Rapid and Sensitive Screening of Sulfamethazine in Porcine Urine with an Enzyme-Linked Immunosorbent Assay and a Field-Portable Immunofiltration Assay

2002 ◽  
Vol 65 (5) ◽  
pp. 820-827 ◽  
Author(s):  
PATRICIA CRABBE ◽  
CARLOS VAN PETEGHEM
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Visual Detection ◽  
Close Contact ◽  
Negative Control ◽  
Mass Spectrometry Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blue Color ◽  
Nylon Membrane ◽  
Spectrometry Analysis

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra-and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ–horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered “negative,” i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered “positive,” i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

scholarly journals Easy Enzyme-Linked Immunosorbent Assay for Spectinomycin in Chicken Plasma

1996 ◽  
Vol 79 (2) ◽  
pp. 426-430 ◽  
Author(s):  
Touichi Tanaka ◽  
Hideharu Ikebuchi ◽  
Jun-Ichi Sawada ◽  
Mariko Okada ◽  
Yasumasa Kido
Keyword(s):  
Detection Limit ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Immunosorbent Assay ◽  
Bovine Serum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carrier Protein ◽  
Bovine Serum Albumin Conjugate

Abstract An easy, sensitive, competitive indirect enzyme- linked immunosorbent assay (CI-ELISA) for specti nomycin in chicken plasma was developed. Preparation of a spectinomycin-bovine serum albumin conjugate in which the hapten is linked to the carrier protein through the C-4 position is described. Antibodies raised against antigens in rabbits had excellent specificity for spectinomycin, exhibiting a cross-reactivity of 44.0% with dihydrospectinomy-cin and 13.8% with tetrahydrospectinomycin. No cross-reactivity was observed with other antibiotics. The detection limit of the CI-ELISA was 2 ng/mL (equivalent into 40 ng/mL undiluted chicken plasma) spectinomycin. Known amounts (0.1-100 μg/mL) of spectinomycin were added to chicken plasma and then analyzed. Average recoveries were 97-110%. This procedure may be used without prior extraction of samples.

scholarly journals Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

2017 ◽  
Vol 8 ◽  
pp. 244-253 ◽  
Author(s):  
Paula Ciaurriz ◽  
Fátima Fernández ◽  
Edurne Tellechea ◽  
Jose F Moran ◽  
Aaron C Asensio
Keyword(s):  
Gold Nanoparticles ◽  
Detection Limit ◽  
Immunosorbent Assay ◽  
Wheat Gluten ◽  
Diagnostic Technique ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rabbit Igg ◽  
Enzyme Molecules ◽  
Main Components ◽  
Limit Sensitivity

The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

scholarly journals Improved Serodiagnostic Performance for Lyme Disease by Use of Two Recombinant Proteins in Enzyme-Linked Immunosorbent Assay Compared to Standardized Two-Tier Testing

2017 ◽  
Vol 55 (10) ◽  
pp. 3046-3056 ◽  
Author(s):  
Gary L. Bradshaw ◽  
R. Kelley Thueson ◽  
Todd J. Uriona
Keyword(s):  
Lyme Disease ◽  
Recombinant Proteins ◽  
Immunosorbent Assay ◽  
Negative Control ◽  
Full Length ◽  
Test Method ◽  
Stage I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Content Type

ABSTRACTThe most reliable test method for the serological confirmation of Lyme disease (LD) is a 2-tier method recommended by the CDC in 1995. The first-tier test is a low-specificity enzyme-linked immunosorbent assay (ELISA), and the second-tier tests are higher-specificity IgG and IgM Western blots. This study describes the selection of twoBorrelia burgdorferirecombinant proteins and evaluation of their performance in a simple 1-tier test for the serological confirmation of LD. These two proteins were generated from (i) the full-lengthdbpAgene combined with the invariable region 6 of thevlsEgene (DbpA/C6) and (b) the full-lengthospCgene (OspC). The expressed DbpA/C6 and OspC proteins were useful in detecting anti-BorreliaIgG and IgM antibodies, respectively. A blind study was conducted on a well-characterized panel of 279 human sera from the CDC, comparing ELISAs using these two recombinant antigens with the 2-tier test method. The two methods (DbpA/C6-OspC versus 2-tier test) were equivalent in identifying sera from negative-control subjects (99% and 100% specificity, respectively) and in detecting stage II and III LD patient sera (100% and 100% sensitivity). However, the DbpA/C6-OspC ELISA was markedly better (80% versus 63%) than the 2-tier test method in detecting anti-Borreliaantibodies in stage I LD patients. The findings suggest that these antigens could be used in a simple 1-tier ELISA that is faster to perform, easier to interpret, and less expensive than the 2-tier test method and which is better at detectingBorrelia-specific antibodies in sera from patients with stage I LD.

scholarly journals Combined Use of Enzyme-Linked Immunosorbent Assay and Flow Cytometry To Detect Antibodies to Trypanosoma cruzi in Domestic Canines in Texas

2004 ◽  
Vol 11 (2) ◽  
pp. 313-319 ◽  
Author(s):  
Sean V. Shadomy ◽  
Stephen C. Waring ◽  
Cynthia L. Chappell
Keyword(s):  
Flow Cytometry ◽  
Trypanosoma Cruzi ◽  
Immunosorbent Assay ◽  
Negative Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Harris County ◽  
Cross Sectional ◽  
Survey Results ◽  
Flow Cytometry Detection ◽  
Negative Controls

ABSTRACT Canines may be sentinels and/or reservoirs for human Trypanosoma cruzi exposures. This study adapted a method originally designed for human diagnostics to detect serum immunoglobulin G to T. cruzi in canines. The method combined an enzyme-linked immunosorbent assay (ELISA) for screening and flow cytometry detection of anti-live trypomastigote antibodies (ALTA) for confirmation. The assays were optimized by using known positive and negative control canine sera, and cutoff values were established. The ELISA and ALTA assay easily distinguished between reactive (positive controls) and nonreactive (negative controls) sera and were used to test sera collected in a cross-sectional seroprevalence survey of 356 domestic canines from Harris County, Tex., and the surrounding area. Fifty-three (14.9%) of 356 asymptomatic canines in the survey were positive by ELISA, and 5 (1.4%) were confirmed positive with the ALTA assay, with an additional 4 (1.1%) canines classified as “suspect positive.” Thus, the overall prevalence of T. cruzi antibodies in this population was 2.6%. This is the first U.S. study to use the combination of ELISA and ALTA to detect serum antibodies to T. cruzi and the first report of the prevalence of T. cruzi infection in domestic canines in the Houston, Tex. (Harris County), region. Our results demonstrate that the combination of ELISA and ALTA has been successfully adapted for use in testing canines for serological evidence of T. cruzi infection. Seroprevalence survey results suggest that T. cruzi antibody-positive domestic canines in the peridomestic setting are present in the Houston, Tex., region and further suggest that T. cruzi is enzootic in the region.

Indirect Enzyme-Linked Immunosorbent Assay for Saxitoxin in Shellfish

1985 ◽  
Vol 68 (1) ◽  
pp. 13-16 ◽  
Author(s):  
Fun S Chu ◽  
Titan S L Fan
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Indirect Elisa ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromogenic Substrate ◽  
Rabbit Igg ◽  
Lower Detection ◽  
Peroxidase Conjugate

Abstract An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of saxitoxin (STX). Antibodies against STX were demonstrated in rabbits 5 weeks after immunizing with STX-bovine serum albumin (STX-HCHO-BSA). In the ELISA, STX-HCHO-BSA or polylysine-STX was coated onto the microtiter plate, followed by incubation with standard toxin and anti-STX antibody. The amount of antibody bound to the solid phase was determined by incubation with goat anti-rabbit IgG peroxidase conjugate and a reaction with chromogenic substrate. Competitive indirect ELISA revealed that the antiserum did not cross-react with either carbamoyl-neo-STX-suIfate or tetrodotoxin. The antibodies for STX cross-reacted with decarbamoyl- STX and neo-STX about 56% and 16% as much as they did with STX, respectively. The lower detection limits for STX, decarbamoyl-STX, and neo-STX in this sytem were about 25, 45, and 156 pg per assay, respectively. When STX added to clams or mussels was assayed, the detection limit for STX was about 50-100 ppb, and recoveries were in the range of 86.8-107%.

Enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone in dried blood spotted on filter paper.

1987 ◽  
Vol 33 (6) ◽  
pp. 761-764 ◽  
Author(s):  
M Maeda ◽  
H Arakawa ◽  
A Tsuji ◽  
Y Yamagami ◽  
A Isozaki ◽  
...  
Keyword(s):  
Detection Limit ◽  
Immunosorbent Assay ◽  
Filter Paper ◽  
Cost Effective ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adrenal Hyperplasia ◽  
Bovine Serum Albumin Conjugate ◽  
Enzyme Conjugate ◽  
Sensitivity Specificity ◽  
Microwell Plate

Abstract In this rapid, cost-effective, enzyme-linked immunosorbent assay for 17 alpha-hydroxyprogesterone (17-OHP) eluted from dried blood spotted on filter paper, second antibody is coated onto the microwell plate and horseradish peroxidase (EC 1.11.1.7) is the label enzyme. Antiserum to 17-OHP was prepared by using 4-(2-carboxymethylthio)-17-OHP-bovine serum albumin conjugate as immunogen. Enzyme conjugate was prepared from 4-(2-carboxymethylthio)-17-OHP and peroxidase. The blood spots are assayed in the microwells without extraction or centrifugation steps. The detection limit of the assay is 1 microgram/L, equivalent to 3.5 pg (10.6 fmol) per disc. Intra- and interassay CVs at two steroid concentrations (7.38 and 22.79 micrograms/L) ranged from 3.74 to 11.90% (n = 5), and 9.49 and 9.83% (n = 5), respectively. Results correlated well (r = 0.91) with those of a fluorescence enzyme immunoassay. The sensitivity, specificity, and precision of this method make it potentially useful in the mass screening of neonates for congenital adrenal hyperplasia.

scholarly journals Role of lgtC in Resistance of Nontypeable Haemophilus influenzae Strain R2866 to Human Serum

2006 ◽  
Vol 74 (11) ◽  
pp. 6226-6235 ◽  
Author(s):  
Alice L. Erwin ◽  
Simon Allen ◽  
Derek K. Ho ◽  
Paul J. Bonthius ◽  
Justin Jarisch ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Human Serum ◽  
Mass Spectrometry Analysis ◽  
Start Codon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Resistance ◽  
Nontypeable Haemophilus Influenzae ◽  
Spectrometry Analysis ◽  
Nthi Strain ◽  
High Level

ABSTRACT We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame (“off”) in most colonies of R3392 but in frame with its start codon (“on”) in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galα1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

scholarly journals Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay

2009 ◽  
Vol 16 (11) ◽  
pp. 1665-1674 ◽  
Author(s):  
Maja Neiman ◽  
Carl Hamsten ◽  
Jochen M. Schwenk ◽  
Göran Bölske ◽  
Anja Persson
Keyword(s):  
Immunosorbent Assay ◽  
False Negative ◽  
Negative Control ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Contagious Bovine Pleuropneumonia ◽  
Mycoplasma Mycoides ◽  
Small Colony ◽  
Suspension Bead Array ◽  
Selection Of

ABSTRACT A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10−6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

scholarly journals Streptococcus mutans Surface α-Enolase Binds Salivary Mucin MG2 and Human Plasminogen

2004 ◽  
Vol 72 (11) ◽  
pp. 6748-6752 ◽  
Author(s):  
Jingping Ge ◽  
Diana M. Catt ◽  
Richard L. Gregory
Keyword(s):  
Streptococcus Mutans ◽  
Mass Spectrometry Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ionization Time ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Salivary Mucin ◽  
Transmission Electron ◽  
A Cell ◽  
Human Plasminogen

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis identified enolase as a cell surface component of Streptococcus mutans, which was confirmed by enzyme-linked immunosorbent assay, Western blotting, and transmission electron microscopy. Surface enolase was demonstrated to bind to human plasminogen and salivary mucin MG2. The results suggested a role for enolase in S. mutans attachment, clearance, or breach of the bloodstream barrier.

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