ELISA-like Method Research Articles

Determination of Xylazine and Its Metabolite in Milk by a Molecularly Imprinted Microsphere Based Pseudo Immunoassay

The residues of xylazine and its metabolite in foods of animal origin are dangerous to consumers, but there has been no enzyme-linked immunosorbent assay (ELISA) or ELISA-like method reported to determine them so far. In this study, molecularly imprinted microspheres with xylazine as the template were synthesized, and 2,6-dimethylaniline (the metabolite of xylazine) was used as a hapten to prepare a streptavidinated horseradish peroxidase-labeled conjugate. A direct competitive pseudo-ELISA was developed on a microplate for xylazine and 2,6-dimethylaniline in milk. Because biotinylated horseradish peroxidase was employed to enhance the signal, the limits of detection for xylazine (0.003 ng/mL) and 2,6-dimethylaniline (0.005 ng/mL) were increased by 16.7- and 14-fold, respectively, compared to a horseradish peroxidase-labeled conjugate. Moreover, this material may be reused three times. The analysis of milk samples was consistent with values obtained by liquid chromatography tandem mass spectrometry. This is the first paper reporting a molecularly imprinted polymer-based method for the determination of xylazine and its metabolites in food.

Highly catalytic and stable Au@AuPt nanoparticles for visual and quantitative detection of E. coli O157:H7.

A visual and quantitative ELISA-like method for Escherichia coli O157:H7 is developed based on highly catalytic and stable Au@AuPt nanoparticles. The proposed enhanced ELISA method can visually detect 100 CFU mL-1 O157:H7 with high specificity and without the need for strict low-temperature reagent storage, thereby increasing the utility. Moreover, it is applicable to spiked tap water and milk tea samples without additional treatments.

Automated liquid handling robot for rapid lateral flow assay development

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.Graphical abstract

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAβ-1-3GlcNAc of the internal disaccharide units with GlcUAβ-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins.

Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named “VIRELISA” (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 105 v.g. to 5 × 109 v.g., with an LOD (limit of detection) of 5 × 104 v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.

Homing peptide-based ELISA-like method for the selective and sensitive determination of fibrin

A novel, homing peptide-based ELISA-like method for the determination of fibrin is presented.

Identification of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome-associated DNA methylation patterns.

BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex condition involving multiple organ systems and characterized by persistent/relapsing debilitating fatigue, immune dysfunction, neurological problems, and other symptoms not curable for at least 6 months. Disruption of DNA methylation patterns has been tied to various immune and neurological diseases; however, its status in ME/CFS remains uncertain. Our study aimed at identifying changes in the DNA methylation patterns that associate with ME/CFS.MethodsWe extracted genomic DNA from peripheral blood mononuclear cells from 13 ME/CFS study subjects and 12 healthy controls and measured global DNA methylation by ELISA-like method and site-specific methylation status using Illumina MethylationEPIC microarrays. Pyrosequencing validation included 33 ME/CFS cases and 31 controls from two geographically distant cohorts.ResultsGlobal DNA methylation levels of ME/CFS cases were similar to those of controls. However, microarray-based approach allowed detection of 17,296 differentially methylated CpG sites in 6,368 genes across regulatory elements and within coding regions of genes. Analysis of DNA methylation in promoter regions revealed 307 differentially methylated promoters. Ingenuity pathway analysis indicated that genes associated with differentially methylated promoters participated in at least 15 different pathways mostly related to cell signaling with a strong immune component.ConclusionsThis is the first study that has explored genome-wide epigenetic changes associated with ME/CFS using the advanced Illumina MethylationEPIC microarrays covering about 850,000 CpG sites in two geographically distant cohorts of ME/CFS cases and matched controls. Our results are aligned with previous studies that indicate a dysregulation of the immune system in ME/CFS. They also suggest a potential role of epigenetic de-regulation in the pathobiology of ME/CFS. We propose screening of larger cohorts of ME/CFS cases to determine the external validity of these epigenetic changes in order to implement them as possible diagnostic markers in clinical setting.

Abstract 39: UDP-N-acetylglucosamine as a regulator of cancer cell signaling and microenvironment

Abstract A common feature to most cancers is their disturbed glucose metabolism: increased glucose and glutamine uptake combined with preference to utilize aerobic glycolysis, a phenomenon called Warburg effect. In addition to providing fast, yet inefficient way to produce energy, Warburg effect increases the availability of glycolysis intermediates that function as starting points of metabolic pathways facilitating the fast cell proliferation. One of the pathways involved is the hexosamine biosynthesis producing UDP-N-acetylglycosamine (UDP-GlcNAc). UDP-GlcNAc is a nucleotide sugar with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAcylation of intracellular proteins. UDP-GlcNAc is linked to cancer through its dependence on glycolysis intermediate availability and by the fact that both hyaluronan and O-GlcNAcylation are closely involved in the development and progression of various cancers. Our hypothesis is that the disturbed sugar metabolism increases the levels of UDP-GlcNAc, which implements its tumorigenic effects through hyaluronan and O-GlcNAcylation, and that these changes manifest themselves also in clinical data. In order to test our hypothesis we collected 33 biopsy samples from breast cancer patients, and 13 healthy control samples. Clinical data and immunohistochemical samples are available from the operated patients. From these samples we have analyzed UDP-sugar content with HPLC, hyaluronan levels with ELISA-like method and by immunohistochemistry, and measured mRNA expression of key enzymes with quantitative RT-PCR. Our results show for the first time that UDP-sugars levels are indeed elevated in breast cancer patients. The level of UDP-GlcNAc shows a 12-times increase, while other UDP-sugars were 4 to 6 times higher. To investigate the cause of differential increment of UDP-GlcNAc and other UDP-sugars, we measured the mRNA levels of genes regulating the synthesis of UDP-GlcNAc. Cancer patients had increased expression of GFAT2 while others (GFAT1, GNPDA1 and 2) remained unchanged. In accordance to our hypothesis, the increased levels of UDP-GlcNAc correlated with changes in the mRNA expression of enzymes involved in both hyaluronan synthesis and O-GlcNAcylation of proteins. Our results show that the increased glucose uptake associated to aerobic glycolysis dramatically increases UDP-sugars in breast cancer, promoting malignant growth by increased hyaluronan synthesis and O-GlcNAc signaling. Citation Format: Sanna Oikari, Satu Tiainen, Tiia Kettunen, Amro Masarwah, Ritva Vanninen, Markku Tammi, Päivi Auvinen. UDP-N-acetylglucosamine as a regulator of cancer cell signaling and microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 39.

Selective and sensitive determination of cypermethrin in fish via enzyme-linked immunosorbent assay-like method based on molecularly imprinted artificial antibody-quantum dot optosensing materials.

Molecularly imprinted silica layers appended to quantum dots (MIP-QDs) with customized selective artificial recognition sites were fabricated in this study by optimizing the ratio of the functional monomer to the template. Scanning electron microscopy, transmission electron microscopy, Brunauer–emmett–teller, Fourier transform infrared spectroscopy, and selectivity assay analyses were also performed. Results demonstrated that the selective fluorescence quenching properties of MIP-QDs toward cypermethrin (CYP) are due to strong interactions between these molecules. An enzyme-linked immunosorbent assay (ELISA)-like method based on the MIP-QDs was established under optimal conditions. The fluorescence quenching observed from this method showed a linear relationship with CYP concentration over the range of 0.05–60.0 mg/kg with a correlation coefficient of 0.9838. Good recovery (82.7–92.4%) and a relative standard deviation of less than 10.1% were obtained from fish samples spiked with three levels of CYP. This method also demonstrated a low detection limit of 1.2 μg/kg. The ELISA-like method based on MIP-QDs can be successfully employed to detect residual of CYP in fish samples.

Sperm global DNA methylation level: association with semen parameters and genome integrity.

Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

Glycosaminoglycan profiles in the uterus of adult ovariectomized rats treated with estrogen and progestagen

To evaluate the effects of conjugated equine estrogens (CE) alone or in combination with medroxyprogesterone acetate (MPA) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Thirty days after ovariectomy, adult rats were randomly divided into four groups: GI, control (treated with drug vehicle); GII, CE (50 μg/kg per day); GIII, MPA (0.2mg/kg per day), and GIV, CE+MPA (doses as in GII and GIII). Drugs and vehicle were given by gavage during 28 days. Afterwards the animals were anesthetized, the cervix and uterine horns were dissected out and the middle portion fixed in 10% formaldehyde solution; other portions were fixed in acetone for histological examination and glycosaminoglycan quantification, respectively. Agarose gel electrophoresis was used for sulfated GAG analyses, and hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student's t test and the Tukey-Kramer test (P<0.05). The cervix and uterine horn structures presented signs of atrophy in the control group (GI). The other groups, mainly groups III and IV, had histological aspects of proliferation. In all groups the concentration of sulfated GAGs (especially dermatan sulfate) was higher than that of non-sulfated GAGs, both in cervix and in uterine horns. Estrogens increased sulfated GAG concentration at the cervix and the horn, whereas in uterine horns the amounts of sulfated GAGs were decreased after estrogens plus MPA treatment. The concentration of hyaluronic acid in uterine horns was higher than in cervices. The profiling and amounts of glycosaminoglycans in the two portions of the rat uterus are uneven. Dermatan sulfate occurs in higher concentrations in both cervix and uterine horns. Sulfated GAGs in rat cervix were increased by estrogens plus MPA, but were decreased by MPA alone in uterine horns.

A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes

Three major difficulties must be overcome to establish a quantitative method for melanosomal transfer analysis: (i) establishing a three-dimensional co-culture reassuring direct melanocyte to keratinocyte transfer, (ii) separation of melanocytes and keratinocytes following co-culture and (iii) melanosome quantification in each cell population. Melanocytes and keratinocytes are cultured on the opposite sides of the porous membrane of hanging cell inserts (1μm pores, 2×10(6) pores/cm(2) ). Cell separation is performed after 3days of co-culture by simple trypsinisation. Melanosome quantification in separated cell populations was accomplished by an ELISA-like method using gp-100 as the antigen. Melanocytes and keratinocytes come into 'direct' contact through the pores, and melanosomal transfer is accomplished without cell passage through the membrane. Cell separation by simple trypsinisation results in pure melanocyte and keratinocyte populations. Melanosome quantification by the ELISA-like method proved to be sensitive and specific to distinguish the known inhibitors and inducers of melanosomal transfer.

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 degrees C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.

Changes in the activity and protein levels of CSF acetylcholinesterases in relation to cognitive function of patients with mild Alzheimer’s disease following chronic donepezil treatment

To evaluate long-term changes in acetylcholinesterase (AChE) activity in CSF and blood following donepezil treatment in relation to the concentration of donepezil and cognition in AD patients. CSF or blood (or both) samples of a total of 104 patients with mild AD were used [MMSE score 23 +/- 0.4; age 75 +/- 1 years (mean +/- SEM); n=53 for CSF and n=51 for plasma/red blood cell (RBC) samples]. The patients were treated with 5 or 10 mg/day donepezil and clinically followed for 2 years. The CSF and RBC AChE activities were measured by the Ellman's direct colorimetric assay. Protein levels of two variants of AChE ("read-through" AChE-R and synaptic AChE-S) were determined by an ELISA-like method. The plasma donepezil concentration was dose-dependent (between 30 and 60 ng/mL in the 5-mg and 10-mg group, respectively). The CSF donepezil concentration was 10 times lower than the plasma level and showed dose- and time-dependent kinetics. The RBC AChE inhibition was moderate (19-29%). CSF AChE-S inhibition was estimated to 30-40% in the 5-mg and 45-55% in the 10-mg group. Positive correlations were observed between the CSF AChE inhibition, an increased protein level of the AChE-R variant and MMSE examination. Patients with high AChE inhibition (>or=45%) showed a stabilized MMSE test result after up to two years, while a significant decline was observed in AD patients with lower AChE inhibition (<or=30%). An increase in the protein level of the AChE-R variant corresponded to a high AChE inhibition in CSF and favored less cognitive deterioration.

Determination of conformational changes in the progesterone receptor using ELISA-like assays

The conformation of proteins often influences their functional activity. The effect of progesterone receptor ligands on the C-terminal conformation of the progesterone receptor affects the recruitment of transcriptional cofactors. These conformations can be studied by differential sensitivity to proteolytic cleavage or immunoprecipitation with a conformation-specific antibody. This study describes an ELISA-like method using conformation-specific antibodies to the C-terminal or an area adjacent to the DNA binding region. Progesterone receptor ligands are shown to influence how the progesterone receptor interacts with these antibodies in a concentration dependent manner. This method allows for quick determination of the potency of agonists as well as mechanistic studies of antagonism. The conformation inducing activity of several standard agonist and antagonist compounds were compared to their binding affinity and ability to induce alkaline phosphatase in T47D cells. This method is useful for screening compounds for functional activity at the progesterone receptor and demonstrates that J867 induces an antagonist conformation of the progesterone receptor similar to the antagonist RU486.

Mannose receptor determination by an ELISA-like method

Mannose receptor determination may be a useful tool in research, because endocytosis via this animal lectin is involved in many functions of macrophage cells, in particular, the scavenger activity, the specific and unspecific defence against infective diseases, the recognition of neoplastic cells and the activation/differentiation process of the monocyte/macrophage and microglial population. To date, available tests required expensive equipment, the use of radioactive material or the availability of a specific antiserum. We describe an ELISA-like assay, based on biotinylated mannose-conjugated bovine serum albumin (BSA), which specifically binds to the cell mannose receptor. Biotin-labelled receptors can be quantified colorimetrically, utilising an avidin–alkaline phosphatase conjugate as the indicator enzyme. This new method is sensitive and reproducible, as well as simple and rapid, and can be performed with standard laboratory equipment.

Elevation of Serum Hyaluronan Level in Werner’s Syndrome

Background: There is a well-established association between Werner’s syndrome (WS) and hyperhyaluronic aciduria; however, to date hyaluronan (HA) in the serum has not been statistically linked with WS. Recently, the gene that causes WS has been defined as a DNA helicase on chromosome 8, and 19 different mutations in WS patients have been identified. It is not known whether the mutation type of the Werner helicase gene affects the levels of serum and urine HA in WS patients. Objective: To evaluate the association of serum HA with WS, and the relationship of the serum and urine HA levels to the mutation type. Methods: HA both in the serum and the urine was measured in 40 patients with WS and 114 normal controls. The serum and urine HA were quantified by sandwich binding protein assay and competitive ELISA-like method, respectively. The muation on WS helicase gene was analyzed by mutant-allele-specific amplification and oligomer ligation assay. Results: WS patients showed significantly higher levels of HA in the serum (mean ± SD: 115.7 ± 119.8 ng/ml, p < 0.001) and urine (1,040.8 ± 777.3 ng/mg creatinine, Cre, p < 0.001) than age-matched controls (serum HA: 15.8 ± 14.2 ng/ml, urine HA: 379.7 ± 124.2 ng/mg Cre). The serum and urine HA levels of WS patients are almost equal to those of normal controls over 80 years (serum HA: 118.5 ± 108.4 ng/ml, urine HA: 914.5 ± 712.1 ng/mg Cre). There was a significant correlation between serum and urine HA levels in WS patients (r = 0.42, p = 0.007). Analysis of the mutation on the helicase gene in 22 WS patients showed that among 44 chromosomes, 3 (6.8%) chromosomes had type 1 mutation, 22 (50.0%) had type 4 mutation, 14 (31.8%) had type 6 mutation, and the rest had other mutations. The serum and urine levels of HA did not show any significant association with the mutation type. Conclusion: The hyperhyaluronic aciduria in WS reflects the high level of serum HA. The serum and urine HA levels are useful biochemical markers for WS irrespective of the muation type of the Werner helicase gene.