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.

Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

“…For testing the kinetics of the antibody constructs, a GLC chip was coated with SEB on two lanes and ricin on two lanes, and the antibodies tested essentially as previously described [11,20,21], except that due to the high affinity of sdAb A3 it was necessary to regenerate the surface between runs using a 36 second exposure to10 mM glycine HCl pH 2.5 with 0.05% SDS. SEB was resistant to these conditions, however ricin was not, so binding to ricin was evaluated first with the surface being regenerated using 10 mM glycine HCl pH 2.5.…”

Structure of a low-melting-temperature anti-cholera toxin: llama VHH domain

“…Thermal denaturation was monitored by circular dichroism (CD) as described previously (Goldman et al, 2011). The protein was diluted to 20 mg ml À1 in deionized water; the average melting temperature (T m ) and standard deviation were calculated from three measurements using protein from two different preparations.…”

A Case of Abrin Toxin Poisoning, Confirmed via Quantitation of l-Abrine (N-Methyl-l-Tryptophan) Biomarker

“…Ribosome-inactivation tests can accurately determine if clinical samples test positive for the toxin abrin [3,8]; however, the test cannot provide a measure of the concentration of abrin in the body which may help in patient management. Abrin antibody detection and radioimmunoassay tests have also been used to detect abrin exposure in clinical samples [2][3][4][5][6][7][8][9].…”

“…Ribosome-inactivation tests can accurately determine if clinical samples test positive for the toxin abrin [3,8]; however, the test cannot provide a measure of the concentration of abrin in the body which may help in patient management. Abrin antibody detection and radioimmunoassay tests have also been used to detect abrin exposure in clinical samples [2][3][4][5][6][7][8][9]. While these tests are sensitive and can provide quantitative results, they require large sample volume, overnight incubation (in the case of radioimmunoassay test), and more invasive procedures to obtain patient samples [2,11].…”