Biotin interference in immunoassays based on biotin-strept(avidin) chemistry: An emerging threat

Biotechnol Adv. 2019 Sep-Oct;37(5):634-641. doi: 10.1016/j.biotechadv.2019.03.007. Epub 2019 Mar 11.


Biotinylated antibodies/antigens are currently used in many immunoassay formats in clinical settings for diversified analytes and biomarkers to offer high detection selectivity and sensitivity. Biotin cannot be synthesized by mammals and must be taken as an essential supplement. Normal intake of biotin from various foods and milk causes no effect on the streptavidin/biotin-based immunoassays. However, overconsumption of biotin (daily doses 100-300 mg) poses a significant problem for immunoassays using the biotin-strept(avidin) pair. Biotin interferences are noted in immunoassays of thyroid markers, drugs, hormones, cancer markers, the biomarker for cardiac function (β-human chorionic gonadotropin), etc. The biotin level required for serious interference in test results varies significantly from test to test and cannot easily be predicted. Immunoassay manufacturers with technologies based on strept(avidin)-biotin binding must investigate the interference from biotin (up to at least 1200 ng/mL or 4.9 μM of biotin) in various formats. There is no concrete solution to circumvent the biotin interference encountered in blood samples, short of biotin removal. Considering the short half-life of biotin in the human body, patients must stop taking biotin supplements for >48 h before the test. However, this scenario is not considered for patients in emergency situations or those with biotinidase deficiency, mitochondrial metabolic disorders or multiple sclerosis. Apparently, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays using strep(avidin)-biotin chemistry. To date, there is no quick and reliable procedure for the detection of biotin at below nanomolar levels in blood and biological samples. Traditional lab-based techniques including HPLC/MS-MS cannot process an enormous number of public samples. Biosensors with high detection sensitivity, miniaturization, low cost, and multiplexing have the potential to address this issue.

Keywords: Avidin/streptavidin; Biotin interference; Biotin removal; Competitive assay; Falsely negative; Falsely positive; Immunoassays; Sandwiched assay.

Publication types

  • Review

MeSH terms

  • Animals
  • Artifacts
  • Biomarkers / analysis
  • Biomarkers / blood
  • Biosensing Techniques / methods
  • Biotin / analysis
  • Biotin / chemistry*
  • Biotin / isolation & purification
  • Biotin / therapeutic use
  • Humans
  • Immunoassay / methods*
  • Sensitivity and Specificity
  • Streptavidin / chemistry*


  • Biomarkers
  • Biotin
  • Streptavidin