Direct enzyme-linked immunosorbent assay and a radioimmunoassay for melatonin compared

1995 ◽  
Vol 41 (3) ◽  
pp. 381-386 ◽  
Author(s):  
S Chegini ◽  
B Ehrhart-Hofmann ◽  
A Kaider ◽  
F Waldhauser
Keyword(s):  
Work Load ◽  
Sample Volume ◽  
Enzyme Linked Immunosorbent Assay ◽  
Procedure Time ◽  
Detection Range ◽  
Melatonin Concentration ◽  
Direct Elisa ◽  
Concentration Step ◽  
Well Correlation ◽  
Serum Effect

Abstract A novel commercially available ELISA for direct measurement of melatonin concentration in serum was evaluated and compared with an RIA routinely used in our laboratory. The direct ELISA is technically simpler, requires a smaller sample volume (0.1 mL), and compares well with RIA in terms of stability of the calibration curve and intra- and interassay CVs. Correlation with RIA measurements is, however, suboptimal (y = 0.39x + 56; r = 0.65, P < 0.001; n = 138), which may be due to a serum effect, as evidenced by dilution studies. Furthermore, the detection range of the ELISA does not cover the physiological daytime melatonin concentrations in humans. Adding an extraction and 10-fold concentration step shifted the detection range of the ELISA to include low physiological concentrations as well. Correlation with RIA measurements also improved significantly (y = 0.97x-23; r = 0.95, P < 0.001; n = 105), probably due to removal of the serum effect. Although extraction increases the required sample volume (1.5 mL), work load, and procedure time, this step is necessary for the ELISA to compete successfully with RIA.

scholarly journals Streptavidin-conjugated gold nanoclusters as ultrasensitive fluorescent sensors for early diagnosis of HIV infection

2018 ◽  
Vol 4 (11) ◽  
pp. eaar6280 ◽  
Author(s):  
Aditya Dileep Kurdekar ◽  
L. A. Avinash Chunduri ◽  
C. Sai Manohar ◽  
Mohan Kumar Haleyurgirisetty ◽  
Indira K. Hewlett ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Limit Of Detection ◽  
Gold Nanoclusters ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Metal Nanoclusters ◽  
Detection Range ◽  
In Silico Studies ◽  
P24 Antigen ◽  
Hiv 1

We have engineered streptavidin-labeled fluorescent gold nanoclusters to develop a gold nanocluster immunoassay (GNCIA) for the early and sensitive detection of HIV infection. We performed computational simulations on the mechanism of interaction between the nanoclusters and the streptavidin protein via in silico studies and showed that gold nanoclusters enhance the binding to the protein, by enhancing interaction between the Au atoms and the specific active site residues, compared to other metal nanoclusters. We also evaluated the role of glutathione conjugation in binding to gold nanoclusters with streptavidin. As proof of concept, GNCIA achieved a sensitivity limit of detection of HIV-1 p24 antigen in clinical specimens of 5 pg/ml, with a detection range up to1000 pg/ml in a linear dose-dependent manner. GNCIA demonstrated a threefold higher sensitivity and specificity compared to enzyme-linked immunosorbent assay for the detection of HIV p24 antigen. The specificity of the immunoassay was 100% when tested with plasma samples negative for HIV-1 p24 antigen and positive for viruses such as hepatitis B virus, hepatitis C virus, and dengue. GNCIA could be developed into a universal labeling technology using the relevant capture and detector antibodies for the specific detection of antigens of various pathogens in the future.

scholarly journals IgM and IgA Antibody Responses in 12 Cases of Human Acquired Toxoplasmosis

1997 ◽  
Vol 39 (6) ◽  
pp. 327-332 ◽  
Author(s):  
Emília E. H. TAKAHASHI ◽  
Cláudio L. ROSSI
Keyword(s):  
Serological Test ◽  
Clinical Manifestations ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igm Antibodies ◽  
Iga Antibodies ◽  
Direct Elisa ◽  
Acute Toxoplasmosis ◽  
Antibody Levels ◽  
Acquired Toxoplasmosis

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels <FONT FACE="Symbol">£</font> 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis

scholarly journals Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles

2009 ◽  
Vol 16 (3) ◽  
pp. 408-413 ◽  
Author(s):  
Shixing Tang ◽  
Mahtab Moayeri ◽  
Zhaochun Chen ◽  
Harri Harma ◽  
Jiangqin Zhao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Laboratory Research ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Serum Samples ◽  
Detection Range ◽  
Edema Factor ◽  
Anthrax Lethal Factor ◽  
Europium Nanoparticles

ABSTRACT We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

scholarly journals The optimization of an ultra-sensitive single molecule assay for the multiplexed detection of SARS-CoV-2 antigens

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S145-S145
Author(s):  
Y Senussi ◽  
Z N Swank ◽  
D R Walt
Keyword(s):  
Single Molecule ◽  
Respiratory Infections ◽  
Sample Volume ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
N Protein ◽  
Children And Young Adults ◽  
Inflammatory Syndrome

Abstract Introduction/Objective SARS-CoV-2 antigens, including the nucleocapsid (N) protein, spike protein, and its S1 subunit have served as key biomarkers for research and diagnostic purposes. We previously developed quantitative single molecule array (Simoa) assays to measure the concentration of spike, S1 subunit and N protein in plasma samples with femtomolar limits of detection. We aimed to test antibodies that were not available early in the pandemic, reduce assay cross-reactivity, develop a multiplexed assay for spike, S1, and N protein in order to minimize the sample volume needed. Methods/Case Report Using the Simoa platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 17 S1 subunit and spike antibodies for a total of 130 antibody-pair combinations, we performed dilution linearity experiments to determine the ideal dilution factor, spike and recovery experiments, tested the assay using S1 subunit from other human coronavirus HKV1, NL63, and 229E, pre-pandemic plasma samples from patients that were sick with viral or bacterial respiratory infections. We then used the best antibody pairs to measure S1 and spike in plasma samples collected from patients with severe SARS-CoV-2. Lastly, we conjugated the best-performing capture antibodies for spike, S1 and N to beads labeled with different fluorophores to test if the assay for all three antigens could be multiplexed. Results (if a Case Study enter NA) We observed no cross-reactivity with S1 from other coronavirus strains, no detection of S1 or spike in a cohort of 30 pre-pandemic samples and successfully developed a multiplexed assay for the detection of spike, S1, and N protein, enabling us to use 50% less sample volume. Conclusion Reduction of necessary sample volume is important for studies involving multisystem inflammatory syndrome in children (MIS-C), and possible adverse effects of SARS-CoV-2 vaccinations on children and young adults. An improved assay with minimal cross-reactivity will also be useful to study individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

scholarly journals Adaptation of ELISA Detection of Plasmodium Falciparum and Plasmodium Vivax Circumsporozoite Proteins in Mosquitoes to a Multiplex Bead-Based Immunoassay

2021 ◽  
Author(s):  
Alice Sutcliffe ◽  
Seth R. Irish ◽  
Eric Rogier ◽  
Micaela Finney ◽  
Sarah Zohdy ◽  
...  
Keyword(s):  
Lower Limit ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Limits Of Detection ◽  
Detection Range ◽  
Cutoff Value ◽  
Sporozoite Rate ◽  
The Impact ◽  
Lower Limits ◽  
Multiplex Bead

Abstract Background: Plasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of P. falciparum (Pf), P. vivax VK210 (Pv210) or P. vivax VK247 (Pv247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.Methods: Recombinant positive controls for Pf, Pv210 and Pv247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cutoff measures were applied to demonstrate how cutoff criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins. Results: Following optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for Pf, Pv and Pv247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for Pf, 425 for Pv210 and 1650 for Pv247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4% and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decreased in the Madagascar sporozoite rate to 2.8-4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cutoff value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cutoff value is determined.Conclusions: The csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to Pf, Pv210 and Pv247 can be added following optimization.

Detection of Zearalenone By Tandem Immunoaffinity-Enzyme-Linked Immunosorbent Assay and Its Application to Milk

1990 ◽  
Vol 53 (7) ◽  
pp. 577-580 ◽  
Author(s):  
JUAN I. AZCONA ◽  
MOHAMED M. ABOUZIED ◽  
JAMES J. PESTKA
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Monoclonal Antibody ◽  
Initial Sample ◽  
Average Recovery ◽  
Milk Samples ◽  
Coefficients Of Variation ◽  
Direct Elisa ◽  
Commercial Milk

A hybridoma-based method utilizing tandem affinity chromatography and enzyme-linked immunosorbent assay (ELISA) was devised to detect zearalenone. A zearalenone specific monoclonal antibody was attached to Sepharose for initial sample clean-up. Zearalenone was eluted with methanol and then quantified by competitive direct ELISA. Average ELISA recoveries from the column for water spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml were 107, 86, 95, 95, and 92%, respectively, with a mean recovery of 95%. Mean interwell and interassay coefficients of variation were 9.7 and 8.9%, respectively. Average recovery by the method from milk spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml was 187, 113, 107, 110, and 112%, respectively, with a mean recovery of 126%. Mean interwell and interassay coefficients of variation were 14.5 and 9.1%, respectively. Zearalenone was not detectable in 12 commercial milk samples assayed by the tandem method.

scholarly journals ELISA for Aging Biomarkers Induced by Telomere Dysfunction in Human Plasma

2010 ◽  
Vol 2010 ◽  
pp. 1-4 ◽  
Author(s):  
Hong Jiang ◽  
Wenqing Chen ◽  
Lihui Qu ◽  
Ying Chen ◽  
Qiang He ◽  
...  
Keyword(s):  
Telomere Length ◽  
Bone Marrow Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antimicrobial Protein ◽  
Human Aging ◽  
Late Generation ◽  
Direct Elisa ◽  
Fresh Plasma ◽  
The Relationship ◽  
Frozen Plasma

Background. We identified cathelicidin related antimicrobial protein (CRAMP) secreted from telomere dysfunctional bone marrow cells of late generation telomerase knockout mice (G4mTerc−/−), increased in blood and various tissues. It can represented human aging and disease. The main aim of this study is to investigate the sensitive direct enzyme-linked immunosorbent assay (ELISA) method to analyze the human aging and disease in plasma and the detailed methods to quantify the direct ELISA of these aging biomarkers.Methods. Telomere lengths of 50 healthy persons are measured with real-time PCR in blood cells. Plasma samples from all subjects are analyzed using direct ELISA.Results. From 25 years old person to 78 years, the telomere length becomes shorter during aging. In blood plasma, the expression levels of CRAMP increases during human aging. There is the reverse correspondence between the telomere length and the plasma CRAMP level. We also find that the fresh plasma, the frozen plasma which thawed less than 3 times, and the plasma kept in the room temperature less than 3 hours are better for the ELISA analyze of CRAMP in the plasma.Conclusion. This CRAMP ELISA could become a powerful tool for investigating the relationship between human aging and telomere length shortening.

Capture immunoassay for the diagnosis of bovine mastitis using a monoclonal antibody to polymorphonuclear granulocytes

1992 ◽  
Vol 59 (2) ◽  
pp. 123-133 ◽  
Author(s):  
Catherine A. O'Sullivan ◽  
Patrick J. Joyce ◽  
Teresa Sloan ◽  
Alan G. Shattock
Keyword(s):  
Monoclonal Antibody ◽  
Somatic Cell ◽  
Bovine Milk ◽  
Bovine Mastitis ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Assay Sensitivity ◽  
Milk Samples ◽  
Cell Counts ◽  
Direct Elisa

SummaryA direct capture enzyme-linked immunosorbent assay (ELISA) was developed to measure elevated polymorphonuclear granulocyte (PMN) antigens using horseradish peroxidase (EC 1.11.1.7) conjugated rabbit polyclonal anti-PMN antisera and a monoclonal antibody specific for PMN cells. Optical densities obtained in the ELISA were used to predict the cell counts of milk samples. Predicted counts were not significantly different from actual somatic cell counts (SCC). In a total of 156 bovine milk samples the correlation coefficient between somatic cell counting, taking > 500000 cells/ml as being indicative of mastitis, and the assay was 0·94, yielding an assay sensitivity of 95·2% and a specificity of 97·3%. In further trials the ELISA could detect elevated PMN antigens in milk with SCC as low as 100000 cells/ml. The results indicate that the monoclonal antibody based direct ELISA has excellent potential in the detection and determination of bovine mastitis.

Use of the enzyme-linked immunosorbent assay (ELISA) in the detection of Rhizobium both in culture and from root nodules of soybeans and cowpeas

1985 ◽  
Vol 31 (6) ◽  
pp. 524-528 ◽  
Author(s):  
S. Asanuma ◽  
G. Thottappilly ◽  
A. Ayanaba ◽  
V. Ranga Rao
Keyword(s):  
Silica Gel ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Root Nodules ◽  
Enzyme Linked Immunosorbent Assay ◽  
Double Antibody ◽  
Laboratory Conditions ◽  
Direct Elisa ◽  
Rhizobium Sp

The enzyme-linked immunosorbent assay (ELISA) was used to detect Rhizobium sp. in the "cowpea" group and R. japonicum both in culture and from nodules of glasshouse and field-grown plants. Double antibody sandwich (direct ELISA) and indirect ELISA were found to be equally sensitive in detecting rhizobia under controlled laboratory conditions. It was found that nodules preserved by freezing or drying over silica gel were equally good. No loss in sensitivity was detected when nodules were crushed directly in the microplates.

scholarly journals An Immunoaffinity Column for the Determination of Peanut Protein in Chocolate

1999 ◽  
Vol 82 (3) ◽  
pp. 666-668 ◽  
Author(s):  
Harvey W Newsome ◽  
Michael Abbott
Keyword(s):  
Detection Limit ◽  
Immunoaffinity Chromatography ◽  
Polyclonal Antiserum ◽  
Peanut Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoaffinity Column ◽  
Rabbit Polyclonal Antiserum ◽  
The Matrix ◽  
Direct Elisa

Abstract An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2–3.2 μg/g of peanut protein averaged 77% (range, 72–84%), and the minimum detection limit was 0.1 μg/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.

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