1. Paralytic Shellfish Poisoning: Preparation of Monoclonal Antibodies to Saxitoxins for use in PSP Detection Thomas N. Stewart 1, Randy Allen 1, R. Wayne Litaker 2 and Pat Tester 2 1 Mercury Science Inc., Raleigh, NC 27607, USA 2 National Ocean Service, NOAA, Beaufort, North Carolina 28516, USA Paralytic Shellfish Poisoning: Preparation of Monoclonal Antibodies to Saxitoxins for use in PSP Detection Thomas N. Stewart 1, Randy Allen 1, R. Wayne Litaker 2 and Pat Tester 2 1 Mercury Science Inc., Raleigh, NC 27607, USA 2 National Ocean Service, NOAA, Beaufort, North Carolina 28516, USA ABSTRACT Paralytic shellfish poisoning (PSP) is a serious public health threat caused by the consumption of shellfish contaminated with saxitoxins (STX). Shellfish become toxic when they consume miroalgae in the genus Alexandrium which are the source of the STXs. Blooms of toxic Alexandrium are spatially and temporally variable. This makes it difficult to predict when shellfish are likely to become contaminated. The logical solution would be to monitor STX levels in shellfish on a regular basis using HPLC or LC_MS methods, but this is not practical given the vast shoreline of Alaska which would need to be sampled, the high cost per assay, and the shipping and processing times involved. What is needed to address this issue is a means of directly testing for saxitoxins using an inexpensive, rapid field test. Such kits generally employ an enzyme-linked immunoassay (ELISA) format. Developing ELISAs for STX, however, is challenging because there are over 50 different structural forms (congeners) of saxitoxin which cause varying degrees of toxicity. Currently there are ELISA test kits on the market, but these kits fail to detect a number of toxic congeners which may be present in shellfish. The goal of this project is to develop monoclonal antibodies with specificities which allow them to fully detect the toxic STX congeners which are not detected using the current kits. Having these antibodies should allow development of novel ELISA assays capable of improved detection of the STX levels in shellfish, better protection of human health and improved utilization of shellfish resources. Our approach is to prepare several STX immu- nogens having different structures that mimic the major classes of STX. We report here the results of immune response to our initial STX immunogen indicating successful immunization and hybridoma production. INTRODUCTION: STX TOXICITY VARIES WITH STRUCTURE INITIAL RESULTS: SERA RESPONSE AND HYBRIDOMA SELECTION CONCLUSIONS METHODS: PREPARATION OF ANTIBODIES 1.IMMUNIZE: Mice are immunized with a derivative of saxitoxin covalently attached to bovine serum albumin. Since potentially toxic metabolites can be produced by the immunogens, additional steps are taken to identify immunization conditions that are not lethal. 2. MEASURE RESPONSE: Mice sera are tested for immune response producing antibodies to saxitoxin. Several mice are immunized since the immune response can vary greatly between mice. 3.PERFORM FUSION: A fusion is performed on the spleen of the mouse showing the best response to the immunogen and hybridoma cells are created. Hybridoma cells are immortal and produce monoclonal antibodies. 4. IDENTIFY CLONES: Hybridoma cells producing anti- STX antibodies are identified and cloned. 5. SELECT BEST ANTIBODIES: Sensitivity and specificity of clones are measured to determine which antibodies are most suitable for STX assay development. Figure 1. The sera of mice immunized with STX-1-BSA were tested for binding to STX-HRP conjugate. In this assay, mouse #8 shows the highest concentration of antibodies to STX. The Control mouse has not been exposed to STX-1-BSA and has no STX antibodies. A novel synthetic method to prepare STX immunogens and enzyme conjugates has been developed. An immune response and recognition of STX-HRP has been demonstrated and monoclonal antibodies to STX have been developed. These antibodies are currently being screened for cross reactivity to different STX congeners. Additional antibodies to different toxic congeners of STX will be produced. Learn more at www.mercuryscience.com email: info@mercuryscience.com The saxitoxins encompass a family of more than 50 related neurotoxins. The main difference among congeners are how various side groups (H, OH, SO 3 -, or OSO 3 - ) are attached to the backbone structure of the toxin. The chart on the right illustrates the specific side group configurations of the most common congeners found in shellfish. Using novel immunogens, a panel of antibodies that can detect STX congeners with different key side groups can be produced. Once developed and tested for cross-reactivity, the resulting antibodies can be combined in an ELISA assay capable of detecting the cumulative toxicity of a wide range of STX congeners. ACKNOWLEDGEMENTS This research was funded by: North Pacific Research Board: Project #1118 Improved Detection Kit for the Toxins Which Cause Paralytic Shellfish Poisoning STX immunogens are synthesized and generously provided by: Dr. Sherwood Hall Food & Drug Administration, College Park, MD 20740, USA STX-1-BSA Figure 2. Hybridoma cells produced during the fusion are assayed for recognition of STX. In thi s photo, eight of the ninety six hybridoma cell lines tested show binding to STX. These hybridomas have been cloned and are currently being evaluated for recognition of STX congeners. CongenerR1R2R3R4 STXHHHH B1HHHSO 3 - GTX2HOSO 3 - HH C1HOSO 3 - HSO 3 - GTX3HHOSO 3 - H C2HHOSO 3 - SO 3 - NeoOHHHH B2OHHHSO 3 - GTX1OHOSO 3 - HH C3OHOSO 3 - HSO 3 - GTX4OHHOSO 3 - H C4OHHOSO 3 - SO 3 - hybridoma cell