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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

Circular dichroism of linked constructs. Representative constructs in which sdAb are joined in each orientation are shown. Blue curve is heating, and green is cooling.
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biosensors-02-00043-f005: Circular dichroism of linked constructs. Representative constructs in which sdAb are joined in each orientation are shown. Blue curve is heating, and green is cooling.

Mentions: To evaluate the melting temperature of the linked sdAb and their ability to re-fold after heat denaturation, we used circular dichroism (CD). First, we confirmed that the component, unlinked sdAb all unfolded on heating and then re-folded when cooled (Figure 4). Then each of the linked sdAb was evaluated to determine its melting temperature and ability to re-gain its secondary structure after heating. Representative spectra are shown in Figure 5; none of the linked sdAb constructs re folded as well as the component sdAb. We had previously demonstrated the ability of the component sdAb to function in assays for the detection of ricin after heat denaturation, and found that there was a good correlation between the recovery of secondary structure as judged by the CD and the ability of the sdAb to function after heating [21,22]. From the CD data, all of the linked constructs appeared to re-fold poorly after denaturation; we did not test their binding ability after heating as we expected them to have lost a large percentage of their binding ability.


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)

Circular dichroism of linked constructs. Representative constructs in which sdAb are joined in each orientation are shown. Blue curve is heating, and green is cooling.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00043-f005: Circular dichroism of linked constructs. Representative constructs in which sdAb are joined in each orientation are shown. Blue curve is heating, and green is cooling.
Mentions: To evaluate the melting temperature of the linked sdAb and their ability to re-fold after heat denaturation, we used circular dichroism (CD). First, we confirmed that the component, unlinked sdAb all unfolded on heating and then re-folded when cooled (Figure 4). Then each of the linked sdAb was evaluated to determine its melting temperature and ability to re-gain its secondary structure after heating. Representative spectra are shown in Figure 5; none of the linked sdAb constructs re folded as well as the component sdAb. We had previously demonstrated the ability of the component sdAb to function in assays for the detection of ricin after heat denaturation, and found that there was a good correlation between the recovery of secondary structure as judged by the CD and the ability of the sdAb to function after heating [21,22]. From the CD data, all of the linked constructs appeared to re-fold poorly after denaturation; we did not test their binding ability after heating as we expected them to have lost a large percentage of their binding ability.

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