Application of Fisetin to the Quantitation of Serum Albumin

Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin–albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.

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SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

Communications Biology ◽

10.1038/s42003-021-01649-6 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Mikail Dogan ◽

Lina Kozhaya ◽

Lindsey Placek ◽

Courtney Gunter ◽

Mesut Yigit ◽

...

Keyword(s):

Specific Antibody ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

High Specificity ◽

Spike Protein ◽

Humoral Immune ◽

Specificity And Sensitivity ◽

Wide Range ◽

Specific Igg ◽

Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

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Microalbuminuria and borderline-increased albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay

Clinical Chemistry ◽

10.1093/clinchem/43.6.996 ◽

1997 ◽

Vol 43 (6) ◽

pp. 996-1002 ◽

Cited By ~ 68

Author(s):

Manfred A Kessler ◽

Andreas Meinitzer ◽

Walter Petek ◽

Otto S Wolfbeis

Keyword(s):

Calibration Curve ◽

Emission Intensity ◽

Clinical Laboratory ◽

Detection Limits ◽

High Specificity ◽

Fluorescence Assay ◽

Urine Samples ◽

Albumin Concentration ◽

Specificity And Sensitivity

Abstract We report a new automated fluorescence assay for determination of albumin in urine. The dye Albumin Blue 580 specifically binds to albumin with exhibition of strong red fluorescence. The albumin concentration is calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. Two Cobas Fara programs cover working ranges of 2–200 and 1–50 mg/L with detection limits of 1.4 and 0.4 mg/L, respectively. Within-run CVs (n = 10) ranged from 1.7% (189 mg/L) to 8.9% (7.2 mg/L) for 2–200 mg/L and from 2.9% (43.3 mg/L) to 5.7% (2.3 mg/L) for the 1–50 mg/L range. A test of urine samples (n = 100) submitted to routine analysis gave results that agreed well with those by the Behring nephelometric assay: AB 580 = 0.922 (± 0.010) BNA + 4.16 (± 0.78). No interference was detected from other urine components, including several proteins and 46 drugs. The high specificity and sensitivity make the method ideal for determination of microalbuminuria. In addition, the method is fast, inexpensive, and well-suited for clinical laboratory application and thus may be used instead of immunoassays.

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DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays

Microbiology Insights ◽

10.4137/mbi.s29736 ◽

2015 ◽

Vol 8 ◽

pp. MBI.S29736 ◽

Cited By ~ 2

Author(s):

Kenjiro Nagamine ◽

Guo-Chiuan Hung ◽

Bingjie Li ◽

Shyh-Ching Lo

Keyword(s):

Safety Concern ◽

Rapid Development ◽

High Sensitivity ◽

High Specificity ◽

Clostridium Tetani ◽

Specificity And Sensitivity ◽

Wide Range ◽

Pcr Assays ◽

Primer Sets ◽

Species Specific

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5–50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

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Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology

Scientific Reports ◽

10.1038/s41598-019-51501-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Kh Shamsur Rahman ◽

Bernhard Kaltenboeck

Keyword(s):

Chlamydia Pneumoniae ◽

High Specificity ◽

Cross Reactivity ◽

Peptide Antigens ◽

Specificity And Sensitivity ◽

Peptide Antibody ◽

Antibody Assays ◽

Human Sera ◽

Composite Reference Standard ◽

Individual Peptide

Abstract Cross-reactivity of classical chlamydial antigens compromises Chlamydia (C.) pneumoniae serology. By testing with 185 human antisera, we expanded 18 previously discovered C. pneumoniae-specific B-cell epitopes to 48 peptide antigens from 12 C. pneumoniae immunodominant proteins. For specific detection of antibodies against C. pneumoniae, we developed novel ELISAs with strongly reactive individual peptide antigens and mixtures of these peptides. By comparison to a composite reference standard (CRS) for anti-C. pneumoniae antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti-C. pneumoniae IgG ELISAs (36-12% sensitivity at 95% specificity). Human C. pneumoniae (Cpn) and C. trachomatis (Ctr) seroreactivity was 54% biased towards co-positivity in commercial Cpn and Ctr ELISAs, but unbiased in Cpn and Ctr peptide antibody assays, suggesting severe cross-reactivity of commercial ELISAs. Using hyperimmune mouse sera against each of 11 Chlamydia spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all Chlamydia spp., but Cpn and Ctr peptide antigens react only with antisera against the cognate chlamydial species. With simultaneously high specificity and sensitivity, and convenient use for non-specialized laboratories, these ELISAs have the potential to improve serodiagnosis of C. pneumoniae infection.

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Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603753113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7088-7093 ◽

Cited By ~ 56

Author(s):

Michael T. Hwang ◽

Preston B. Landon ◽

Joon Lee ◽

Duyoung Choi ◽

Alexander H. Mo ◽

...

Keyword(s):

Personalized Medicine ◽

High Specificity ◽

Practical Implementation ◽

Strand Displacement ◽

Snp Detection ◽

Single Nucleotide ◽

Specificity And Sensitivity ◽

Dna Strand Displacement ◽

Wide Range ◽

Dna Strand

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

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Serum Proteomic Fingerprints of Adult Patients with Severe Acute Respiratory Syndrome

Clinical Chemistry ◽

10.1373/clinchem.2005.061689 ◽

2006 ◽

Vol 52 (3) ◽

pp. 421-429 ◽

Cited By ~ 62

Author(s):

Ronald TK Pang ◽

Terence CW Poon ◽

KC Allen Chan ◽

Nelson LS Lee ◽

Rossa WK Chiu ◽

...

Keyword(s):

Viral Load ◽

Severe Acute Respiratory Syndrome ◽

Roc Curve ◽

Serum Proteins ◽

Early Stage ◽

High Specificity ◽

Roc Curves ◽

Roc Curve Analysis ◽

Outbreak Period ◽

Specificity And Sensitivity

Abstract Background: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus strain, SARS-CoV. Specific proteomic patterns might be present in serum in response to the infection and could be useful for early detection of the disease. Methods: Using surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, we profiled and compared serum proteins of 39 patients with early-stage SARS infection and 39 non-SARS patients who were suspected cases during the SARS outbreak period. Proteomic patterns associated with SARS were identified by bioinformatic and biostatistical analyses. Features of interest were then purified and identified by tandem mass spectrometry. Results: Twenty proteomic features were significantly different between the 2 groups. Fifteen were increased in the SARS group, and 5 were decreased. Their concentrations were correlated with 2 or more clinical and/or biochemical variables. Two were correlated with the SARS-CoV viral load. Hierarchical clustering analysis showed that a majority of the SARS patients (95%) had similar serum proteomic profiles and identified 2 subgroups with poor prognosis. ROC curve analysis identified individual features as potential biomarkers for SARS diagnosis (areas under ROC curves, 0.733–0.995). ROC curve areas were largest for an N-terminal fragment of complement C3c α chain (m/z 28 119) and an internal fragment of fibrinogen α-E chain (m/z 5908). Immunoglobulin κ light chain (m/z 24 505) positively correlated with viral load. Conclusions: Specific proteomic fingerprints in the sera of adult SARS patients could be used to identify SARS cases early during onset with high specificity and sensitivity.

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Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

10.1101/2020.07.07.20148106 ◽

2020 ◽

Cited By ~ 7

Author(s):

Mikail Dogan ◽

Lina Kozhaya ◽

Lindsey Placek ◽

Courtney L. Gunter ◽

Mesut Yigit ◽

...

Keyword(s):

Specific Antibody ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

High Specificity ◽

Spike Protein ◽

Humoral Immune ◽

Specificity And Sensitivity ◽

Wide Range ◽

Specific Igg ◽

Negative Controls

AbstractDevelopment of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

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SELEX-Based Direct Enzyme-Linked Aptamer Assay (DELAA) for Diagnosis of Toxoplasmosis by Detection of SAG1 Protein in Mice and Humans

10.21203/rs.3.rs-207045/v1 ◽

2021 ◽

Author(s):

Jilong Shen ◽

Wei Wang ◽

Yuanhong Xu ◽

Xuhang Shen ◽

Wen Cui ◽

...

Keyword(s):

Acute Infection ◽

Pcr Amplification ◽

High Specificity ◽

Laboratory Diagnosis ◽

Specificity And Sensitivity ◽

Recognition Probe ◽

Human Sera ◽

Aptamer Assay ◽

Oligonucleotide Library ◽

Major Surface

Abstract Background: Toxoplasma gondii is a single-celled parasite commonly found in mammals. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma-derived DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key marker for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology.Methods: Recombinant truncated SAG1（r-SAG1）of Toxoplasma WH3 strain (type Chinese 1) was prokaryotically expressed and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamer-2 was screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 obtained from tachyzoite lysates (n-SAG1), mouse sera of acute infection, and human sera that had been verified to be positive for Toxoplasma DNAs by PCR amplification. Results: The soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, and then labelled with biotin. The selected aptamers were amplified by PCR, followed by DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for Toxoplasma circulating antigen test.Conclusions: A new direct enzyme-linked aptamer assay (DELAA), with aptamer-2 as the recognition probe, was developed for detection of native SAG1 protein of Toxoplasma. With increased sensitivity and specificity, stability, easy and cheap preparation, the aptamer-based technology is considered as a efficient method for the diagnosis of active and reactivated toxoplasmosis.

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SERUM PROTEIN EVALUATION OF PIT BULL, BULLMASTIFF AND CANE CORSO BREEDS OF DOMESTIC DOG (CANIS LUPUS FAMILIARIS)

International Journal of Advanced Research ◽

10.21474/ijar01/12145 ◽

2020 ◽

Vol 8 (12) ◽

pp. 256-260

Author(s):

Chuku L.C. ◽

◽

Chinaka N.C. ◽

Keyword(s):

Serum Albumin ◽

Serum Protein ◽

Reference Range ◽

Serum Proteins ◽

Domestic Dog ◽

Canis Lupus Familiaris ◽

Male And Female ◽

Normal Reference ◽

Wide Range ◽

G Ratio

The diversity of proteins, their metabolism and protein functions are attributed to a wide range of responses in cells, organs and tissues of animals. The study evaluated specific serum proteins from male and female gender of 3 dog breeds (Pit bull, Bullmastiff and Cane corso). Cellulose acetate electrophoresis technique was used to ascertain the concentrations (g/dL) of the individual serum proteins and compared to the respective normal reference values for domestic dog. Results obtained indicate that the Pit bull (male and female) and the Bullmastiff (female) had serum albumin concentrations that were higher than the normal reference range. An increase above normal in serum globulin (Î±1-globulin, Î±2-globulin and Î²2-globulin) concentrations was observed in the female Cane corso breed, as other dog gender and breed fell within reference range. A comparison based on serum albumin/globulin (A/G) ratio of the dog breeds revealed a normal A/G concentration except for the female Cane corso (0.36 g/dL) which was lower than normal, and the female Pit bull (1.19 g/dL) which was found to be higher. Such abnormal decrease and or increase in these respective serum protein concentrations could be attributed to prolong dehydration due to the dogs regular activity and low fluid (water) replacement and or incidences of mild to acute inflammatory response/disease due to consumption of a certain diet type over time.

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Density of nerve fibres in eutopic endometrium in women with endometriosis

Open Medicine ◽

10.2478/s11536-012-0122-9 ◽

2013 ◽

Vol 8 (2) ◽

pp. 141-145 ◽

Cited By ~ 1

Author(s):

Ruta Liutkeviciene ◽

Zana Bumbuliene ◽

Jolita Zakareviciene

Keyword(s):

Clinical Practice ◽

Minimally Invasive ◽

High Specificity ◽

Nerve Fibers ◽

Endometrial Biopsy ◽

Nerve Fibres ◽

Exclusion Criteria ◽

Eutopic Endometrium ◽

Specificity And Sensitivity ◽

Wide Range

AbstractEndometriosis is the disease which usually takes a wide range of time to be diagnosed. Recently, the investigators found that endometriosis could be diagnosed using the density of nerve fibers in eutopic endometrium after taking endometrium bioptate. The aim of the article is to summarize the existing literature on density of nerve fibers in eutopic endometrium in women with and without endometriosis. In this review were involved only those studies which used the same exclusion criteria and the same technology to detect nerve fibers in eutopic endometrium. Our review confirmed the position of all studies’ results that detection of specific nerve fibers within eutopic endometrium using minimally invasive endometrial biopsy technique could be widely used in clinical practice to diagnose endometriosis with high specificity and sensitivity.

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