Enzyme-Linked Immunosorbent Assay for T-2 Toxin Metabolites in Urine

1989 ◽  
Vol 72 (2) ◽  
pp. 345-348
Author(s):  
Rachel C Lee ◽  
Ru-Dong Wei ◽  
Fun S Chu
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Portion ◽  
Standard Curve ◽  
Sample Extract ◽  
Direct Elisa ◽  
Minimum Detection Level ◽  
Elisa Plate

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate- bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15- triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2- 4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol- 4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'- OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.

Enzyme-Linked Immunosorbent Assay for Microcystins in Blue-Green Algal Blooms

1990 ◽  
Vol 73 (3) ◽  
pp. 451-456 ◽  
Author(s):  
Fun S Chu ◽  
Xuan Huang ◽  
R D Wei
Keyword(s):  
Liquid Chromatography ◽  
Immunosorbent Assay ◽  
Algal Blooms ◽  
Ammonium Bicarbonate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Standard Curve ◽  
Green Algal ◽  
Recovery Study ◽  
Minimum Detection Level ◽  
Elisa Plate

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) for the freshwater blue-green algal toxin mlcrocystln (MCYST) In algae and water was developed. The assay Involves coating antl-MCYST-variant leuclne-arglnine (LR) antibody to the ELISA plate and the use of MCYST-LRperoxidase as the enzyme marker. The linear portion of the standard curve for MCYST in phosphate buffer containing saline (PBS) was 0.5-10.0 ng/mL (25-500 pg/assay). The minimum detection level for MCYST-LR was 0.20 ng/mL (10 pg/assay). Contaminated water could be directly used In the ELISA. The overall analytical recoveries for MCYST-LR added to water at levels of 1-20 ng/mL was 83.4%. For analysis of cellular MCYST, the toxin was first extracted from the algae with 0.1M ammonium bicarbonate, diluted with PBS to less than 0.5 mg dried algae/mL (<5.0 mg wet welght/mL) and directly used in the ELISA. C-18 reverse-phase Sep-Pak cartridges effectively adsorbed MCYST from the toxln-containlng solutions. The toxin could be recovered from the cartridge by elutlng with 60% methanol. Using this approach, an algae extract that was relatively free of MCYST was prepared and was used in a recovery study. The overall analytical recovery of MCYST added to the algae extract In the range of 0.25-20 ppm was 83% with a coefficient of variation of 11.9%. The detection limit for MCYST In dried algae was about 0.25-0.5 pg/g (0.25-0.5 ppm) lyophlllzed algae sample. This method was applied for the analysis of several naturally occurring algal blooms. Limited samples were also analyzed for MYCST by liquid chromatography. ELISA data were in general agreement with those obtainedby liquid chromatography. MCYST concentrations from 0.006 to 2.9 fig/g (6 to 2900 ppb) and from 26 to 5200 /ig/g (26 ppm to 5200 ppm) were found In water and algae (dried weight), respectively

Determination of Zearalenone and Related Metabolites in Porcine Urine by Modified Enzyme-Linked Immunosorbent Assay

1990 ◽  
Vol 73 (1) ◽  
pp. 65-68
Author(s):  
Ormond A Macdougald ◽  
Andrew J Thulin ◽  
James J Pestka
Keyword(s):  
Immunosorbent Assay ◽  
Excretion Rate ◽  
Urine Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Portion ◽  
Average Recovery ◽  
Standard Curve ◽  
Coefficients Of Variation ◽  
Peroxidase Conjugate

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) was modified for the determination of zearalenone and zearalenols in porcine urine. In the modified procedure, standard or unknown concentrations of zearalenone were added to all wells, followed by rapid addition of zearalenonehorseradlsh peroxidase conjugate, whereas in previous methods, zearalenone and zearalenone-horseradish peroxidase were mixed and then added to microtiter wells. The modification increased the number of urine samples that could be analyzed per assay. The linear portion of the modified ELISA standard curve covered the range of 10-500 ng zearalenone/mL. Average recovery of zearalenone from spiked urine was 91,101,95,107,136,112, and 120% for 1, 10, 25, 50, 100, 250, and 500 ng zearalenone/mL urine, respectively. Mean within-assay coefficient of variation for each concentration of zearalenone in 5 standard curves was 5.95%. Between-assay coefficients of variation for concentrations of zearalenone equivalents in lower and upper regions of the standard curve were 10.9% (n = 18) and 8.8% (n = 20), respectively. Analysis of urine samples showed that a gilt excreted 5.3% of ingested zearalenone in the 8 h following ingestion, with an average excretion rate of about 100 /ug zearalenone equivalents/h.

Development and Characterization of Anti-Estradiol Polyclonal Antibody

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang
Keyword(s):  
Polyclonal Antibody ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Absorbance ◽  
Standard Curve ◽  
Ic50 Value ◽  
New Zealand White Rabbits

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

Diarrhetic Shellfish Poisoning: Evaluation of Enzyme-Linked Immunosorbent Assay Methods for Determination of Dinophyslstoxin-2

1995 ◽  
Vol 78 (6) ◽  
pp. 1403-1407 ◽  
Author(s):  
Eoin P Carmody ◽  
Kevin J James ◽  
Seán S Kelly
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Regulatory Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Diarrhetic Shellfish Poisoning ◽  
Elisa Method ◽  
Multiple Samples ◽  
Liquid Chromatographic

Abstract Dinophysistoxin-2 (DTX-2), an isomer of okadaic acid (OA), recently has been found in Irish waters. DTX-2 was the predominant toxin during prolonged infestations in cultivated mussels along the southwest coast of Ireland. Substantial variations in toxin levels may exist both horizontally and vertically in the water column. The need to take multiple samples and the ethical concern about the use of mammals for routine quality control of shellfish prompted examination of 2 commercially available enzyme-linked immunosorbent assay (ELISA) methods, designed to detect OA, for determination of both OA and DTX-2. One ELISA method (DSPCheck, Sceti Co. Ltd., (Tokyo, Japan) showed good cross-reactivity (40 ± 5%) with standard DTX-2. This study showed that both ELISA methods show good correlation with the liquid chromatographic analysis of 9-anthryldiazomethane derivatives when OA is the predominant toxin present. The sensitivity was also good for OA determination using both methods, which allowed toxin measurement at 10 ng/mL (0.5 ng/well). This level is equivalent to 0.03 μg/g mussel meat. Blank mussel samples spiked with DTX-2 standards gave a good linear correlation (r = 0.997) with this ELISA method when toxin levels were 0.03-0.3 μg/g mussel meat. This range is appropriate for regulatory control of diarrhetic shellfish poisoning.

scholarly journals Single-Laboratory Validation of the Biosense Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA) for Determination of Domoic Acid Toxins in Shellfish

2007 ◽  
Vol 90 (4) ◽  
pp. 1000-1010 ◽  
Author(s):  
Hans Kleivdal ◽  
Sven-Inge Kristiansen ◽  
Mona V Nilsen ◽  
Lyn Briggs
Keyword(s):  
Immunosorbent Assay ◽  
Domoic Acid ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Relative Standard

Abstract Method validation was conducted for an enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins, known to give amnesic shellfish poisoning (ASP) symptoms, in shellfish. The calibration curve range of the assay is approximately 10260 pg/mL, with a dynamic working range for DA toxins in shellfish from 0.01 to at least 250 mg/kg. The ASP ELISA showed no significant cross-reactivity to structural analogs, and proved to be robust to deliberate alterations of the optimal running conditions. The shellfish matrix effects observed with mussels, oysters, and scallops were eliminated by diluting shellfish extracts 1:200 prior to analysis, leading to a limit of detection at 0.003 mg/kg. Thirteen blank shellfish homogenates were spiked with certified mussel material containing DA to levels in the range of 0.125 mg DA/kg, and analyzed in quadruplicate on 3 different days. The relative standard deviation (RSD) under intra-assay repeatability conditions ranged from 6.5 to 13.1%, and under interassay repeatability conditions the RSD ranged from 5.7 to 13.4%, with a mean value of 9.3%. The recoveries ranged from 85.5 to 106.6%, with a mean recovery of 102.2%. A method comparison was conducted with liquid chromatography with ultraviolet detection, using naturally contaminated scallop samples (n = 27) with DA levels at 0244 mg/kg. The overall correlation coefficient was 0.960 and the slope of the regression was 1.218, indicating a good agreement between the methods.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay Based on the Multiepitope Peptide for the Synchronous Detection of Staphylococcal Enterotoxin A and G Proteins in Milk

2015 ◽  
Vol 78 (2) ◽  
pp. 362-369 ◽  
Author(s):  
MINGYAN LIANG ◽  
TINGTING ZHANG ◽  
XUELAN LIU ◽  
YANAN FAN ◽  
SHENGLIN XIA ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Signal To Noise Ratio ◽  
Food Poisoning ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synchronous Detection ◽  
Standard Curve ◽  
Stability And Accuracy

Staphylococcal food poisoning (SFP), one of the most common foodborne diseases, results from ingestion of staphylococcal enterotoxins (SEs) in foods. In our previous studies, we found that SEA and SEG were two predominant SE proteins produced by milk-acquired S. aureus isolates. Here, a tandemly arranged multiepitope peptide (named SEAGepis) was designed with six linear B-cell epitopes derived from SEA or SEG and was heterologously expressed. The SEAGepis-specific antibody was prepared by immunizing rabbit with rSEAGepis. Then, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on rSEAGepis and the corresponding antibody was developed to simultaneously detect SEA and SEG. Under the optimized conditions, the ic-ELISA standard curve for rSEAGepis was constructed in the concentration range of 0.5 to 512 ng/ml, and the average coefficients of variation of intra-and interassay were 4.28 and 5.61% during six standard concentrations. The average half-maximal inhibitory concentration was 5.07 ng/ml, and the limit of detection at a signal-to-noise ratio of 3 was 0.52 ng/ml. The anti-rSEAGepis antibody displayed over 90% cross-reactivity with SEA and SEG but less than 0.5% cross-reactivity with other enterotoxins. Artificially contaminated milk with different concentrations of rSEAGepis, SEA, and SEG was detected by the established ic-ELISA; the recoveries of rSEAGepis, SEA, and SEG were 91.1 to 157.5%, 90.3 to 134.5%, and 89.1 to 117.5%, respectively, with a coefficient of variation below 12%. These results demonstrated that the newly established ic-ELISA possessed high sensitivity, specificity, stability, and accuracy and could potentially be a useful analytical method for synchronous detection of SEA and SEG in milk.

Enzyme-linked immunosorbent assay for chromogranin A.

1989 ◽  
Vol 35 (9) ◽  
pp. 1934-1938 ◽  
Author(s):  
L Dillen ◽  
J De Block ◽  
L Van Lear ◽  
W De Potter
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Immunosorbent Assay ◽  
Coefficient Of Variation ◽  
Chromogranin A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromaffin Granules ◽  
Standard Curve

Abstract This is an enzyme-linked immunosorbent assay (ELISA) for determining chromogranin A (CGA) with use of a monoclonal antibody. CGA was isolated from bovine chromaffin granules. The analytical ELISA procedure for bovine CGA was developed and optimized. Typical standard curves ranged from 500 pg to 500 ng of CGA. We then studied human plasma CGA-immunoreactivity as measured by this assay. The curve for dilutions of human plasma paralleled the standard curve for bovine CGA. The intra-assay coefficient of variation for determination of human plasma CGA was 4.56%, indicating that reliable determinations can be performed for human plasma. However, further study revealed the presence of two CGA-immunoreactive substances in human plasma, one of which corresponds to the native CGA. The nature of the second immunoreactive substance still remains unknown. Nevertheless the measured CGA concentrations (ranging from 0.19 to 0.35 mg/L) in plasma are comparable with previously reported values.

scholarly journals Simultaneous Determination of Florfenicol and Its Metabolite Florfenicol Amine in Swine Muscle Tissue by a Heterologous Enzyme-Linked Immunosorbent Assay

2009 ◽  
Vol 92 (3) ◽  
pp. 981-988 ◽  
Author(s):  
Pengjie Luo ◽  
Haiyang Jiang ◽  
Zhanhui Wang ◽  
Caimao Feng ◽  
Fangyang He ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
Muscle Tissue ◽  
Immunosorbent Assay ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coefficients Of Variation ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic

Abstract A sensitive and heterologous enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) in swine muscle tissue was developed. FFA was conjugated to bovine serum albumin by a formaldehyde coupling method as an immunogen to immunize rabbits. FFA, thiamphenicol glycinate, and modified FF were conjugated to ovalbumin as coating antigens. The effect of different types of hapten heterology on the sensitivity and specificity of the ELISA was evaluated. Using FF glutaric anhydride ester as a coating hapten and antibody raised against modified FFA, an ELISA was developed that showed an IC50 value of 0.48 ng/mL. The antibody showed a cross-reactivity of 100 with FFA, 97 with FF, 6 with thiamphenicol, and a negligible value with chloramphenicol. From fortified swine muscle samples at levels of 4320 ng/g, the average recoveries of FF and FFA ranged from 58.2 to 96.8, with coefficients of variation less than 14. Analysis of incurred samples by the ELISA gave similar results to those by a previously developed liquid chromatographic method. The ELISA could be used as a rapid method for the simultaneous determination of FF and FFA in swine muscle tissue.

scholarly journals Enzyme-linked immunosorbent assay determination of specific rubella antibody levels in micrograms of immunoglobulin G per milliliter of serum in clinical samples

1978 ◽  
Vol 8 (4) ◽  
pp. 419-423
Author(s):  
P O Leinikki ◽  
I Shekarchi ◽  
P Dorsett ◽  
J L Sever
Keyword(s):  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Solid Phase ◽  
Laboratory Diagnosis ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Standard Curve ◽  
Antibody Levels

A "microgram assay" is described in which solid-phase enzyme-linked immunosorbent assay is used for the determination of specific rubella immunoglobulin G (IgG) antibody levels in micrograms per milliliter of serum. The quantitation was based on a standard curve obtained by using a reference serum, for which the specific IgG content was assayed by immunochemical purification. IgG was first purified and specific rubella antibodies were separated by an immunoadsorbent prepared by linking rubella virus antigens to Sepharose 4B. By using IgG-specific conjugate, the levels of specific rubella IgG antibodies could then be determined from clinical samples. Seronegative samples showed antibody levels less than 1 microgram/ml, whereas levels up to several hundred micrograms per milliliter were detected in some postinfection sera. The correlation between microgram antibody levels and hemagglutination inhibition titers was linear. The method offers a simple and sensitive antibody assay which could be used both for the laboratory diagnosis of acute rubella and for the evaluation of immunity.

scholarly journals Enzyme Immunoassay for Direct Determination of Microcystins in Environmental Water

1997 ◽  
Vol 80 (2) ◽  
pp. 408-417 ◽  
Author(s):  
Satoshi Nagata ◽  
Tomoaki Tsutsumi ◽  
Akihiro Hasegawa ◽  
Fuyuko Yoshida ◽  
Yoshio Ueno ◽  
...  
Keyword(s):  
Tap Water ◽  
Direct Determination ◽  
Environmental Water ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Powerful Method ◽  
Standard Curve ◽  
Liquid Chromatographic ◽  
Reliable Correlation

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed for direct quantitation of microcys- tins (MCs), a group of freshwater cyanobacterial toxins. An anti-MC monoclonal antibody exhibiting broad cross-reactivity to major MC derivatives was used. The detection limit and linear range of the ELISA standard curve with microcystin-(leucine-ar-ginine) (MCLR), a variant of MCs, were 20 and 20–500 pg/mL, respectively. For analysis of MC released from cyanobacterial cells, water sample filtered through a glass fiber filter was applied directly to ELISA. For analysis of total MC (released MC plus intracellular MC), intracellular toxin was extracted by freeze-thawing twice before filtration. Mean recovery of MCLR added to tap water and toxin-free environmental water was 101%, with a coefficient of variation (CV) of 7.3% at toxin levels of 20–500 pg/mL. Mean recovery of MCLR added to toxin-free cyanobacterial extracts was 93%, with a CV of 12.5% at toxin levels of 50–500 pg/mL. At 20 pg/mL, an increasing matrix effect on assay variance was observed; therefore, both released MC and total MC were measured in the range 50–500 pg/ mL. Comparative studies with a liquid chromatographic (LC) method showed that the ELISA gives a reliable correlation with LC for analysis of MC in water extracts of natural blooms and cultured cyanobacterial cells (r = 0.98). The ELISA was applied to water samples collected from lakes and ponds in Japan. In 4 of 13 and 12 of 17 samples, 81–800 pg released MC/mL and 64–94 000 pg total MC/mL were detected, respectively. By LC separation followed by the ELISA analysis, the presence of MCLR, microcystin-arginine-arginine, and micro-cystin-tyrosine-arginine were confirmed in 4 ELISA-positive samples selected randomly. The newly developed ELISA is a reliable and powerful method for mass monitoring of MC levels in environmental water.

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