Llama-derived single domain antibodies specific for Abrus agglutinin

Abstract

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations.

Keywords: abrin; reversible refolding; single domain antibody.

Figure 1

Luminex direct binding, representatives of the first six sequence families isolated through panning two rounds on commercial abrin. The sdAb were examined for binding to commercial abrin (abrin), abrax, and commercial abrin toxoid (abrin-td) as well as the related toxin ricin, ricin a chain, ricin b chain, and RCA120, and unrelated proteins staphylococcal enterotoxin B (SEB), and bovine serum albumin (BSA).

Figure 2

Luminex direct binding, representatives of the two sequence families isolated after panning three rounds on commercial abrin and two anti-abrin mAbs. The sdAb and mAbs were examined for binding to commercial abrin (abrin), abrax, and commercial abrin toxoid (abrin-td) as well as the related toxin ricin, ricin a chain, ricin b chain, and rca120. Note the difference in the scale of the Y axis between the mAbs and sdAb.

Figure 3

Circular dichroism on the three sdAb that showed high affinity and specificity to the commercial abrin toxin. Each sdAb was monitored through one heating and cooling cycle.

Figure 4

Luminex direct binding, representatives of two clones isolated after panning on the abrin fractions. Binding data is shown for commercial abrin (abrin), the abrin fractions (Frac I, Frac II, and Frac III), the commercial abrin toxoid (abrin td), the Abrus agglutinin (Abrus Agg), ricin b chain (RB), ricin a chain (RA), Abrax, ricin, and RCA120.

Figure 5

Sandwich assay using the Luminex for commercial abrin (panel A), abrin fractions I II and III (panel B), and Abrus agglutinin (panel C) with a sdAb and monoclonal captures paired with a llama polyclonal tracer. Data is reported as the ratio of signal over background, with a value of 5 considered positive.