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Linking Single Domain Antibodies that Recognize Different Epitopes on the Same Target.

Glaven RH, Anderson GP, Zabetakis D, Liu JL, Long NC, Goldman ER - Biosensors (Basel) (2012)

Bottom Line: Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks.Through this work we determined that the order of genetically linked sdAb seems more important than the linker length.The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.

View Article: PubMed Central - PubMed

Affiliation: Nova Research Inc., 1900 Elkin Street, Suite 230, Alexandria, VA 22308, USA. rglaven@nmic.navy.mil.

ABSTRACT
Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.

No MeSH data available.


Related in: MedlinePlus

Sequential binding to ricin surface towards demonstrating simultaneous binding by sdAb in the H1-31-B4 construct. The left panel shows binding of a linked B4-31-B4 to a ricin surface that has been coated with sdAb-H1. The sdAb-H1 was applied over the ricin surface at a concentration of 100 nM for 3 min to approach saturation. Next the B4-31-B4 was applied in increasing concentrations as indicated on the figure. The right panel shows the B4-31-B4 applied to a ricin surface that had been coated with the H1-31-B4 construct to approach saturation using the same conditions as with the sdAb-H1. The same range of B4-31-B4 concentrations was applied.
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biosensors-02-00043-f002: Sequential binding to ricin surface towards demonstrating simultaneous binding by sdAb in the H1-31-B4 construct. The left panel shows binding of a linked B4-31-B4 to a ricin surface that has been coated with sdAb-H1. The sdAb-H1 was applied over the ricin surface at a concentration of 100 nM for 3 min to approach saturation. Next the B4-31-B4 was applied in increasing concentrations as indicated on the figure. The right panel shows the B4-31-B4 applied to a ricin surface that had been coated with the H1-31-B4 construct to approach saturation using the same conditions as with the sdAb-H1. The same range of B4-31-B4 concentrations was applied.

Mentions: It was more difficult to determine if both sdAb were binding target at the same time. Assays in which components were added to the ricin surface in series to see if binding was blocked seemed to indicate that, for at least the H1-B4 constructs, the sdAb are binding ricin simultaneously. As can be seen in Figure 1, the sdAb-B4 has a fast off rate when bound to a ricin surface. The fact that the off rate of sdAb-H1 and that of the linked construct are much slower indicates that the sdAb-H1 is binding to the ricin target. In order to do these experiments we prepared a construct in which B4 was linked to itself through the 31 amino acid linker, giving a slower off rate. Figure 2 shows that when the sdAb H1 is bound to the ricin surface that the B4-31-B4 construct can also bind, indicating that when the sdAb-H1 binds ricin, it does not block sdAb-B4’s epitope. The right panel of Figure 2 shows that when H1-31-B4 binds to the ricin surface, the binding of the B4-31-B4 construct is blocked, suggesting that the sdAb-B4 component of the linked construct is also actively binding target. These experiments, however, cannot distinguish if each sdAb in the linked constructs is binding to the same ricin, or neighboring ricins on the surface.


Linking Single Domain Antibodies that Recognize Different Epitopes on the Same Target.

Glaven RH, Anderson GP, Zabetakis D, Liu JL, Long NC, Goldman ER - Biosensors (Basel) (2012)

Sequential binding to ricin surface towards demonstrating simultaneous binding by sdAb in the H1-31-B4 construct. The left panel shows binding of a linked B4-31-B4 to a ricin surface that has been coated with sdAb-H1. The sdAb-H1 was applied over the ricin surface at a concentration of 100 nM for 3 min to approach saturation. Next the B4-31-B4 was applied in increasing concentrations as indicated on the figure. The right panel shows the B4-31-B4 applied to a ricin surface that had been coated with the H1-31-B4 construct to approach saturation using the same conditions as with the sdAb-H1. The same range of B4-31-B4 concentrations was applied.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263546&req=5

biosensors-02-00043-f002: Sequential binding to ricin surface towards demonstrating simultaneous binding by sdAb in the H1-31-B4 construct. The left panel shows binding of a linked B4-31-B4 to a ricin surface that has been coated with sdAb-H1. The sdAb-H1 was applied over the ricin surface at a concentration of 100 nM for 3 min to approach saturation. Next the B4-31-B4 was applied in increasing concentrations as indicated on the figure. The right panel shows the B4-31-B4 applied to a ricin surface that had been coated with the H1-31-B4 construct to approach saturation using the same conditions as with the sdAb-H1. The same range of B4-31-B4 concentrations was applied.
Mentions: It was more difficult to determine if both sdAb were binding target at the same time. Assays in which components were added to the ricin surface in series to see if binding was blocked seemed to indicate that, for at least the H1-B4 constructs, the sdAb are binding ricin simultaneously. As can be seen in Figure 1, the sdAb-B4 has a fast off rate when bound to a ricin surface. The fact that the off rate of sdAb-H1 and that of the linked construct are much slower indicates that the sdAb-H1 is binding to the ricin target. In order to do these experiments we prepared a construct in which B4 was linked to itself through the 31 amino acid linker, giving a slower off rate. Figure 2 shows that when the sdAb H1 is bound to the ricin surface that the B4-31-B4 construct can also bind, indicating that when the sdAb-H1 binds ricin, it does not block sdAb-B4’s epitope. The right panel of Figure 2 shows that when H1-31-B4 binds to the ricin surface, the binding of the B4-31-B4 construct is blocked, suggesting that the sdAb-B4 component of the linked construct is also actively binding target. These experiments, however, cannot distinguish if each sdAb in the linked constructs is binding to the same ricin, or neighboring ricins on the surface.

Bottom Line: Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks.Through this work we determined that the order of genetically linked sdAb seems more important than the linker length.The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.

View Article: PubMed Central - PubMed

Affiliation: Nova Research Inc., 1900 Elkin Street, Suite 230, Alexandria, VA 22308, USA. rglaven@nmic.navy.mil.

ABSTRACT
Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.

No MeSH data available.


Related in: MedlinePlus