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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

Fluid array assay for the detection of ricin using linked constructs, mAbs and an sdAb as capture reagents combined with a llama polyclonal anti-ricin reporter reagent. The linked constructs and mAbs gave similar absolute signals, whereas the sdAb gave lower signal. Error bars represent the variation between replicate experiments (left). The linked sdAb have lower background and perform better than the mAbs or sdAb when looking at signal versus background (right).
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biosensors-02-00043-f003: Fluid array assay for the detection of ricin using linked constructs, mAbs and an sdAb as capture reagents combined with a llama polyclonal anti-ricin reporter reagent. The linked constructs and mAbs gave similar absolute signals, whereas the sdAb gave lower signal. Error bars represent the variation between replicate experiments (left). The linked sdAb have lower background and perform better than the mAbs or sdAb when looking at signal versus background (right).

Mentions: We incorporated the linked sdAb as capture elements in Luminex fluid array assays for the detection of ricin and compared limits of detection obtained using the new reagents to those using conventional mAbs and sdAb. Results are shown in Figure 3 for detection of ricin where linked sdAb along with two mAbs and an unlinked sdAb were immobilized on Luminex microspheres. Dilutions of ricin were applied to the microspheres and binding was detected through use of polyclonal llama anti-ricin antibody. The family of H1-B4 constructs all performed about equivalently in the detection of ricin and gave approximately the same magnitude of signal as the monoclonal antibodies. However, the linked constructs are very specific and have a much lower background signal than the mAbs in the absence of ricin. This can be seen when one plots signal over background. In repeated studies, these linked constructs provided for lower limits of detection than the mAbs. Setting threshold for detection at a signal to background ratio of 4, the linked constructs are able to detect at least 0.064 ng/mL ricin while the mAbs are reliably able to detect 0.32 ng/mL.


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)

Fluid array assay for the detection of ricin using linked constructs, mAbs and an sdAb as capture reagents combined with a llama polyclonal anti-ricin reporter reagent. The linked constructs and mAbs gave similar absolute signals, whereas the sdAb gave lower signal. Error bars represent the variation between replicate experiments (left). The linked sdAb have lower background and perform better than the mAbs or sdAb when looking at signal versus background (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00043-f003: Fluid array assay for the detection of ricin using linked constructs, mAbs and an sdAb as capture reagents combined with a llama polyclonal anti-ricin reporter reagent. The linked constructs and mAbs gave similar absolute signals, whereas the sdAb gave lower signal. Error bars represent the variation between replicate experiments (left). The linked sdAb have lower background and perform better than the mAbs or sdAb when looking at signal versus background (right).
Mentions: We incorporated the linked sdAb as capture elements in Luminex fluid array assays for the detection of ricin and compared limits of detection obtained using the new reagents to those using conventional mAbs and sdAb. Results are shown in Figure 3 for detection of ricin where linked sdAb along with two mAbs and an unlinked sdAb were immobilized on Luminex microspheres. Dilutions of ricin were applied to the microspheres and binding was detected through use of polyclonal llama anti-ricin antibody. The family of H1-B4 constructs all performed about equivalently in the detection of ricin and gave approximately the same magnitude of signal as the monoclonal antibodies. However, the linked constructs are very specific and have a much lower background signal than the mAbs in the absence of ricin. This can be seen when one plots signal over background. In repeated studies, these linked constructs provided for lower limits of detection than the mAbs. Setting threshold for detection at a signal to background ratio of 4, the linked constructs are able to detect at least 0.064 ng/mL ricin while the mAbs are reliably able to detect 0.32 ng/mL.

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