ELISA validation and determination of cut-off level for chloramphenicol (CAP) residues in shrimp and fish

An analytical validation of enzyme-linked immunosorbent assay (ELISA) screening for detection of chloramphenicol (CAP) in shrimp and fish was conducted according to the Commission Decision 2002/657/EC and guidelines for the validation of screening methods for residues of veterinary medicines. The analyte was extracted from shrimp and fish with ethyl acetate mixture, and CAP concentrations were measured photometrically at 450 nm. The recovery rate of the analyte from spiked samples was 80%. For the laboratory the cut-off level of CAP in fish and shrimp as the minimum recovery was established along with detection capability (CCβ). No relevant interferences between matrix effects and structurally related substances including florfenicol and thiamphenicol were observed. The experimental results were quite satisfactory and ELISA method was found very useful for determination of CAP residues in shrimp and fish monitoring.

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ELISA validation and determination of cut-off level for chloramphenicol residues in honey

Bulletin of the Veterinary Institute in Pulawy ◽

10.1515/bvip-2015-0052 ◽

2015 ◽

Vol 59 (3) ◽

pp. 353-356 ◽

Cited By ~ 2

Author(s):

Bogumił Biernacki

Keyword(s):

Matrix Effects ◽

Screening Methods ◽

Analytical Validation ◽

Detection Capability ◽

Elisa Method ◽

Related Substances ◽

Veterinary Medicines ◽

Acetate Mixture ◽

Ethyl Acetate Mixture

Abstract An analytical validation of a screening ELISA for detection of chloramphenicol (CAP) in honey was conducted according to the Commission Decision 2002/657/EC and Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. The analyte was extracted from honey with a water and ethyl acetate mixture, and CAP concentrations were measured photometrically at 450 nm. The recovery rate of the analyte from spiked samples was 79%. The cut-off level of CAP in honey as the minimum recovery (0.17 units) was established. Detection capability (CCβ) was fixed at 0.25 μg kg−1. No relevant interferences between matrix effects and structurally related substances including florfenicol and thiamphenicol were observed. The ELISA method should be useful for determination of CAP residues in honey monitoring.

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Валідація методу кількісного визначення нітрофурану (АОЗ) і хлорамфеніколу у зразках меду натурального за допомогою тест-системи RidaScreen®

Ukrainian Journal of Ecology ◽

10.15421/2018_290 ◽

2018 ◽

Vol 8 (1) ◽

pp. 892-897

Author(s):

K.S. Myagka ◽

S.A. Tkachuk ◽

N.A. Mezhenska

Keyword(s):

Screening Method ◽

Test System ◽

Screening Methods ◽

Detection Capability ◽

Initial Validation ◽

Veterinary Medicines ◽

Allowable Level ◽

Validation Parameters ◽

Natural Honey

We presented the validation method of the quantitative determination of Nitrofuran (AOZ) and Chloramphenicol in honey samples by the RidaScreen® test system (according to Commission Decision 2002/657/EC and Guidelines for the validation of screening methods for residues of veterinary medicines (initial validation and transfer) of 20/01/2010). We revealed several validation indicators like ccβ (detection capability), accuracy (precision), recovery and some others during the initial validation (specificity and selectivity). We determined the minimum detection values of Nitrofuran (AOZ) in honey, the highest value (the highest response) for blank samples is 0.066 μg/kg and the lowest value for enriched samples (lowest response) is 0.401 μg/kg. Based on the obtained results none of the responses for enriched samples do not coincide with the range of replies of blank samples. Hence, it follows that the detection capability (CCβ) for this screening method is less than or equal to 0.4 μg/kg. For the determination of Chloramphenicol, the highest response is noted for blank samples – 0.021 μg/kg (21 ng/kg) and the lowest response noted for enriched samples is 0.048 μg/kg (48 ng/kg). None of the responses for enriched samples do not coincide with the range of replies of blank matrix (samples), therefore we could conclude that the CCβ of this screening method is less than or equal to 0.05 μg/kg (β-error < 5 %) and the cut-off value of this test is 0.048. According to the validation the lowest value of substances that could detected by ELISA using the following test systems for competitive ELISA (enzyme-linked immunoassay) for Nitrofuran (AOZ) – 0.4 μg/kg, for Chloramphenicol – 0.05 μg/kg. Described in State Standard 4497:2005 “Natural Honey. Technical conditions” the maximum allowable level for Chloramphenicol – 0.3 μg/kg and Nitrofuran (AOZ) – 0.6 μg/kg requirecorrection according to the established validation parameters.

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Validation of the method of quantitative determination of florphenicol in muscle samples by the method of immuno-enzyme analysis

VETERINARY SCIENCE TECHNOLOGIES OF ANIMAL HUSBANDRY AND NATURE MANAGEMENT ◽

10.31890/vttp.2020.06.07 ◽

2020 ◽

pp. 40-45

Author(s):

M. V. Kostyuk ◽

◽

K. S. Myagka ◽

G. S. Kochetova ◽

◽

...

Keyword(s):

Immunosorbent Assay ◽

Screening Method ◽

Enzyme Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Laboratory Diagnostics ◽

Screening Methods ◽

Detection Ability ◽

Wide Range ◽

Control Samples

Introduction. Amphenicols are a group of chemical compounds with antibacterial activity, including chloramphenicol (HAF), thiamphenicol (TAF), florfenicol (FF) and their derivatives. Florfenicol (FF) is a synthetic antimicrobial agent with a broad spectrum of action and is one of the most commonly used drugs in poultry, and was developed specifically for veterinary medicine. Given the wide range of activity of florfenicol, the high therapeutic effect combined with low toxicity makes it important for use in animal husbandry. The known method of enzyme-linked immunosorbent assay (ELISA) differs favorably from other screening methods by high sensitivity, specificity, simplicity and speed of performance, availability and stability of reagents, the ability to computer processing of measurement results and automation of test steps, which provides high test efficiency. The aim of the work. To validate the method of enzyme-linked immunosorbent assay to determine the residues of florfenicol in the samples of months of different species of animals (cattle, pigs, chickens, geese, turkeys, rabbits) and fish. Materials and methods. The research was conducted on the basis of the State Research Institute for Laboratory Diagnostics and Veterinary Sanitary Expertise. The material for the study was a solution of florfenicol with a concentration of 1 mg/liter. Results of research and discussion. It was found that the highest value (highest response) for control samples is 0.24 μg/kg and the lowest value for enriched samples (lowest response) - 3.62 μg/kg. According to the obtained results, none of the answers for the enriched samples coincides with the range of answers for the control samples. It follows that the detection ability (CCβ) for this screening method reduces or decreases 5.0 μg/kg The cut-off rate of this test is 3.62 μg/kg. Conclusions and prospects for further research. Validation characteristics have been established for the determination of florfenicol residues in muscle samples, such as: detection ability (CCβ) is 5.0 μg/kg, cut-off level is 3.62 μg/kg. The lowest content of florfenicol that can be determined is 0.2 μg/kg. The percentage of return for enriched samples of both groups is 93 %, which corresponds to the specified production.

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

Journal of AOAC International ◽

10.1093/jaoac/90.4.1000 ◽

2007 ◽

Vol 90 (4) ◽

pp. 1000-1010 ◽

Cited By ~ 9

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.

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Determination of Thiabendazole in Fruit Juices by a New Monoclonal Enzyme Immunoassay

Journal of AOAC International ◽

10.1093/jaoac/84.1.156 ◽

2001 ◽

Vol 84 (1) ◽

pp. 156-161 ◽

Cited By ~ 11

Author(s):

Antonio Abad ◽

Juan J Manclús ◽

María J Moreno ◽

Angel Montoya

Keyword(s):

Matrix Effects ◽

Simple Procedure ◽

Fruit Juices ◽

Enzyme Linked Immunosorbent Assay ◽

Dilution Method ◽

Sample Throughput ◽

Banana Juice ◽

Routine Monitoring ◽

Low Levels

Abstract A competitive, indirect enzyme-linked immunosorbent assay (ELISA) for thiabendazole has been developed and applied to the analysis of fruit juices spiked with this fungicide. The immunoassay is based on a new monoclonal antibody derived from a hapten functionalized at the nitrogen atom in the 1-position of the thiabendazole structure. To our knowledge, such a structure has not been previously used to obtain antibodies to thiabendazole. The l50 value and the detection limit of the ELISA for standards were 0.2 and 0.05 ng/mL, respectively. Fruit juices were analyzed by diluting samples in assay buffer, without extraction or cleanup. Samples were not even centrifuged or filtered to remove fruit pulp. Under these conditions, the immunoassay was able to accurately determine thiabendazole down to 1 ng/mL in orange and grapefruit juices, down to 5 ng/mL in banana juice, and down to 20 ng/mL in apple and pear juices. Sensitivity differences of the ELISA were caused by the minimum dilution required by each juice to minimize matrix effects: 1/10 for orange and grapefruit juices, 1/50 for banana juice, and 1/100 for apple and pear juices. In an attempt to further increase the sensitivity of the immunoassay for matrixes showing the strongest interferences, apple and pear juices spiked with thiabendazole at low levels (1–20 ng/mL) were extracted with ethyl acetate before analysis. This simple procedure entailed a significant reduction of matrix effects, which in fact allowed us to determine accurately as low as 5 ng/mL thiabendazole in apple and pear juices. Irrespective of whether samples were analyzed by the direct dilution method or after extraction, the simplicity, sensitivity, and sample throughput of this monoclonal immunoassay makes it a very convenient method for the routine monitoring of thiabendazole residues in fruit juices.

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A New Lc-Ms/Ms Method for Multiple Residues/Contaminants In Bovine Meat

10.21203/rs.3.rs-35730/v1 ◽

2020 ◽

Author(s):

Zehra Hajrulai-Musliu ◽

Risto Uzunov ◽

Stefan Jovanov ◽

Dean Jankuloski ◽

Velimir Stojkovski ◽

...

Keyword(s):

Coefficient Of Variation ◽

Matrix Effects ◽

Limit Of Detection ◽

Veterinary Drug ◽

Detection Capability ◽

Limit Of Quantification ◽

Internal Standards ◽

Anabolic Hormones ◽

Residual Matrix

Abstract A multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, β-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the coefficient of correlation (R2) > 0.98. The decision limit (CCα) values were in the range from 0.067 to 2103.84 μg/kg, while the range for detection capability (CCβ) was from 0.083 to 2482.13 μg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 μg/kg, and 0.081 to 328.13 μg/kg, respectively. The recovery of analytes ranged from 61.28 % to 116.20 %. The intra-day coefficient of variation (CV) was from 0.97 to 25.93 % and the inter-day CV was 2.30-34.04 %. The method has been used for the determination of 49 residues/contaminants in bovine meat.

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A new LC–MS/MS method for multiple residues/contaminants in bovine meat

BMC Chemistry ◽

10.1186/s13065-021-00788-5 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Zehra Hajrulai-Musliu ◽

Risto Uzunov ◽

Stefan Jovanov ◽

Dean Jankuloski ◽

Velimir Stojkovski ◽

...

Keyword(s):

Coefficient Of Variation ◽

Matrix Effects ◽

Limit Of Detection ◽

Veterinary Drug ◽

Detection Capability ◽

Limit Of Quantification ◽

Internal Standards ◽

Anabolic Hormones ◽

Residual Matrix

AbstractA multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, β-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the R2 > 0.98. The decision limit (CCα) values were in the range from 0.067 to 2103.84 μg/kg, while the range for detection capability (CCβ) was from 0.083 to 2482.13 μg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 μg/kg, and 0.081 to 328.13 μg/kg, respectively. The recovery of analytes ranged from 61.28% to 116.20%. The intra-day coefficient of variation (CV) was from 0.97 to 25.93% and the inter-day CV was 2.30–34.04%. The method has been used for the determination of 49 residues/contaminants in bovine meat. Application of the method in routine analysis in bovine samples, revealed in limited samples the presences of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively.

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An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665299 ◽

1983 ◽

Vol 50 (03) ◽

pp. 740-744 ◽

Cited By ~ 139

Author(s):

Nils Bergsdorf ◽

Torbjörn Nilsson ◽

Per Wallén

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Specific Activity ◽

Thromboembolic Disease ◽

Tissue Type ◽

Enzyme Linked Immunosorbent Assay ◽

Type Plasminogen Activator ◽

Tissue Plasminogen ◽

Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

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