What is Biotin?
Biotin is a water-soluble necessary for several biological reactions. This small molecule has the ability to non covalently bind to streptavidin. This interaction is exploited in laboratory techniques such as western blotting, flow cytometry, immuno-histochemistry, and immunoassays. But biotin or B vitamin is also a common component of daily supplement diet and its ingestion is becoming an important growing phenomenon.
The issue with Biotin : Alert on biotin interference for immunoassays using sera
The increasing population of patients and consumers receiving biotin has highlighted the potential for exogenous biotin interference in routine clinical laboratory assays that use a biotin–streptavidin link- age in their design.
The Food & Drug Administration (FDA) alerted clinical laboratory developers that exogenous biotin can significantly interfere with different immunoassays platforms and cause incorrect test results: biotin-induced interference is likely to affect many assays using Streptavidin-Biotin coupling and causes lower values (sandwich type assays) or higher values (competition type assays).
As recommended by the FDA, the easiest solution is to use an alternative assay that does not utilize biotin as a reagent.
The solution to Biotin interference: Replacing biotin/streptavidin by DNP/anti-DNP monoclonal
At SYnAbs, we figure that DNP seems to be the adequate molecule to do the job.
DNP stands for Di-Nitro-Phenol, 2,4-Dinitrophenol (2,4-DNP or simply DNP), an organic compound with the formula HOC6H3(NO2)2. Dinitrophenyl (DNP) is a hapten, not found endogenously in tissues, so serves as a control for immuno-assay.
But as passionated scientists we were eager to prove what we were saying.
So we decide to verify that a biotinylated mAb revealed with streptavidin -HRP can be replaced by a DNP labelled mAb revealed with the mouse anti DNP MADP2–HRP.
SYnAbs results with anti-DNP to solve biotin interference
- The antigen (here a mouse IgG2a) was coated on an ELISA plate that was saturated with gelatin. As a negative control, the antigen was omitted.
- Various concentrations of the rat anti mouse IgG2a LO-MG2a biotinylated or DNP labelled were added (the DNP labelling was performed with the N-Succinimidyl N-(2,4-dinitrophenyl-6-aminocaproate from Sigma).
- The biotinylated or DNP labelled LO-MG2a were revealed respectively with Streptavidin-HRP or MADNP2-HRP at 1 µg/ml.
These are the ELISA outcomes we obtained.
Labelled antibodies and probes with DNP
The detection of a biotinylated monoclonal antibody with streptavidin HRP can be advantageously replaced by the detection of a DNP labelled mAb with an anti DNP-HRP.
DNP is an excellent alternative to biotin for use in applications requiring biotin-free systems. Probes or antibodies labeled with DNP can be detected using the SYnAbs anti-DNP antibodies.
SYnAbs has developed anti-DNP in mice (MA-DNP) and in rats (LO-DNP), offering 16 different monoclonal antibodies references. Since small molecules (hormones, antibiotic, peptide, chemical therapeutic drug…) induce a better immune response in rat specie, LO-DNP are high-performance and well-known references in Pharma and IVD industries. LO-DNP-2 is part of SYnAbs Top3 sales chart.