Development of a Biotin-Avidin Mediated Immunoassay for Simultaneous Detection of Danofloxacin, Enrofloxacin and Ciprofloxacin in Milk

2013 ◽  
Vol 781-784 ◽  
pp. 1630-1636 ◽  
Author(s):  
Jie Biao Guo ◽  
Xuan Xin ◽  
Rui Min Zhong ◽  
Xing Ping Li
Keyword(s):  
Simultaneous Detection ◽  
Antigenic Site ◽  
Extraction Process ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Monoclonal Antibody ◽  
Common Structure ◽  
Coefficients Of Variation ◽  
Sample Extraction ◽  
The Common

To generate a group-specific monoclonal antibody (McAb) against danofloxacin (DANO), enrofloxacin (ENR) and ciprofloxacin (CIP), glutaraldehyde was used to link the presentative hapten of CIP to the immunogen and coating antigen, respectively, leaving the common structure of these 3 drugs exposed in both conjugates as a major antigenic site. Consequently, a McAb (6D3) with high cross-reactivity to this three antibiotics has been obtained by using hybridomas technique. In a biotin-avidin mediated enzyme-linked immunosorbent assay, the IC50values for DANO, ENR and CIP were 5.1, 4.5 and 4.2 ng mL-1, respectively. The ELISA was used for the detection of spiked DANO and ENR+CIP in milk. The recoveries ranged from 74.1 to 92.4% and coefficients of variation were in a range of 6.6-11.9%. The accuracies and sensitivity of the method were good for simultaneous analysis of the 3 drugs in milk after a simple sample extraction process.

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 for Simultaneous Detection of Two Fungicides Kresoxim-methyl and Trifloxystrobin in Oranges

2016 ◽  
Vol 34 (No. 5) ◽  
pp. 429-438 ◽  
Author(s):  
Yang Wei ◽  
Zhu Jie ◽  
Liu Ming-Qi ◽  
Dai Xian-Jun
Keyword(s):  
Immunosorbent Assay ◽  
Reaction Rate ◽  
Polyclonal Antibodies ◽  
Simultaneous Detection ◽  
Acid Methyl Ester ◽  
Enzyme Linked Immunosorbent Assay ◽  
Strobilurin Fungicides ◽  
Acid Methyl ◽  
Recovery Study ◽  
The Common

To assemble an indirect competitive enzyme-linked immunosorbent assay (ELISA) for estimating two strobilurin fungicides at the same time, the hapten was synthesised which contained the common active group, (E)-2-(2-bromo-phenyl)-2-(methoxyimino) acetic acid methyl ester (OEBr) in kresoxim-methyl and trifloxystrobin. The immunogen and coating antigen were respectively prepared through conjugating the above-mentioned hapten with BSA and OVA by the mixed anhydride and activated ester methods, and polyclonal antibodies were produced by immunised rabbits. An enzyme-linked immunosorbent assay was developed for simultaneous detection of kresoxim-methyl and trifloxystrobin. In ELISA, the antiserum showed high affinity and sensitivity to kresoxim-methyl and trifloxystrobin, and their IC<sub>50</sub> value and detection limit (expressed as IC<sub>10</sub>) were 14.7 and 0.0044 µg/ml, respectively, for kresoxim-methyl, and 22.9 and 0.014 µg/ml, respectively for trifloxystrobin. The cross-reaction rate was below 0.1% for other strobilurin fungicides. Recovery study of ELISA from spiked samples of homogenised peeled oranges (final concentrations of 100, 10, and 1 µg/ml) resulted in recovery levels in the range of 82–104%.

scholarly journals Evaluation of a laboratory-developed test for simultaneous detection of norovirus and rotavirus by real-time RT-PCR on the Panther Fusion® system

2019 ◽  
Vol 39 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Robert K. Kulis-Horn ◽  
Carsten Tiemann
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Extraction Process ◽  
Clinical Samples ◽  
Preparation Methods ◽  
Virus Assay ◽  
Sample Extraction ◽  
Stool Samples ◽  
Development And Validation ◽  
Result Interpretation

Abstract The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.

Detection of Zearalenone By Tandem Immunoaffinity-Enzyme-Linked Immunosorbent Assay and Its Application to Milk

1990 ◽  
Vol 53 (7) ◽  
pp. 577-580 ◽  
Author(s):  
JUAN I. AZCONA ◽  
MOHAMED M. ABOUZIED ◽  
JAMES J. PESTKA
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Monoclonal Antibody ◽  
Initial Sample ◽  
Average Recovery ◽  
Milk Samples ◽  
Coefficients Of Variation ◽  
Direct Elisa ◽  
Commercial Milk

A hybridoma-based method utilizing tandem affinity chromatography and enzyme-linked immunosorbent assay (ELISA) was devised to detect zearalenone. A zearalenone specific monoclonal antibody was attached to Sepharose for initial sample clean-up. Zearalenone was eluted with methanol and then quantified by competitive direct ELISA. Average ELISA recoveries from the column for water spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml were 107, 86, 95, 95, and 92%, respectively, with a mean recovery of 95%. Mean interwell and interassay coefficients of variation were 9.7 and 8.9%, respectively. Average recovery by the method from milk spiked with zearalenone at levels of 1, 5, 10, 25, and 50 ng/ml was 187, 113, 107, 110, and 112%, respectively, with a mean recovery of 126%. Mean interwell and interassay coefficients of variation were 14.5 and 9.1%, respectively. Zearalenone was not detectable in 12 commercial milk samples assayed by the tandem method.

scholarly journals Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 770 ◽  
Author(s):  
Jian-Xin Huang ◽  
Chan-Yuan Yao ◽  
Jin-Yi Yang ◽  
Zhen-Feng Li ◽  
Fan He ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Water Samples ◽  
Simultaneous Detection ◽  
Aldehyde Group ◽  
Cross Reactivity ◽  
Side Chain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibition Concentration ◽  
Ic50 Values ◽  
Optimized Conditions

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples.

scholarly journals Detection of Pork in Heat-Processed Meat Products by Monoclonal Antibody-Based ELISA

2000 ◽  
Vol 83 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Fur-Chi Chen ◽  
Y-H Peggy Hsieh
Keyword(s):  
Monoclonal Antibody ◽  
Muscle Protein ◽  
Meat Products ◽  
Antibody Test ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Meat ◽  
Coefficients Of Variation ◽  
Test Kit ◽  
Meat Samples

Abstract An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to a porcine thermal-stable muscle protein was developed for detection of pork in cooked meat products. The assay specifically detects porcine skeletal muscle, but not cardiac muscle, smooth muscle, blood, and nonmuscle organs. No cross-reactivity was observed with common food proteins. Validity of the assay was evaluated with laboratory formulated and commercial meat samples. The detection limit was determined as 0.5% (w/w) pork in heterologous meat mixtures. Overall, intra- and inter-assay coefficients of variation were 5.8 and 7.9%, respectively. The accuracy in analyzing market samples was 100% as verified by product labeling and confirmed by a commercial polycolonal antibody test kit.

scholarly journals Simultaneous Detection of Haemophilus influenzae Type b Polysaccharide-Specific Antibodies and Neisseria meningitidis Serogroup A, C, Y, and W-135 Polysaccharide-Specific Antibodies in a Fluorescent-Bead-Based Multiplex Immunoassay

2009 ◽  
Vol 16 (3) ◽  
pp. 433-436 ◽  
Author(s):  
Richarda M. de Voer ◽  
Rutger M. Schepp ◽  
Florens G. A. Versteegh ◽  
Fiona R. M. van der Klis ◽  
Guy A. M. Berbers
Keyword(s):  
Haemophilus Influenzae ◽  
Neisseria Meningitidis ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Haemophilus Influenzae Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Specific Antibodies ◽  
Multiplex Immunoassay

ABSTRACT We expanded the meningococcal serogroup A, C, Y, and W-135 multiplex immunoassay (MIA) to simultaneously detect immunoglobulin type G antibodies directed toward Haemophilus influenzae type b polysaccharide (HibPS). The monoplex HibPS assay was compared to a HibPS-specific competitive enzyme-linked immunosorbent assay and showed a good correlation (R = 0.96). Furthermore, no cross-reactivity between HibPS and the four meningococcal serogroups was detected. This pentaplex meningococcal Hib MIA is a useful tool to investigate serological responses toward different childhood PS vaccines.

Evaluation by Enzyme-Linked Immunosorbent Assay of Cleanup for Thin-Layer Chromatography of Aflatoxin B 1in Corn, Peanuts, and Peanut Butter

1988 ◽  
Vol 71 (5) ◽  
pp. 953-956 ◽  
Author(s):  
Fun S Chu ◽  
Rachel C Lee ◽  
Mary W Trucksess ◽  
Douglas L Park
Keyword(s):  
Thin Layer ◽  
Peanut Butter ◽  
Collaborative Study ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coefficients Of Variation ◽  
Final Interpretation ◽  
The Cross ◽  
Aflatoxin B ◽  
Layer Chromatography

Abstract A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B, added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B, levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.

Development of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virus

2015 ◽  
Vol 61 (11) ◽  
pp. 811-817 ◽  
Author(s):  
Yufeng Li ◽  
Fangyuan Zheng ◽  
Baochao Fan ◽  
Hassan Mushtaq Muhammad ◽  
Yao Zou ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Virus Neutralization ◽  
Enzyme Linked Immunosorbent Assay ◽  
S Protein ◽  
Diarrhea Virus ◽  
Coefficients Of Variation ◽  
Porcine Epidemic Diarrhea ◽  
The Relationship

Porcine epidemic diarrhea (PED) is a highly contagious, enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV). To find a suitable ELISA method to assess the infection of PEDV and the effectiveness of vaccines, we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA) based on a truncated recombinant spike (S) protein expressed in Escherichia coli. The parameters of the iELISA were optimized, and the cutoff value determined as 0.259 by analyzing optical density (OD) values of 80 PEDV negative sera confirmed by western blot. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were both less than 10%. Cross-reactivity assays demonstrated that iELISA was PEDV-specific. A virus neutralization test with sera of 7 different OD values showed a positive correlation between the OD values and virus neutralization. The results suggest this iELISA is specific, sensitive, and repeatable. Further studies should focus on the relationship between OD values of sera and its virus neutralization.

scholarly journals 753. Performance Characteristics of Diagnostic Assays in Blastomycosis

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S424-S424
Author(s):  
Timothy O’Dowd ◽  
Jack McHugh ◽  
Nancy Wengenack ◽  
Elitza Theel ◽  
Paschalis Vergidis
Keyword(s):  
Granulomatous Inflammation ◽  
Cross Reactivity ◽  
Performance Characteristics ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fungal Culture ◽  
Noninvasive Test ◽  
Serum Antigen ◽  
Urine Antigen ◽  
Antigen Tests ◽  
Urine And Serum

Abstract Background Blastomycosis has historically been a difficult diagnosis to establish, often initially misdiagnosed as bacterial pneumonia. Serologic assays and polymerase chain reaction (PCR) tests are available, but their performance is not well defined. The objective of this study was to characterize their performance. Methods Subjects were identified via chart review of patients diagnosed with blastomycosis from 2005 to 2020. A definitive diagnosis was based on fungal culture, histopathology, or cytology. Performance characteristics of the Blastomyces antibody enzyme linked immunosorbent assay (ELISA), immunodiffusion (ID), complement fixation (CF), urine and serum antigen ELISAs, and PCR were evaluated in patients with confirmed blastomycosis. Data on patient demographics, location of disease, and mortality was also collected. Results We identified 193 patients with blastomycosis. The mean age was 51.8 years (range, 11-84) and 73.6% of patients were male. 42.5% resided in Minnesota, 18.1% in Wisconsin, and 12.9% in Iowa. Diagnosis was based on culture in 142 (73.2%) or histopathology/cytology in 67 (34.7%) patients. Granulomatous inflammation was present in 73.1% (38/52) while 21.2% (41/193) had evidence of extrapulmonary dissemination. The antibody, ID, and CF assays were positive in 43.5% (37/85), 35.1% (33/94) and 20.5% (8/39) of patients, respectively. Sensitivity of Blastomyces PCR was 40% (4/10) in sputum and 75% (21/28) in bronchoalveolar lavage (BAL) fluid. Blastomyces urine and serum antigen tests were positive in 68% (34/50) and 50% (9/18) of cases, respectively, while the urine antigen was positive in 63.6% (7/11) of disseminated cases. Patients had a positive Histoplasma urine antigen test in 54.1% (20/37) and Aspergillus galactomannan in BAL in 34.8% (8/23) of cases. Serum beta-D-glucan test was positive in 16.7% (2/12). 90-day mortality was 21/193 (10.9%) and median time from diagnosis to death was 18 days. Conclusion In this cohort, Blastomyces urine antigen was the most sensitive noninvasive test, with similar performance in pulmonary and disseminated disease. However, its utility is limited by poor specificity due to cross-reactivity. Blastomyces PCR from BAL fluid demonstrated the highest sensitivity. Blastomyces antibody, ID, and CF had poor sensitivity. Disclosures All Authors: No reported disclosures

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