Serum Protein Research Articles

Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of GFAP in serum.

Glial fibrillary acidic protein (GFAP) in serum has been shown as a biomarker of traumatic brain injury (TBI) which is a significant global public health concern. Accurate and rapid detection of serum GFAP is critical for TBI diagnosis. In this study, a time-resolved fluorescence immunochromatographic test strip (TRFIS) was proposed for the quantitative detection of serum GFAP. This TRFIS possessed excellent linearity ranging from 0.05 to 2.5 ng/mL for the detection of serum GFAP and displayed good linearity (Y = 598723X + 797198, R2 = 0.99), with the lowest detection limit of 16 pg/mL. This TRFIS allowed for quantitative detection of serum GFAP within 15 min and showed high specificity. The intra-batch coefficient of variation (CV) and the inter-batch CV were both < 4.0%. Additionally, this TRFIS was applied to detect GFAP in the serum samples from healthy donors and patients with cerebral hemorrhage, and the results of TRFIS could efficiently discern the patients with cerebral hemorrhage from the healthy donors. Our developed TRFIS has the characteristics of high sensitivity, high accuracy, and a wide linear range and is suitable for rapid and quantitative determination of serum GFAP on-site.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites

BackgroundGlycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.MethodsWe enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS.ResultsHuman albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.ConclusionsAlbumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Correction of Nitrosative Stress During Reparative Regeneration of the Nasal Cavity Mucosa in an Experimental

Introduction. Nitric oxide metabolites play an important role in prolongation of the inflammatory reaction, disorders of epithelial regeneration in diseases of the upper respiratory tract. One of the promising approaches to regulating the bioactivity of nitric oxide is the use of antioxidants.The objective of the study is to evaluate the effectiveness of antioxidant therapy for the correction of nitrosative stress during reparative regeneration of the nasal mucosa in an experiment.Methods. A preclinical randomized experimental study is conducted on 160 rats, divided into 4 groups. The control group (n = 40) consists of intact animals. Animals in experimental group 1 (n = 40) do not receive treatment aſter injury; in group 2 (n = 40) anti-inflammatory treatment is prescribed. The third group consists of laboratory rats (n = 40), which are additionally injected into the nasal cavity with an antioxidant — a 15 % solution of 1,1-dimethyl-3-oxobutylphosphonic acid dimethyl ester. On days 2, 5, 10, and 14 of observation, the concentration of nitrates and nitrites and C-reactive protein in the blood of animals are determined.Results. In group 3, aſter an increase in the level of nitric oxide metabolites on day 2 aſter injury on day 5, a significant (p &lt; 0.05) decrease was noted in comparison with other groups, and by day 10— normalization of the indicator. An increase in the level of C-reactive protein in blood serum correlated with the concentration of nitric oxide metabolites.Discussion. The optimal therapeutic effect when taking antioxidants develops when administered in the first hours aſter injury to reduce the excessive formation of reactive nitrogen radicals. However, the duration of administration should not exceed 4–5 days to avoid a significant decrease in the level of nitric oxide metabolites and deterioration of mucosal regeneration.Conclusions. Assessment of nitric oxide metabolitesin the blood at different times aſter injury is an important marker of inflammatory activity. The use of antioxidants helps to reduce the concentration of active nitrogen radicals.

Exposure-response relationship of guselkumab and the potential of serum proteomics in identifying predictive biomarker candidates in psoriasis.

Response to biologics in psoriasis varies in real-world settings. Serum biomarkers could aid biologic selection and dose modifications to improve patient outcomes while encouraging cost-effective care. To explore the exposure-response relationship for guselkumab (GUS), to define a GUS concentration target for optimal response and to evaluate the potential of serum protein levels as predictive biomarker candidates. This is a prospective, multicentric, cohort study in psoriasis patients treated with GUS. Serum GUS trough concentrations (TCs) collected at multiple timepoints were measured using an in-house immunoassay. Next, proximity extension assay technology (Target 96 Inflammation Panel Olink®) was used to measure serum protein levels in a subcohort including 38 GUS patients (week 0 and week 4), six psoriasis patients naive for systemic treatment and four healthy controls. Seventy-five patients participated and 400 samples were collected. Guselkumab TCs and clinical response were correlated at week 4, week 12 and in steady-state (≥20 weeks). Optimal responders (Psoriasis Area and Severity Index [PASI] ≤ 2) had significantly higher TCs than suboptimal responders from week 4 onwards in treatment. An optimal steady-state TC of 1.6 μg/mL was defined. Although TC and absolute PASI were lower and worse, respectively, in patients weighing ≥90 kg, clinical outcomes referred to desirable to excellent PASI ranges. Therefore, we do not recommend systematically higher GUS doses in obese patients. We could not reveal early differentially expressed proteins to distinguish future optimal from suboptimal responders. We demonstrated an exposure-response relationship for GUS and an optimal steady-state TC of 1.6 μg/mL in real-world psoriasis patients. Hereby, we deliver more evidence that therapeutic drug monitoring poses a promising strategy in optimizing GUS treatment. No biomarker candidates were identified through serum proteomics. We propose protein screening should be repeated in larger cohorts to continue the quest for predictive biomarkers.

GOAT rs10096097 and CREB1 rs6740584 single nucleotide polymorphisms are associated with type 2 diabetes mellitus in Egyptians.

Diabetes mellitus (DM) is a chronic disorder that affects nearly half a billion people around the world and causes millions of deaths annually. Treatment of diabetes or related complications represents an economic burden not only for developing countries but also for the developed ones. Hence, new efficient therapeutic and preventive strategies and screening tools are necessary. The current work aimed to assess the potential association of single nucleotide polymorphisms (SNPs) in ghrelin O-acyltransferase (GOAT) rs10096097, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) rs6740584, and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) rs62521874 genes with type 2 DM susceptibility in Egyptians. A total of 96 patients with type 2 DM along with 72 healthy individuals participated in this study. Genotyping was executed via real-time polymerase chain reaction (PCR), and the serum protein levels of GOAT, CREB, and MafA were measured by enzyme-linked immunosorbent assay (ELISA). Genotyping revealed a significant association of GOAT rs10096097 and CREB1 rs6740584 SNPs with type 2 diabetes risk, with significantly higher GOAT rs10096097 G allele and CREB1 rs6740584 T allele frequencies in diabetic patients than in controls. However, insignificant association was identified between the MafA rs62521874 SNP and diabetes in the examined sample of the Egyptian residents. Serum GOAT, CREB1, and MafA protein levels did not vary significantly between diabetic and control individuals. Yet, significant variation in serum GOAT and CREB1 levels was detected between CREB1 rs6740584 genotypes within the diabetic group, with CT and TT genotype carriers showing higher levels than AA genotype patients. GOAT rs10096097 and CREB1 rs6740584, but not MafA rs62521874, SNPs are associated with type 2 diabetes risk in the studied Egyptians.

Inhibition of RNA Phosphodiester Backbone Cleavage in the Presence of Organic Cations of Different Sizes.

Living cells contain various types of organic cations that may interact with nucleic acids.In order to understand thenucleic acid-binding properties oforganic cations of different sizes, we investigated the ability ofsimple organic cations to inhibit the RNA phosphodiester bond cleavage promoted by Mg2+, Pb2+, and RNA-cleaving serum proteins. Kinetic analysis using chimeric DNA-RNA oligonucleotides showed that the cleavage at ribonucleotide sites was inhibited in the presence of monovalent cations comprising alkyl chains or benzene rings. The comparison of thecleavage rates in the presence ofquaternary ammonium and phosphonium ionsindicated thatthe steric hindrance effect of organic cations on their binding tothe RNA backboneis significant when the cation size is larger than the phosphate-phosphate distance of a single-stranded nucleic acid.The cleavage inhibition was also observed for ribonucleotides located in long loops but not in short loopsof oligonucleotide structures, indicating less efficient binding of bulky cationsto structurally constrained regions.Theseresults revealtheunique nucleic acid-binding properties of bulky cations distinct from those of metal ions.

Hypercalcaemia in a dog with hypothyroidism

AbstractA 5‐year‐old, entire, female Greenland dog was presented with polyuria and polydipsia. A mildly increased circulating concentration of ionised calcium, mild non‐regenerative anaemia, mild hypercholesterolemia and mild increases in liver enzyme activity were detected. Parathyroid hormone, plasma parathyroid hormone‐related protein and vitamin D were analysed in a blood sample, with all results below the lower limit of the reference range. Further investigation with abdominal ultrasonography, fine‐needle aspiration for cytology of the liver and spleen, thoracic radiography, adrenocorticotropic hormone‐stimulation and serum protein electrophoresis could not detect any disease. During the next months, the dog developed coat changes that clinically corresponded to an endocrinopathy. Analysis of thyroxine and thyroid‐stimulating hormone was performed. Thyroxine was less than 6 nmol/L and thyroid‐stimulating hormone was 1.5 µg/L, which indicated primary hypothyroidism. Treatment with l‐thyroxine resulted in normalisation of the hypercalcaemia. Moreover, the polyuria and alopecia resolved.

Assessment of glial fibrillary acidic protein and anti-glial fibrillary acidic protein autoantibody concentrations and necrotising meningoencephalitis risk genotype in dogs with pug dog myelopathy.

Pugs commonly present with thoracolumbar myelopathy, also known as pug dog myelopathy (PDM), which is clinically characterised by progressive signs involving the pelvic limbs, no apparent signs of pain and, often, incontinence. In addition to meningeal fibrosis and focal spinal cord destruction, histopathology has confirmed lymphohistiocytic infiltrates in the central nervous system (CNS) in a considerable number of pugs with PDM. Lymphohistiocytic CNS inflammation also characterises necrotising meningoencephalitis (NME) in pugs. This study aimed to investigate the potential contribution of an immunological aetiology to the development of PDM. The concentrations of glial fibrillary acidic protein (GFAP) in serum and CSF and of anti-GFAP autoantibodies in CSF were measured with an ELISA. In addition, a commercial test was used for genetic characterisation of the dog leukocyte antigen class II haplotype, which is associated with NME susceptibility. This study included 87 dogs: 52 PDM pugs, 14 control pugs, four NME pugs and 17 dogs of breeds other than pugs that were investigated for neurological disease (neuro controls). Anti-GFAP autoantibodies were present in 15 of 19 (79%) of the PDM pugs tested versus six of 16 (38%) of the neuro controls tested (p = 0.018). All 18 PDM pugs evaluated had detectable CSF GFAP. Serum GFAP was detected in two of three (67%) of the NME pugs and in two of 11 (18%) of the control pugs but not in any of the 40 tested PDM pugs. Male pugs heterozygous for the NME risk haplotype had an earlier onset of clinical signs (70 months) compared to male pugs without the risk haplotype (78 months) (p = 0.036). The study was limited bythe lack of healthy dogs of breeds other than pugs and the small numbers of control pugs and pugs with NME. The high proportion of PDM pugs with anti-GFAP autoantibodies and high CSF GFAP concentrations provide support for a potential immunological contribution to the development of PDM.

Methscore: a comprehensive R function for DNA methylation-based health predictors.

DNA methylation-based predictors of various biological metrics have been widely published and are becoming valuable tools in epidemiologic studies of epigenetics and personalized medicine. However, generating these predictors from original source software and web servers is complex and time consuming. Furthermore, different predictors were often derived based on data from different types of arrays, where array differences and batch effects can make predictors difficult to compare across studies. We integrate these published methods into a single R function to produce 158 previously published predictors for chronological age, biological age, exposures, lifestyle traits and serum protein levels using both classical and principal component-based methods. To mitigate batch and array differences, we also provide a modified RCP method (ref-RCP) that normalize input DNA methylation data to reference data prior to estimation. Evaluations in real datasets show that this approach improves estimate precision and comparability across studies. The function was included in software package ENmix, and is freely available from Bioconductor website (https://www.bioconductor.org/packages/release/bioc/html/ENmix.html). Supplementary data are available at Bioinformatics online.

Cytosolic sulfotransferases in endocrine disruption.

The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of endocrine hormones as well as a broad array of drugs, environmental chemicals, and other xenobiotics. Many endocrine-disrupting chemicals (EDCs) interact with these SULTs as substrates and inhibitors, and thereby alter sulfation reactions responsible for metabolism and regulation of endocrine hormones such as estrogens and thyroid hormones. EDCs or their metabolites may also regulate expression of SULTs through direct interaction with nuclear receptors and other transcription factors. Moreover, some sulfate esters derived from EDCs (EDC-sulfates) may serve as ligands for endocrine hormone receptors. While the sulfation of an EDC can lead to its excretion in the urine or bile, it may also result in retention of the EDC-sulfate through its reversible binding to serum proteins and thereby enable transport to other tissues for intracellular hydrolysis and subsequent endocrine disruption. This mini-review outlines the potential roles of SULTs and sulfation in the effects of EDCs and our evolving understanding of these processes.

Uncommon detection of monoclonal proteins in alpha 2 region on serum protein electrophoresis: A case series

Uncommon detection of monoclonal proteins in alpha 2 region on serum protein electrophoresis: A case series

Bisalbuminemia: A rare discovery during serum protein electrophoresis

Bisalbuminemia: A rare discovery during serum protein electrophoresis

Ustekinumab in serum protein electrophoresis: Defining forthcoming potential artifacts

Ustekinumab in serum protein electrophoresis: Defining forthcoming potential artifacts

A cell-based assay to measure the activity of the complement convertases

IntroductionThe complement system serves as a crucial defense mechanism against invading pathogens, but dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical/lectin and the alternative pathway convertases. Aberrant regulation of the convertases is often implicated in the development of rare complement-related diseases. However, analyzing convertase activity poses a significant challenge due to their labile nature and intricate interactions with serum proteins. MethodsIn this study, we propose a novel assay for the functional evaluation of these complexes. Our approach leverages a widely available human lymphoma cell line, which, when sensitized with antibodies, triggers activation of the classical pathway with a substantial amplification by the alternative pathway. The combined action of two C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors are washed away and guinea pig serum in EDTA buffer supports the development of lytic sites on the platform of pre-existing convertases. ResultsThe assay detects recombinant gain-of-function components of both convertase types within human serum or plasma. Furthermore, we demonstrate the assay's practical utility in analyzing nephrological patients harboring C3 genetic variants and illustrate its capacity to distinguish between patients and asymptomatic relatives carrying the same pathogenic C3 variant. ConclusionsWe provided a proof of concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or these of unknown significance in ubiquitous complement proteins like C3.

Influence of para-substituted benzaldehyde derivatives with different push/pull electron strength groups on the conformation of human serum albumin and toxicological effects in zebrafish

Excessive intake of benzaldehyde and its derivatives can cause irreversible damage to living organisms. Hence, benzaldehyde derivatives with different para-substitutions of push/pull electronic groups were chosen to investigate the effect of different substituent properties on the structure of human serum albumin (HSA). The binding constants, number of binding sites, major interaction forces, protein structural changes, and binding sites of benzaldehyde (BzH) and its derivatives (4-BzHD) with HSA in serum proteins were obtained based on multispectral and molecular docking techniques. The mechanism of BzH/4-BzHD interaction on HSA is mainly static quenching and is accompanied by the formation of a ground state complex. BzH/4-BzHD is bound to HSA in a 1:1 stoichiometric ratio. The interaction forces for the binding of BzH/4-BzHD to HSA are mainly hydrogen bonding and hydrophobic interaction, which are also accompanied by a small amount of electrostatic interactions. The effect of BzH/4-BzHD on HSA conformation follows: 4-Diethylaminobenzaldehyde (4-DBzH) > 4-Nitrobenzaldehyde (4-NBzH) > 4-Hydroxybenzaldehyde (4-HBzH) > 4-Acetaminobenzaldehyde (4-ABzH) > BzH, which means that the stronger push/pull electronic strength of the para-substituted benzaldehyde derivatives has a greater effect on HSA conformation. Furthermore, the concentration-lethality curves of different concentrations for BzH/4-BzHD on zebrafish verified above conclusion. This work provides a scientific basis for the risk assessment of benzaldehyde and its derivatives to the ecological environment and human health and for the environmental toxicological studies of benzaldehyde derivatives with different strengths of push/pull electron substitution.

Anti-Myelin-associated Glycoprotein Neuropathy

Anti-myelin-associated glycoprotein (MAG) neuropathy, which occurs secondary to immunoglobulin (Ig)M paraproteinemia such as monoclonal gammopathy of undetermined significance, is characterized by slow progression, sensory or sensorimotor disturbances, and ataxia. The estimated prevalence of this neuropathy in Japan is 0.28 per 100,000 population with male preponderance. This neuropathy is diagnosed based on the detection of M protein and anti-MAG antibodies in patients' serum. Nerve conduction studies show prolonged distal latency, and histopathological evaluation of sural nerve biopsies shows widely spaced myelin on electron microscopy. Usually, immunotherapy, including administration of intravenous Ig and corticosteroids, is ineffective, and rituximab is beneficial in approximately 50% of patients. Novel therapies, such as administration of Bruton's tyrosine kinase inhibitors are expected to benefit patients with the MYD88L265P mutation.

The effect of “Teepol” residues on the estimation of serum electrolytes, protein, and cholesterol in sample collection tubes: an interference study

The effect of “Teepol” residues on the estimation of serum electrolytes, protein, and cholesterol in sample collection tubes: an interference study

One year review of serum protein electrophoresis and immunofixation tests performed at a central laboratory in a low-middle income setting: A demand management perspective

One year review of serum protein electrophoresis and immunofixation tests performed at a central laboratory in a low-middle income setting: A demand management perspective

Reporting practices of serum protein electrophoresis in Pakistan – A multicentre survey

Reporting practices of serum protein electrophoresis in Pakistan – A multicentre survey

A liposome encapsulated methylene blue-mediated electrochemical and UV–visible dual mode split-type immunoassay for the detection of 17β-estradiol

Herein, we fabricated an electrochemical (EC) and UV–visible absorption (UV–vis) dual mode split-type immunoassay for the detection of 17β-estradiol (E2), which was mediated by liposome encapsulated methylene blue (MB@lip). MB molecule acted as the probe in the EC and UV–vis absorption dual mode detections, and its release was controlled by liposome. The competitive immune recognition was conducted between the E2 in the sample and E2 conjugated bovine serum protein (E2-BSA) adsorbed on the 96-wells plate in combining with E2 antibody labeled with MB@lip (E2-Ab/MB@lip). MB molecule could be released from the resulting immune composite of E2-BSA/E2-Ab/MB@lip in the presence of Triton X-100, and quantified by UV–vis and EC methods. The three-dimensional cross-linked reduced graphene oxide/Ti3C2 (3D-rGO/Ti3C2) aerogel was prepared through hydrothermal method, then complexed with the electroactive anthraquinone (AQ) and used as the electrode modified material. The AQ/3D-rGO/Ti3C2 composite had high surface area and provided abundant adsorption sites for MB, and the displacement/competitive behavior between AQ and MB could dexterously achieve the ratiometric EC detection of E2. In addition, the inherent blue color of MB allowed it to be analyzed by UV–vis absorption method. The proposed dual mode detection method exhibited broad linear ranges of 0.1 pg mL−1 to 50 ng mL−1 (by UV–vis) and 0.03 pg mL−1 to 50 ng mL−1 (by EC) for E2 detection, and the detection limits were 0.023 pg mL−1 (S/N = 3) and 8.0 fg mL−1 (S/N = 3), respectively. Moreover, the proposed immunoassay exhibited good practicability and was applied to monitor E2 in milk and serum successfully.