The monitoring of infliximab drug levels aids in the management of several autoimmune diseases, notably inflammatory bowel disease. Several commercial kits are now available and approved by the Therapeutic Goods Administration (TGA) for the measurement of infliximab levels, but there have been limited verification or comparison studies to date. Finding an assay that most accurately measures infliximab is essential for optimal drug titration and patient management.
We performed this study to compare the performance of the Grifols Promonitor, Theradiag Lisatracker and R-Biopharm Ridascreen enzyme linked immunosorbent assay (ELISA) kits. Preparations of serum containing known concentrations of infliximab were assayed using each kit, including in the presence of interference from anti-infliximab antibodies, autoantibodies and other biological agents.
The Lisatracker kit provided the most accurate determination of infliximab drug levels, however it yielded false positive results at low concentrations of infliximab. The average coefficients of variation (CVs) for the kits were 8% for Lisatracker, 5% for Ridascreen and 11% for Grifols. Infliximab measurements across all kits were affected by interference from antibodies to infliximab (ATI).
This study identified the Lisatracker kit as the most accurate in quantifying infliximab levels, although it was limited by false positive results at low concentrations of infliximab as well as interference from ATI. This has important implications for the monitoring and management of patients receiving infliximab therapy.
Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.
Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients.
All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development.
The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.
Group detection of DON and its modified forms by an ELISA kit.
Deoxynivalenol (DON) and its modified forms (3-, and 15-acetyl-DON, DON-3-glucoside) are commonly analysed by chromatographic methods. Indeed, coupled with proper extraction and clean-up, LC-MS represents the best approach for multi-mycotoxin measurements. On the other hand, immunochemistry-based methods are possibly able to detect a family of structurally related compounds, although the determination of single contributions is not possible so far.
However, ELISA methods often lead to an apparent overestimation of the mycotoxins content because modified forms and matrix components can potentially cross-react with the antibodies (designed for the parent toxin). Several data about the possible cross-reactivity of commercial DON-detecting ELISA kit are reported in the literature so far. Data are commonly obtained in buffer solutions or in matrix-matched solutions, but comparison of a set of naturally incurred samples has never been reported.
In the present work the accuracy of a commercial DON-detecting ELISA kit was evaluated on naturally incurred soft wheat (n = 15) and maize (n = 15), taking into account the matrix effect. Recovery was calculated considering the DON concentration found by LC-MS/MS and the total DON concentration, expressed as the sum of DON and its modified forms found by LC-MS/MS.
The obtained data clearly show that, when 3-modified forms of DON occur in the sample, the ELISA kit does actually detect them, thus returning an apparent overestimation if only DON content is considered. When the ELISA recovery is calculated on the total DON content, the accuracy of the analysis increases and the variability decreases. According to our data, the ELISA kit seems to be a promising group detection tool for the accurate evaluation of DON and its modified forms, expressed as sum of DON, DON-3Glc and 3Ac-DON, for soft wheat and maize samples.
Case study on human α1-antitrypsin: Recombinant protein titers obtained by commercial ELISA kits are inaccurate.
Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme-linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1-antitrypsin (rø1AT) titer by Coomassie-stained SDS-PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit.
This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed-phase high-performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off-the-shelf ELISA kits and found that the ELISA kits led to inconsistent results.
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The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted per se. Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.