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%.

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.

An Indirect Enzyme-Linked Immunosorbent Assay for T-2 Toxin in Biological Fluids

1984 ◽  
Vol 47 (12) ◽  
pp. 964-967 ◽  
Author(s):  
TITAN S. L. FAN ◽  
GUANG S. ZHANG ◽  
F. S. CHU
Keyword(s):  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Microtiter Plate ◽  
Reversed Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sample Extract ◽  
Rabbit Igg ◽  
Cleanup Procedure ◽  
Significant Interference ◽  
Urine Serum

An indirect enzyme-linked immunosorbent assay (ELISA) which can detect 0.2 to 1 ng of T-2 toxin per ml in urine, serum and milk was developed. T-2 hemisuccinate was conjugated to polylysine which was then coated to a microtiter plate and incubated with rabbit anti-T-2 antibody and sample extract. The amount of anti-T-2 antibody bound to the plate was then determined by reaction with goat anti-rabbit IgG-peroxidase complex and by subsequent reaction with the substrate. Samples spiked with T-2 toxin were subjected to a simple cleanup procedure by passing them through a reversed-phase Sep-Pak catridge (C18). The recoveries of tritiated T-2 toxin added to the urine, serum and milk samples were between 71 to 90% after the cleanup step. In the ELISA, significant interference was observed when more than 5 μl of sample, without cleanup treatment, were used in each analysis. After cleanup, extracts equivalent to 50 μl of serum, urine or milk per well did not significantly interfere with the assay. The recoveries of T-2 toxin added to serum (1 to 10 ng/ml), urine (0.2 to 10 ng/ml) and milk (0.2 to 10 ng/ml) after cleanup treatment as determined by the indirect ELISA were found to be 51 to 82%, 73 to 82% and 80 to 83%, respectively.

Enzyme-Linked Immunosorbent Assay of Aflatoxin Bi in Naturally Contaminated Corn and Cottonseed

1986 ◽  
Vol 69 (5) ◽  
pp. 904-907 ◽  
Author(s):  
Bhanu P Ram ◽  
L Patrick Hart ◽  
Odette L Shotwell ◽  
James J Pestka
Keyword(s):  
Correlation Coefficient ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extraction Solvent ◽  
Sample Extract ◽  
Coefficients Of Variation ◽  
Layer Chromatography ◽  
Duplicate Sample

Abstract Naturally contaminated corn and cottonseed samples were screened for aflatoxin B1 (AFB1) by a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were blended 5 min in an extraction solvent of methanol-water-dimethylformamide (70 + 29 + 1) and filtered. Filtrates were assayed by direct competition between AFBi in the corn and cottonseed samples and AFB1-peroxidase conjugate for binding to specific antibody adsorbed to a solid phase microtiter plate. Standard curves prepared using the extract of AFB1-free corn and cottonseed samples, and extraction solvent only, showed negligible interference by the sample extract in the performance of ELISA. The AFBi content in corn and dehulled cottonseed samples as determined by ELISA ranged from 7 to 422 μg/kg and 7 to 3258 μg/kg, respectively. When ELISA estimates of AFB1 in corn were compared with values obtained by thin layer chromatography (CB method), the correlation coefficient (n = 10) was 0.95. Average interassay and subsample coefficients of variation for ELISA in corn were 21.4 and 22.0%, respectively. When ELISA estimates of AFB1 in cottonseed were compared with values obtained by liquid chromatography (Pons method), the correlation coefficient (n = 15) was 0.96. Using this ELISA, 36 duplicate sample extracts can be screened for AFB1 in less than 2 h.

Prostatic inhibin-like peptide quantified in urine of prostatic cancer patients by enzyme-linked immunosorbent assay.

1989 ◽  
Vol 35 (7) ◽  
pp. 1376-1379 ◽  
Author(s):  
T R Teni ◽  
A H Bandivdekar ◽  
A R Sheth ◽  
N A Sheth
Keyword(s):  
Cancer Patients ◽  
Immunosorbent Assay ◽  
Prostatic Cancer ◽  
Solid Phase ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Recovery ◽  
The Status ◽  
Polystyrene Microtiter Plate ◽  
The Mean

Abstract This is a highly specific enzyme-linked immunosorbent assay (ELISA) for measuring prostatic inhibin-like peptide (PIP) in urine, in which we use penicillinase (EC 3.5.2.6) conjugated with PIP and, as solid phase, a polystyrene microtiter plate. We used this ELISA to measure PIP in 24-h urine specimens from men with prostatic cancer (PCa) and from age-matched controls. For prostatic cancer patients the mean +/- SEM urinary PIP of 36.1 +/- 5 micrograms/24 h was significantly (P less than 0.001) lower than the mean of 127.1 +/- 9 micrograms/24 h for the age-matched controls. PIP values for 30 samples measured by both ELISA and RIA correlated well (r = 0.985). We could detect as little as 1.56 ng of PIP in a sample. Analytical recovery of added PIP ranged from 91% to 104%. Mean CVs were 8.9% within-assay and 12.7% between-assay. We believe that this ELISA will be useful in assessing the status of PIP in men with normal and diseased prostates and in examining the function of the hypothalamus-pituitary-prostate axis.

A New Technique in Quantitative Immunohematology: Solid-Phase Kinetic Enzyme-Linked Immunosorbent Assay

Vox Sanguinis ◽  
1983 ◽  
Vol 45 (6) ◽  
pp. 440-448 ◽  
Author(s):  
S. Spitalnik ◽  
J. Cowles ◽  
M.T. Cox ◽  
D. Baker ◽  
J. Holt ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Solid Phase ◽  
New Technique ◽  
Enzyme Linked Immunosorbent Assay ◽  
A New Technique

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

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