Evaluation of ELISA kits for the screening of four nitrofuran metabolites in aquaculture products according to the European guideline for the validation of screening methods

  • The administration of nitrofurans to livestock to treat or prevent animal diseases has been banned in the EU for the production of food of animal origin. The corresponding marker residues are tissue-related metabolites AMOZ, AHD, SEM, and AOZ. The MRPL (minimum required performance limit)/RPA (Reference point for action) was set at 1 µg kg-1 in the EU. Thus, all the laboratories involved in the control of nitrofuran metabolites must detect at least at this analytical limit of performance.
  • The objectives of the work reported here were to evaluate the performance of ELISA kits from two different manufacturers (R-Biopharm, Germany; Europroxima, the Netherlands) for the individual screening of the four nitrofuran metabolites (AOZ, AMOZ; AHD; and SEM) in aquaculture products (fish, shrimps), and then to validate the kits according to the European Decision EC/2002/657 and to the European guideline for the validation of screening methods. The false positive rates were below 9 % for the kits from both manufacturers.
  • The detection capabilities CCβ determined were all below the current RPA (1 µg/kg). However, regarding the updated RPA at 0.5 µg/kg that shall apply in 2022, the AMOZ and SEM kits from R-Biopharm and the SEM kit from Europroxima will not be able to reach it.

Performance evaluation of five ELISA kits for detecting anti-SARS-COV-2 IgG antibodies

Objectives: To evaluate and compare the performances of five commercial ELISA assays (EDI, AnshLabs, Dia.Pro, NovaTec, and Lionex) for detecting anti-SARS-CoV-2 IgG.
Methods: 70 negative control samples (collected before the COVID-19 pandemic) and samples from 101 RT-PCR-confirmed SARS-CoV-2 patients (collected at different time points from symptoms onset: ≤7, 8-14, and >14 days) were used to compare the sensitivity, specificity, agreement, positive and negative predictive values of each assay with RT-PCR. A concordance assessment between the five assays was also conducted. Cross-reactivity with other HCoV, non-HCoV respiratory viruses, non-respiratory viruses, and nuclear antigens was investigated.
Results: Lionex showed the highest specificity (98.6%, 95%CI: 92.3-99.8), followed by EDI and Dia.Pro (97.1%, 95%CI: 90.2-99.2), NovaTec (85.7%, 95%CI: 75.7-92.1), then AnshLabs (75.7%, 95%CI: 64.5-84.2). All ELISA kits cross-reacted with one anti-MERS IgG positive sample except Lionex. The sensitivity was low during the early stages of the disease but improved over time. After 14 days from symptoms onset, Lionex and NovaTec showed the highest sensitivity at 87.9% (95%CI: 72.7-95.2) and 86.4% (95%CI: 78.5-91.7), respectively. The agreement with RT-PCR results based on Cohen’s kappa was as follows: Lionex (0.89)> NovaTec (0.70)> Dia.Pro (0.69)> AnshLabs (0.63)> EDI (0.55).
Conclusion: The Lionex ELISA, which measures antibodies solely to the S1 protein, demonstrated the best performance.

Simultaneous quantification of rituximab and eculizumab in human plasma by liquid chromatography-tandem mass spectrometry and comparison with rituximab ELISA kits

Objectives: Specific and sensitive analytical techniques to quantify therapeutic monoclonal antibodies (mAbs) are required for therapeutic drug monitoring. The quantification of mAbs has been historically performed using enzyme-linked immunosorbent assays (ELISAs), for which the limitations in terms of specificity have led to the development of alternative analytical strategies.
Methods: Here, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous quantification of rituximab (RTX – anti-CD20) and eculizumab (ECU – anti-C5). Sample preparation was based on our previously published method, using protein G purification and trypsin digestion. A new specific peptide for RTX, containing an N-terminal pyroglutamine and a trypsin miss-cleavage, enables better sensitivity, while peptide of ECU was chosen thanks to an in silico trypsin digestion and the Skyline® software. Full-length stable-isotope-labeled adalimumab was added to plasma samples as an internal standard. RTX in 50 human serum samples was quantified by LC-MS/MS and the concentrations obtained compared to those obtained with two commercial ELISA kits (Lisa Tracker® and Promonitor®).
Results: Calibration curves were linear from 1 to 200 µg.mL-1 for RTX and 5 to 200 µg.mL-1 for ECU, and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Comparison of the LC-MS/MS method with ELISA showed a negligible bias with the Lisa Tracker® kit (4%), but significant bias with the Promonitor® assay (mean underestimation of 69% for the Promonitor® assay).
Conclusions: This new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring.

Comparison of an Immunochromatographic Assay Kit with DAS-ELISA for Large-Scale Diagnosis and Molecular Discrimination of Satsuma Dwarf Virus Collected from Citrus Orchards

Satsuma dwarf virus (SDV) seriously damages citrus production by reducing the quality and yield of fruit. To avoid contamination with SDV, mother trees are checked to be SDV-free in advance of nursery tree distribution. In this study, we compared an immunochromatographic assay (ICA) kit with double-antibody sandwich enzyme-linked immunosorbent assay (DASELISA) for large-scale diagnosis of SDV in orchardgrown trees in Shizuoka Prefecture, Japan. The two methods gave conflicting results for 11 of 1,705 samples, all of which were negative by DAS-ELISA but positive by ICA.
The samples scored as positive by either DASELISA or ICA were analyzed by reverse transcription polymerase chain reaction and all were confirmed to be positive. These results validate the use of ICA as a screening method for large-scale diagnosis. Strain discrimination revealed that 16 of 22 isolates belonged to SDV, while citrus mosaic virus (CiMV) infection only and co-infection (SDV and CiMV) were in a minority.

T-Pro Plasmid Mini Kit (100)

RB94-YPD100 T-Pro Biotechnology 100preps/Kit 193.2 EUR

T-Pro Plasmid Mini Kit (250)

RB94-YPD250 T-Pro Biotechnology 250preps/Kit 266.4 EUR

T-Pro Plasmid Midi Kit (20)

RB94-YPI020 T-Pro Biotechnology 20preps/Kit 255.6 EUR

T-Pro Plasmid Maxi Kit (10)

RB94-YPM010 T-Pro Biotechnology 10preps/Kit 266.4 EUR

T-Pro BCA Protein Assay kit

JB04-D001 T-Pro Biotechnology 500assay/KIT 244.8 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008L T-Pro Biotechnology 5ml*20/BT 1000 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008M T-Pro Biotechnology 5ml*2/BT 120 EUR

T-Pro Cell Counting Kit (CCK8)

JK95-C008S T-Pro Biotechnology 1ml*5/BT 80 EUR

T-Pro LumiDura Chemiluminescence Detection Kit (ECL Kit)

JT96-K006M T-Pro Biotechnology 250ml*2/Kit 200 EUR

T-Pro LumiDura Chemiluminescence Detection Kit (ECL Kit)

JT96-K006S T-Pro Biotechnology 100ml*2/Kit 100 EUR

T-Pro Genomic DNA Mini Kit (100)

RB94-NGS100 T-Pro Biotechnology 100preps/kit 255.6 EUR

T-Pro Genomic DNA Midi Kit (20)

RB94-NGM020 T-Pro Biotechnology 20preps/kit 266.4 EUR

T-Pro Bradford Protein Assay kit(1X)

JB04-D002 T-Pro Biotechnology 500ml/BT 193.2 EUR

T-Pro Bradford Protein Assay kit (5X)

JB04-D003 T-Pro Biotechnology 500ml/BT 120 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002M T-Pro Biotechnology 250ml*2/Kit 244.8 EUR

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K002S T-Pro Biotechnology 100ml*2/Kit 182.4 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004M T-Pro Biotechnology 250ml*2/Kit 286.8 EUR

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)

JT96-K004S T-Pro Biotechnology 100ml*2/Kit 204 EUR

T-Pro Plant Genomic DNA Mini Kit (100)

RB94-PGS100 T-Pro Biotechnology 100preps/kit 266.4 EUR

T-Pro Gradient Gel Solution kit 4-12%

JB02-B110M T-Pro Biotechnology 250ml+100ml/kit 60 EUR

T-Pro Plant Genomic DNA Midi Kit (20)

RB94-PGM020 T-Pro Biotechnology 20preps/kit 266.4 EUR

T-Pro Endotoxin Removal Plasmid Midi kit (25)

RB94-EPI020 T-Pro Biotechnology 20preps/Kit 297.6 EUR

T-Pro Endotoxin Removal Plasmid Maxi kit (10)

RB94-EPM010 T-Pro Biotechnology 10preps/Kit 318 EUR

T-Pro Gel/PCR DNA Purification Mini Kit (100)

RB94-NES100 T-Pro Biotechnology 100preps/Kit 193.2 EUR

T-Pro Gel/PCR DNA Purification Mini Kit (250)

RB94-NES250 T-Pro Biotechnology 250preps/Kit 266.4 EUR

T-Pro Gel/PCR DNA Purification Maxi Kit (10)

RB94-NEL010 T-Pro Biotechnology 10preps/Kit 193.2 EUR

T-Pro Stacking Buffer

JB03-C002 T-Pro Biotechnology 500ml/BT 182.4 EUR

T-Pro MycoClean spray

JT90-R002 T-Pro Biotechnology 500ml/BT 172.8 EUR

How Reliable are Commercially Available Glypican4 ELISA Kits?

Objective: Glypican4 is an interesting new adipokine, which seems to play an important role in developmental processes and is potentially associated with metabolic changes in obesity and type 2 diabetes mellitus. Currently, only a few studies examined glypican4 in human blood, mainly in adults.
Design, patients and measurements: The aim of our study was to investigate glypican4 serum levels in lean, overweight, and obese children and adolescents, to unravel a possible association between glypican4 serum levels and parameters of obesity and insulin resistance. In order to determine a suitable method for investigating glypican4 serum levels, we validated two commercially available human glypican4 ELISA kits, using serum and plasma samples of an obese, insulin-resistant patient, and a healthy control subject, a human recombinant glypican4 protein fragment and glypican4-overexpressing cell lysate.
Results: Using ELISA kit #1 we were not able to detect values above background level, apart from standard curve values. ELISA kit #2 initially seemed suitable to measure glypican4, but further validation experiments showed non-linearity of serial dilutions, no recognition of a human recombinant glypican4 protein fragment and non-linearity in the recovery of glypican4-overexpressing cell lysate. In addition, there was a considerable decrease (approx. 68%) of measured values between two experiments, performed at different time points with aliquots of the same serum sample. Contrary to that, further experiments found sample stability not to be compromised.
Conclusions: Extensive evaluation of the performance of two commercially available ELISA kits led to the conclusion that none of them is applicable for the measurement of glypican4 in human blood samples.

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