Matched sandwich pairs of affinity molecules that bind simultaneously to an antigen (target) can validate the identity of a target molecule, enable greater assay specificity, improve assay sensitivity, and enhance research results.
Using the X-Aptamer Selection Kit, you can readily and cost effectively develop a binding partner for your existing antibody.
X-Aptamer and antibodies complement each other, each having their particular advantages. Antibodies are comprised of amino acids, while X-Aptamers are primarily nucleic acids, which are about one-tenth the size of most antibodies. X-Aptamers and antibodies perform a similar function but bind with a target differently – in an orthogonal manner.
It is cumbersome and expensive to develop a sandwich pair of Abs to a target, which is one reason why so many antigens (targets) do not have a matched sandwich pair. The X-Aptamer Selection Kit enables virtually any scientist with access to basic laboratory equipment to develop a matched X-Aptamer sandwich partner for an existing antibody (polyclonal or monoclonal). It is very likely that the selected X-Aptamer will bind to a different epitope than an Ab. The XA:Ab pair can then be used in many types of assays, such as an Enzyme Linked Oligonucleotide Assay (ELONA) as depicted in the diagram below, which is the equivalent of an ELISA that uses two sandwich Abs.
Selecting an X-Aptamer binding partner for an Ab is straightforward. It is easiest if the purified target protein (or a reasonable portion of it) is available or can be synthesized/expressed. However, if the protein target is not available, then an existing Ab may enable isolation of the native target from a biofluid sample, which could then be immediately used in an X-Aptamer selection.
Examples of ELONA using one Ab and one X-Aptamer
FAQ: Sandwich Ab:XA Pair
Basic laboratory equipment (such as a thermocycler, magnet or magnetic stand, centrifuge, pipettes, etc.) and common supplies (such as buffers, BSA, magnetic particles, etc.). Please download the kit protocol for more information.
Probably not. X-Aptamers (as well as traditional aptamers) usually do not bind very well to denatured proteins. They usually need the protein target (antigen) in native conformation.
Any assay that does not denature the protein target (antigen). ELONA (ELISA using at least one oligonucleotide instead of an Ab), Luminex style assays, flow cytometry, immunohistochemistry, etc.
Please see our page dedicated to the kit. Click Here
Absolutely! The X-Aptamer Selection Kit protocol is straightforward and has been successfully performed by undergraduate students. This is NOT the cumbersome SELEX process for selecting aptamers.
Yes. We can perform the selection for you. Click here for details.
Generally, the kit protocol can be completed in a few (2 to 4) days. Subsequent AM Biotech processing of the kit sample and synthesis of the putative X-Aptamers for you to test takes about three to five weeks.
An X-Aptamer is an affinity agent (similar in function to an antibody) that is comprised of a single strand of DNA with one or more (usually several) non-DNA chemical modifications attached to one or more of the DNA nucleobases and/or backbone. The most common modifications used in an X-Aptamer are amino acid functional groups such as indole (tryptophan), phenol (tyrosine), and amine (lysine). Other modifications are also used. The unique chemistry of X-Aptamers significantly enhances interaction with a target for better binding affinity and better specificity than unmodified DNA or RNA aptamers.
It would be best to have the entire protein that the Ab recognizes (many Abs antigens are actually only a portion of the amino acid sequence of the targeted protein). However, having a reasonable portion of the full length of the targeted protein that has a good likelihood of folding into the native conformation or at least a folded domain of the targeted protein is sufficient.
X-Aptamer selections require a way to isolate the target using magnetic particles. This can be accomplished by chemically biotinylating the target, by using a target expressed with a His tag, or by just using an existing tagged Ab to the targeted protein.
Generally, no. X-Aptamers and Abs are orthogonal affinity molecule technologies and thus bind to proteins differently. Ab’s bind to convex surfaces of proteins while X-Aptamers often bind to clefts. While it is possible that an X-Aptamer will bind to the same protein epitope as an Ab, we have found that in the vast majority of cases, X-Aptamers and Abs will bind to different protein epitopes.
Virtually any label, functional group, dye, linker, etc. that you can think of. X-Aptamers are synthetic and thus functionalization is typically straightforward. X-Aptamers can also be functionalized to attach to virtually any material.
Since X-Aptamers are synthetic, then AM Biotech can synthesize as much X-Aptamer as you need in perpetuity. It will always be available. The price for additional amounts of X-Aptamers is comparable to the price of an equivalent molar amount of Ab.