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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

Binding of H1-31-B4, and parental sdAb-B4 and sdAb-H1 to ricin (left), RCA120 (middle) and abrin (right). The H1-31-B4 binds to all three targets, showing that each of the component sdAb is able to recognize target. The X axis is time in seconds, while the Y is the SPR signal reported in resonance units (RU). The different colored curves represent dilutions of the sdAb and linked construct as indicated in the figure. The vertical lines represent the beginning and ending of the injection of linked or parental sdAb.
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biosensors-02-00043-f001: Binding of H1-31-B4, and parental sdAb-B4 and sdAb-H1 to ricin (left), RCA120 (middle) and abrin (right). The H1-31-B4 binds to all three targets, showing that each of the component sdAb is able to recognize target. The X axis is time in seconds, while the Y is the SPR signal reported in resonance units (RU). The different colored curves represent dilutions of the sdAb and linked construct as indicated in the figure. The vertical lines represent the beginning and ending of the injection of linked or parental sdAb.

Mentions: After expressing the linked sdAb constructs, we used SPR to evaluate their binding specificities to ricin, RCA120, ricin A chain, and abrin. These targets allowed us to verify that each of the sdAb in the linked construct was functioning. For example, Figure 1 shows binding of H1-31-B4, along with its component sdAb, to ricin, RCA120, and abrin. RCA120 is a protein that shares high homology to ricin, but is much less toxic [26]. Abrin, like ricin, is a potent ribosome inhibiting toxin; the two toxins have high structural homology as well as some sequence homology [27,28,29]. The sdAb-H1 binds RCA120 but not abrin while sdAb-B4 binds abrin and not RCA120. The H1-31-B4 binds to RCA120 as well as to abrin, confirming that each of the sdAb in the linked construct was still able to bind target. Similarly, constructs consisting of the same sdAb in the opposite orientation as well as those containing different sdAb were evaluated to verify that each sdAb was able to bind target (data not shown).


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)

Binding of H1-31-B4, and parental sdAb-B4 and sdAb-H1 to ricin (left), RCA120 (middle) and abrin (right). The H1-31-B4 binds to all three targets, showing that each of the component sdAb is able to recognize target. The X axis is time in seconds, while the Y is the SPR signal reported in resonance units (RU). The different colored curves represent dilutions of the sdAb and linked construct as indicated in the figure. The vertical lines represent the beginning and ending of the injection of linked or parental sdAb.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00043-f001: Binding of H1-31-B4, and parental sdAb-B4 and sdAb-H1 to ricin (left), RCA120 (middle) and abrin (right). The H1-31-B4 binds to all three targets, showing that each of the component sdAb is able to recognize target. The X axis is time in seconds, while the Y is the SPR signal reported in resonance units (RU). The different colored curves represent dilutions of the sdAb and linked construct as indicated in the figure. The vertical lines represent the beginning and ending of the injection of linked or parental sdAb.
Mentions: After expressing the linked sdAb constructs, we used SPR to evaluate their binding specificities to ricin, RCA120, ricin A chain, and abrin. These targets allowed us to verify that each of the sdAb in the linked construct was functioning. For example, Figure 1 shows binding of H1-31-B4, along with its component sdAb, to ricin, RCA120, and abrin. RCA120 is a protein that shares high homology to ricin, but is much less toxic [26]. Abrin, like ricin, is a potent ribosome inhibiting toxin; the two toxins have high structural homology as well as some sequence homology [27,28,29]. The sdAb-H1 binds RCA120 but not abrin while sdAb-B4 binds abrin and not RCA120. The H1-31-B4 binds to RCA120 as well as to abrin, confirming that each of the sdAb in the linked construct was still able to bind target. Similarly, constructs consisting of the same sdAb in the opposite orientation as well as those containing different sdAb were evaluated to verify that each sdAb was able to bind target (data not shown).

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