Charles... Your answer is a bit off... There are four biotin-binding sites on any single avidin molecule; four biotins will bind to a single streptavidin (SA). In the case of biotinylated antibodies, it is theoretically possible that many conjugated SAs could bind a single multiply-biotinylated antibody. In reality, the number of SAs that can fit around a single antibody is probably pretty small, and when the SA is conjugated to PE (which is huge) or APC (also large), this number becomes even smaller (maybe only 1 or 2). Hence, the amplification noted for biotin-SA systems is theoretically best for FITC-avidin (or other small fluorochromes, such as Cy3, Cy5, TexasRed, Alexa, etc.). That said, why is it that amplifications with SA-PE or SA-APC can be much more than 3x when it is sterically nearly impossible that even 3 SAs are binding a single IgG? The answer is that PE-SA and APC-SA are almost never simple complexes of 1 SA with 1 PE (or APC). While such 1:1 complexes can be made (and are commercially available), most manufacturers sell complexes that are mixtures of much larger multimers--e.g., 2:2, 3:3, 4:4, .... This is because of the chemistry--it is easier to manufacture larger complexes than 1:1 complexes. And customers prefer these "high order" complexes! Why? Because for a single biotin, you can now have a large multi-avidin/PE complex that has as many as 3 or more PE molecules. Binding only two of these complexes to a single antibody can increase fluorescence 6-fold. (Incidentally, this is why the name "tetramer" for the MHC-HLA complexes first made at Stanford is a misnomer. These "tetramers" are probably complexes that include as many as a dozen HLA-molecules--"MHC Multimer" is a far more accurate term. Unfortunately, the term "tetramer" has become so common as to be irreplaceable... however, it is important to realize that these are large-order complexes, because binding studies that purport to measure affinity become far less meaningful (if any meaning is left) due to the high order valency of these complexes). For those interested in more amplification than the SA-biotin system (or rather, interested in more fluorescence signal per antigen), you might want to consider a system devised by Peter Lansdorp (at the Terry Fox Cancer Center in Vancouver). He isolated anti-PE monoclonal antibody, and conjugated it to biotin. Thus, the sequence of steps is: (1) Biotinylated primary (2) Streptavidin-PE (3) Biotinylated anti-PE (binds to the SA-PE that is bound to primary) (4) Strepatavidin-PE and repeating steps (3) and (4) as many times as you wish... making bigger and bigger complexes. Using 3 rounds, we saw amplifications of about 5x over a single round (but didn't really try to optimize it too much). It's a bit painful because of the number of staining/washing steps involved, but for really rare antigens, it may just do the trick. mr At 1:16 PM -0600 2/25/00, ckuszyns@UNMC.EDU wrote: >I think your question may be a bit off. the object of using the Biotin >-avidin system is to be able to label a primary antibody on the cell >surface. You do not use a secondary antibody rather avidin conjugated >to a >fluorochrome. > > The theoretical increase in fluorescence that is tauted for this >complex is based on the chemistry of biotin. the biotin molecule is >capable of binding several (I believe 7) avidin molecules. This is what >gives you the increased signal since now every biotin molecule on your >primary antibody is capable of binding 7 molecules of avidin ie 7 >fluorochrome molecules. This is often more molecules than are attached >to >the secondary antibodies used for primary detection. > > One should remember however, that more is not always better. >Fluorescent molecules may be quenched when in close proximity to other >fluorescent molecules and therefore the net result may be lower intensity >signals. > > We have used biotinylated antibodies in three and four color analyses >since this gives us the option to use the second laser to detect the >avidin >binding using APC. in our hands, the biotin avidin antibody staining is >often at least 1 log brighter than the same antibody directly conjugated >to >the same fluorochrome. > > There are clearly experts in this society who have a better >understanding of this area than I so you will want to assess all >answers to >this question. > >Charles A. Kuszynski, Ph.D. >Assistant Professor/Director >University of Nebraska Medical Center >986495 Nebraska Medical Center >Cell Analysis Facility, WH 3024 >Omaha, NE 68198-6495 >402.559.6267 voice >402.559.4077 fax >ckuszyns@unmc.edu
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