Integrating scFv into xMAP Assays for the Detection of Marine Toxins

Abstract

Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication; environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv). Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to “preserve” monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated.

Recombinant antibodies and their use for food immunoanalysis

“…Recombinant scFvs against two marine toxins, saxitoxin and domoic acid, have been produced using the sequence information of the corresponding mAbs [119]. The assays were implemented in a microsphere-based competitive immunoassay, and the detection limits were similar to those reported with other antibodies, although the method requires further implementation.…”

Food-Borne Biotoxin Neutralization in Vivo by Nanobodies: Current Status and Prospects

“…When shellfish consume algae containing PSTs, they accumulate and concentrate these toxins, posing a risk to humans when consumed as seafood. To date, several antibodies against PSTs have been identified, including mAbs , and scFv . However, Nbs for all the aforementioned marine toxins have not yet been successfully developed.…”

“…To date, several antibodies against PSTs have been identified, including mAbs 117,118 and scFv. 119 However, Nbs for all the aforementioned marine toxins have not yet been successfully developed. Future research is anticipated to lead to the creation of Nbs with exceptional neutralization capabilities, offering hope for effective countermeasures against these toxins.…”

Current Trends and Challenges for Rapid SMART Diagnostics at Point-of-Site Testing for Marine Toxins

“…This technology enables us to discover completely new molecules, such as peptides, other binding proteins or scaffolds, which can be used for a selected target and applied on sensor platform with high efficacy [ 176 ]. An interesting approach was reported by Shriver-Lake et al for the detection of two marine biotoxins (DA and STX) where a recombinant antibody (scFv) was applied for the sensitive and specific detection within an xMAP assay (microsphere-based competitive immunoassay) [ 177 ]. Another promising approach using recombinant antibodies and a Lab-On-A-Disc (LOAD) platform was reported by Maguire et al for the detection of DA and STX.…”