Abstract
(Strept)avidin–biotin technology is frequently used in immunoassay systems to improve their analytical properties. It is known from clinical practice that many (strept)avidin–biotin-based tests provide false results when analyzing patient samples with a high content of endogenous biotin. No specific investigation has been carried out regarding possible interferences from avidin (AVI) and biotin (B7) contained in food matrices in (strept)avidin–biotin-based immunoanalytical systems for food safety. Two kinds of competitive ELISAs for bacitracin (BT) and colistin (COL) determination in food matrices were developed based on conventional hapten–protein coating conjugates and biotinylated BT and COL bound to immobilized streptavidin (SAV). Coating SAV–B7–BT and SAV–B7–COL complexes-based ELISAs provided 2- and 15-times better sensitivity in BT and COL determination, corresponding to 0.6 and 0.3 ng/mL, respectively. Simultaneously with the determination of the main analytes, these kinds of tests were used as competitive assays for the assessment of AVI or B7 content up to 10 and 1 ng/mL, respectively, in food matrices (egg, infant milk formulas enriched with B7, chicken and beef liver). Matrix-free experiments with AVI/B7-enriched solutions showed distortion of the standard curves, indicating that these ingredients interfere with the adequate quantification of analytes. Summarizing the experience of the present study, it is recommended to avoid immunoassays based on avidin–biotin interactions when analyzing biosamples containing these endogenous factors or enriched with B7.
Highlights
IntroductionThe interaction of (strept)avidin protein and biotin (vitamin B7) is widely used in various bioanalytical systems, in particular, in immunoassays [1]
The interaction ofavidin protein and biotin is widely used in various bioanalytical systems, in particular, in immunoassays [1]
In a wide panel of commercialavidin–biotin-based immunoassay systems used in clinical practice for diagnostic purposes, it has been found that the presence of endogenous biotin in patients’ biofluids can interfere with the analysis, leading to false results and, to misdiagnosis and patient mismanagement [8,9]
Summary
The interaction of (strept)avidin protein and biotin (vitamin B7) is widely used in various bioanalytical systems, in particular, in immunoassays [1]. Due to the high affinity (KD ≈ 10−15 M) and binding valence between this protein (four binding sites) and the vitamin, they are successfully applied as immunoreagent labels to provide an additional functionality [2], for oriented immobilization/presentation [3], as well as to accelerate the interaction [4] or enhance the output signal in immunoassays of various designs [5,6] In this regard, the involvement of the (strept)avidin–biotin system is one of the main strategies for increasing the sensitivity of immunoassay [7]. Excessive consumption as a result of therapy for a number of disorders (multiple sclerosis, phenylketonuria, biotinidase deficiency) or incorporation in “Hair, Skin, and Nails” cosmetic formulas may lead to μM B7 blood levels, which can significantly distort the results of (strept)avidin–biotin-based tests [9]
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