induced IL-10. Splenic phagocytes from Py infected mice exhibit an impaired ability to restrict growth of intracellular Lm, and neutrophils from Py-infected mice produce less reactive oxygen species (ROS) in response to Lm or Sp. Analysis also identified a defect in a serum component in Py-infected mice that contributes to reduced production of ROS in response to Sp. Finally, treating naïve mice with Plasmodium-derived hemozoin containing naturally bound bioactive molecules, excluding DNA, impaired clearance of Lm. Collectively, we have demonstrated that Plasmodium infection impairs host immunity to diverse bacteria, including S. pneumoniae, through multiple effects on innate immunity, and that a parasite-specific factor (Hz+bound bioactive molecules) directly contributes to Plasmodium-induced suppression of antibacterial innate immunity.

Physical study of Nickel Serum Level in blood samples from Iraqi populations

Trace elements examining of basic biological components in the human and animal body is essential due to the increase and the decrease in them may lead to toxicity. In this study, 32 patients who were diagnosed with lung cancer in 32 healthy volunteers were compared. Serum level concentrations of Fe trace elements were measured using Atomic Absorption Spectrometry. There was a statistical significant difference in nickel serum levels when comparing the mean of healthy with patient concentration, where the value of P (0.05 > p) was found. The level of serum components in the patient may be important in causing lung cancer.

P0301FIT FOR PURPOSE VALIDATION OF A CLINICAL ASSAY FOR THE DETECTION OF SERUM LEVELS OF GALACTOSE-DEFICIENT IMMUNOGLOBULIN A1 (GD-IGA1) IN PATIENTS WITH IGA NEPHROPATHY (IGAN)

Abstract Background and Aims Analysis of serum or plasma from patients with IgA nephropathy (IgAN) has confirmed the presence of elevated levels of circulating immune complexes containing Gd-IgA1 (Czerkinsky 1986). New sensitive and reasonably specific noninvasive tests are emerging to guide the therapeutic strategy that is applicable to all stages of IgAN (Suzuki 2014). Here we are reporting the fit for purpose validation of an ELISA method for the quantitative determination of Gd-IgA1 in human serum samples to support biomarker investigations in clinical studies of Merck KGaA, Darmstadt. Method The assay was developed based on a commercially available immunoassay kit. The dynamic range of the calibration curve was determined from 1.56 ng/mL (LLOQ) to 100 ng/mL (ULOQ). With a minimum required dilution of 200-fold and standard assay volume of 50.0 μL, the range of the method in matrix was from 312 ng/mL to 20, 000 ng/mL. In assay validation phase, multiple validation parameters were evaluated, which included minimum required dilution (MRD), calibration curve, matrix effect, Intra- & Inter run accuracy & precision, selectivity, and parallelism. Additional validation parameters include sample stability (short/long term, freeze-thaw) and batch-to-batch comparison. Results All samples measured for intra & Inter - assay precision, accuracy, fulfilled the specifications according to the acceptance criteria. The selectivity was assessed using blank serum matrix from 10 individuals: the result indicated that matrix components in serum did not interfere with the detection of Gd-IgA1. Parallelism assessment was performed successfully for both samples from healthy donors and IgAN patient samples up to dilution factor (DF) 3200 (serum samples from healthy donors were determined up to DF 1600). All DF-corrected results within the assay range were determined with %CV ≤ 30.0%. Batch to batch comparison was assessed successfully based on the known shelf life of the kit. Short term stability using QC samples were given for up to 24hrs at room temperature. Freeze-thaw stability was given for up to 3 cycles at -20°C±5°C and -75°C±15°C. The investigations were performed according to general guidelines for method validation and applicable regulations. The results of investigated validation parameters fulfilled the requirements and recommendations, generally accepted for bioanalytical projects. Conclusion The present validation qualified the method for the quantitative determination of Gd-IgA1 in human serum samples from clinical studies.

Using Fourier Transform Infrared (FTIR) Spectroscopy Coupled with Multivariate Analysis to Diagnose Polycystic Ovary Syndrome (PCOS)

The biochemical diagnostic test is important in the identification of patients with polycystic ovary syndrome (PCOS). Fourier Transfer Infrared (FTIR) measurement which analyzes the overall metabolic profiling of serum may provide more biochemical information. Here we applied the FTIR spectroscopy with multivariate analysis for a PCOS diagnosis. Sixty patients and thirty one healthy subjects were recruited in the present study. FTIR measurement was carried out on the subjects’ serum. The most significant variables obtained from FTIR data were nominated by variable importance in the projection (VIP) value following an OPLS-DA model creating from three different spectral regions: first region/ 900-1200 cm-1, second region/ 1500-1700 cm-1, and third region/ 2800-3100 cm-1. An OPLS-DA of the FTIR data showed variances in the components of patients’ serum. All OPLS-DA models showed validation (P-value < 0.05) based on CV- ANOVA, 900-1200 cm-1 region (R2Y(cum)= 0.926; Q2(cum)= 0.57), 1500-1700 cm-1 (R2Y(cum)= 0.828; Q2(cum)= 0.594), and 2800-3100 cm-1 (R2Y(cum)= 0.927; Q2(cum)= 0.717). These results showed that spectral biomarkers separated patients’ serum. In summary, our work proves that vibrational spectroscopy (FTIR) combined with multivariate analysis (OPLS-DA) showing the possibility to become a chemical reagent-free approach for biochemical assessment of bio-fluid.