Sensitive and rapid colorimetric immunoenzymometric assay of ferritin in biological samples

1990 ◽  
Vol 36 (6) ◽  
pp. 837-840 ◽  
Author(s):  
G A Ramm ◽  
L R Duplock ◽  
L W Powell ◽  
J W Halliday
Keyword(s):  
Detection Limit ◽  
Shelf Life ◽  
Biological Samples ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Average Recovery ◽  
Specific Antibodies ◽  
Chlorophenol Red ◽  
Liver Ferritin ◽  
Beta Galactosidase

Abstract We describe a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying ferritin in human and rat biological fluids. We used chlorophenol red beta-D-galactopyranoside as the colorimetric substrate of beta-galactosidase (EC 3.2.1.23), which is coupled to specific antibodies to either human or rat liver ferritin. The assay is sensitive (detection limit for human assay = 0.58 micrograms/L and for rat assay = 0.37 micrograms/L), accurate (average recovery for human assay = 93% and for rat assay = 92%), and precise (total CVs for human assay = 2.3-12.2% and for rat assay = 5.6-11.3%). The results correlated well with those of an established immunoradiometric technique (r = 0.99691). This assay has a prolonged shelf-life, is inexpensive, and utilizes a stable colorimetric substrate that requires relatively short incubation.

Immunoassays for Analysis of Mycotoxins

1984 ◽  
Vol 47 (7) ◽  
pp. 562-569 ◽  
Author(s):  
FUN SUN CHU
Keyword(s):  
Ochratoxin A ◽  
Aflatoxin B1 ◽  
Recent Progress ◽  
Biological Fluids ◽  
Aflatoxin M1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Protein Conjugates ◽  
Advantages And Disadvantages ◽  
The Past

During the past few years, several laboratories have prepared specific antibodies against aflatoxins B1, M1, B2a and Q1, ochratoxin A, T-2 toxin, and zearalenone. These antibodies were obtained from rabbits after immunizing with various mycotoxin-protein conjugates. With the availability of these antibodies, specific, simple and sensitive radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) procedures for monitoring mycotoxins and their metabolites in foods, feeds and body fluids have been developed. In this review, details are presented for the preparation of antibodies and the application of RIA and ELISA to determine aflatoxins B1 and M1, ochratoxin A and T-2 toxin in corn, peanuts, milk and other biological fluids. The sensitivity of ELISA for analysis of these mycotoxins in foods varied from 0.1 μg/L for aflatoxin M1 in milk to 5 μg/kg of aflatoxin B1 in peanuts. The advantages and disadvantages of ELISA for monitoring mycotoxins in foods and feeds are discussed. In addition, a description of recent progress on simplified clean-up procedures which may increase the sensitivity of immunoassays is presented.

Determination of Captan in Water, Peaches, and Apple Juice by a Magnetic Particle-Based Immunoassay

1994 ◽  
Vol 77 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Jeanne A Itak ◽  
Michele Y Selisker ◽  
David P Herzog ◽  
James R Fleeker ◽  
Edward R Bogus ◽  
...  
Keyword(s):  
Detection Limit ◽  
Apple Juice ◽  
Magnetic Particle ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Electron Capture Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extraction Column ◽  
Average Recovery ◽  
Step Procedure

Abstract A magnetic particle-based enzyme-linked immunosorbent assay (EUSA) was used to determine captan residues in water, peaches, and apple juice. Precision studies of captan-spiked water samples showed coefficients of variation (CVs) of 24% at 0.1 ppm and 15% at 2.0 ppm. Average recoveries from water were 110–118%, with an estimated sensitivity of 0.01 ppm for captan. Low cross-reactivity was seen with the related phthalimide fungicides capta-fol and folpet. A least-detectable dose (LDD) of 1 ppm was estimated for captafol in the immunoassay; an LDD of 8.6 ppm was estimated for folpet. Apple juice samples were analyzed by a 3-step procedure: the sample was concentrated on a Cis bonded silica solid-phase column, the eluate was diluted in buffer, and the sample was assayed by ELISA. The detection limit in apple juice was estimated to be 0.1 ppm on the basis of the detection limit observed in water and the required calculation factors; the average recovery was 115%. Acetone extraction, Cis bonded silica solid-phase extraction column, and the immunoassay were used consecutively to analyze peach samples. The detection limit in peaches was estimated to be 0.15 ppm on the basis of the detection limit observed in water and the required calculation factors. Captan residues were found in all but one of the peach samples and ranged from 0.15 to 1.12 ppm (|ig/g). Recovery of added captan from peaches averaged 98% for both spike levels tested. Results of analysis of captan in peaches by immunoassay and gas chromatography with electron capture detection (GC/ECD) were compared. GC/ECD gave an average recovery of 75%; immunoassay gave an average recovery of 112%.

scholarly journals Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.

1996 ◽  
Vol 40 (11) ◽  
pp. 2592-2597 ◽  
Author(s):  
F S Buckner ◽  
C L Verlinde ◽  
A C La Flamme ◽  
W C Van Voorhis
Keyword(s):  
Trypanosoma Cruzi ◽  
Screening Method ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Toxicity ◽  
Microtiter Plates ◽  
Chlorophenol Red ◽  
New Compounds ◽  
Beta Galactosidase

A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli beta-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red beta-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 microM in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with beta-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

scholarly journals Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples

Antioxidants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1160
Author(s):  
Shingo Kasamatsu ◽  
Yuki Kakihana ◽  
Taisei Koga ◽  
Hisashi Yoshioka ◽  
Hideshi Ihara
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Hydrogen Sulfide ◽  
Biological Samples ◽  
Biological Fluids ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biological Processes ◽  
Pathological Conditions ◽  
Colorimetric Immunoassay ◽  
Hybridoma Cell Lines

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

scholarly journals PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 303
Author(s):  
Wei-Ting Hsu ◽  
Chia-Yu Chang ◽  
Chih-Hsuan Tsai ◽  
Sung-Chan Wei ◽  
Huei-Ru Lo ◽  
...  
Keyword(s):  
Recombinant Baculovirus ◽  
Immunocytochemical Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Perfect Agreement ◽  
Specific Antibodies ◽  
Diarrhea Virus ◽  
Serological Studies ◽  
A Cell ◽  
Infected Insect

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

Liquid Chromatographic Determination of Sulfamoyldapsone in Swine Tissues

1987 ◽  
Vol 70 (6) ◽  
pp. 1031-1032
Author(s):  
Yuuko S Endoh ◽  
Ryozo Yamaoka ◽  
Nobuo Sasaki
Keyword(s):  
Detection Limit ◽  
Quantitative Determination ◽  
Chromatographic Determination ◽  
Alumina Column ◽  
Average Recovery ◽  
Diaminodiphenyl Sulfone ◽  
Alumina Column Chromatography ◽  
Liquid Chromatographic Determination ◽  
Liquid Chromatographic

Abstract A liquid chromatographic (LC) method is described for the quantitative determination of sulfamoyldapsone (2-sulfamoyl-4,4'-diaminodiphenyl sulfone) in swine muscle, liver, kidney, and fat. Sulfamoyldapsone was extracted from tissues with acetonitrile saturated with n-hexane. The extract was washed with n-hexane saturated with acetonitrile, concentrated, and cleaned up by alumina column chromatography. Sulfamoyldapsone was separated on an ODS column by using acetonitrile-methanol-water (6 + 18 + 76) and was detected at 292 nm. Overall average recovery of sulfamoyldapsone added to tissues at levels of 0.1 and 0.5 /μg/g was 93.3% ± 6.0. Detection limit was 0.02 μg/g in these tissues.

scholarly journals High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Markus H. Kainulainen ◽  
Eric Bergeron ◽  
Payel Chatterjee ◽  
Asheley P. Chapman ◽  
Joo Lee ◽  
...  
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Negative Results ◽  
A Value ◽  
Elisa Testing ◽  
The World ◽  
Antibody Titers ◽  
Qualitative Assay ◽  
Homogeneous Assay ◽  
Asymptomatic Infections

AbstractSARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

scholarly journals Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

1992 ◽  
Vol 34 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Eide Dias Camargo ◽  
Paulo Mutuko Nakamura ◽  
Adelaide José Vaz ◽  
Marcos Vinícius da Silva ◽  
Pedro Paulo Chieffi ◽  
...  
Keyword(s):  
Low Cost ◽  
Clinical Signs ◽  
Toxocara Canis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Specific Antibodies ◽  
Normal Individuals ◽  
Before And After ◽  
Dot Elisa ◽  
Stability Time

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

scholarly journals Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation

2005 ◽  
Vol 49 (9) ◽  
pp. 3770-3775 ◽  
Author(s):  
Catherine François ◽  
Isabelle Bernard ◽  
Sandrine Castelain ◽  
Bryan Charleston ◽  
Martin D. Fray ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Biological Samples ◽  
Pegylated Interferon ◽  
Biologically Active ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hepatitis C Virus Infection ◽  
Promoter Activation ◽  
Antiviral Protein

ABSTRACT Alpha interferons (α-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of α-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of α-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by α-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other α-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.

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