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Contrary to what one might first think, a biotin antibody is not a substance that is raised against biotin. Rather, a biotin antibody is an antibody that has been conjugated to biotin. Biotin binds strongly to a ligand — avidin — which can, in turn, be conjugated to light-detectable molecules such as fluorophores. Biotin and avidin form one of the strongest biologically relevant covalent bonds known, making biotin- and avidin-conjugated ligand/receptor units especially useful in scientific studies.
Biotin is a B-complex vitamin — vitamin B7. It is such a small molecule that multiple biotin molecules can be conjugated to a single monoclonal or polyclonal antibody. The fact that biotin is also water-soluble allows for greater versatility in buffer systems than if it were insoluble. For greater specificity, monoclonal antibody conjugation to biotin is often recommended over polyclonal antibody conjugation. The biotin molecule can be directly bonded with the antibody of interest, or it can be linked through a molecular tail to allow for more freedom of movement.
The biotin ligand avidin is found in egg albumin. It can be cheaply made and easily isolated. Multiple avidin molecules will bind to each biotin receptor, amplifying a single antibody detection signal. When a biotin antibody adheres to an antigen of interest, an experimenter can then expose the complex to avidin conjugated to fluorophores, making the complex detectable. Through fluorescent imaging, the biotin antibody and avidin-conjugated ligand can be detected with light microscopy, and antigens of interest can be studied in situ.
Although the creation and use of a biotin antibody and a fluorescently tagged avidin molecule is a common solution for antigen detection, alternatives exist that still make use of the biotin/avidin complex. Binding biotin to a ligand like a neurotoxin can be advantageous in detecting placement of neuronal receptors affected by the neurotoxin in question. It also is possible to conjugate biotin to a nucleotide sequence such as messenger ribonucleic acid (mRNA) to find where these nucleotide sequences are being translated into proteins or to detect cellular mRNA regulation.
It is possible to buy biotinylated polyclonal or monoclonal antibodies, though biotinylated antibody production is within the ability of many experimenters. Protocols can be found online suggesting antibody biotinylation methods, though most easily followed protocols directly link biotin to an antibody rather than suggest how to intercalate a molecular spacer. Molecular spacers between biotin and its associated antibody allow for greater spacing to expose more molecules to binding sites, and these biotin antibodies usually are purchased rather than made.
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