1.  The need for quantitative, real time, shipboard detection methods for risk management of shellfish poisons is eminent. Harvesting resources are more effective when they have the ability to accurately determine the level of toxins that may be present in shellfish. Current semi-quantitative, lateral flow, shipboard procedures for Paralytic Shellfish Poisoning (PSP) have been shown to have a false positive rate in excess of 30%. Harvesting costs have increased as product, whose samples were determined to be positive by the shipboard procedure, were passed by and later determined by dockside mouse bioassay (MBA) to be safe to harvest. A quantitative, microtiter plate format, Saxitoxins (STX) ELISA procedure has been adapted and customized for shipboard testing for PSP. The PSP assay range is from 20 µg to 120 µg (STX equivalent)/100 g composite sample of shellfish, with regulatory interests at 40 µg and 80 µg/100 g. The application employs a handheld, durable photometric analyzer with LCD readout. Absorbances are entered into a tailored program in a mini-laptop computer and concentrations calculated. Crews have achieved timely, reproducible results while at sea with both ease and precision. With samples obtained off the coast of Northeastern United States, a false positive rate, based on MBA results, is less than 1%. The shipboard protocol can be applied to other marine toxins in shellfish such as ASP, DSP and NSP. The Abraxis shipboard applications allows shellfish harvesting operations to make specific, measurable, actionable, realistic and timely decisions enabling improved cost efficiency while managing the public health risks associated with shellfish poisons. ABSTRACT  This work describes the efficacy, precision, and portability for quantitative analysis of Saxitoxins at sea.  The ELISA exhibits within and between assay precision of less than 10% and accuracy between 82- 110% which provides for consistent monitoring of environmental samples.  The ELISAs are rapid (results within 60 minutes) and very sensitive, allowing for the monitoring of shellfish samples to comply with regulatory levels. (i.e. harvesting level at 80 µg/100g). The shellfish extraction minimized solvents use to increase safety onboard ship. CONCLUSIONS CONCLUSIONS  Abraxis PSP Shipboard application is intended for the screening of PSP toxins in the range of 20µg/100g to 120µg/100g composite shellfish tissue. Samples requiring regulatory action should be confirmed by alternative methods such as HPLC, GCMS, Mouse Bioassay or other conventional method.  Development of a portable system to extract and dilute samples, perform ELISA, and analyze tissue concentrations at the ppb level. OBJECTIVES OBJECTIVES Application of a Quantitative ELISA Screening Procedure Suitable for Shipboard Analysis of Marine Algal Toxins in Shellfish Tissue Fernando M. Rubio, David C. Deardorff, Jaclyn A. Pitt Abraxis LLC, Warminster, PA, USA METHODS METHODS Assay Performance RESULTS AND DISCUSSION  Very sensitive PSP toxins ELISA has been developed in a portable and user-friendly application. Shellfish tissue sensitivity for Saxitoxin is 0.010 ppb or STX eq. 20µg/100g.  Saxitoxin Shipboard ELISA kits show good correlation to Mouse Bioassay, while the currently used lateral flow technology yielded >99% false positive indication.  Upon training, the crews demonstrated ease of use with reproducible and accurate results. Acceptable B/Bos and average absorbances were provided for QA self-check.  The ELISAs exhibits good recoveries of algal toxins from spiked water samples: Saxitoxin 86%.  The ELISA showed a relatively low intra and inter assay variance: Saxitoxin < 9%.  Saxitoxin Cross-Reactivity: The ELISA has good cross-reactivity against Saxitoxin (STX), decarbamoyl STX, GTX 2 & 3, GTX 5B and less cross- reactivity towards other saxitoxin congeners. However, due to the great sensitivity of the assay (0.010 ppb), monitoring of total saxitoxin in water in addition to shellfish tissue is possible. References References 1) M. G. Weller, A. Zeck, A. Eikenberg, S. Nagata, Y. Ueno, and R. Niessner, Development of a Direct Competitive Microcystins Immunoassay of Broad Specificity. Analytical Sciences. 17, 2001, 1445-1448. 2) Worldwide Patenting PCT WO 01/18059 A2. 3) U.S. Patent Number 6,967,240 4) W. J. Fischer, I. Garthwaite, C.O. Miles, K.M. Ross, J.B. Aggen, A.R. Chamberlin, N.A. Towers, and D.R. Dietrich, Congener-Independent Immunoassay for Microcystins and Nodularins. Environ. Sci. Technol. 35, 2001, 4849-4858. ELISA Assay Procedure Saxitoxin Ship Board Microtiter Plate Components Saxitoxin Cross-Reactivity Precision The following results were obtained: Control 1 2___ _ 3__ Replicates 3 3 3 Days 4 4 4 n 9 9 9 Mean (ppb) 0.023 0.073 0.153 % CV (within assay) 8.7 2.5 3.2 % CV (between assay) 8.4 2.5 2.6 ______________________________ ____ Recovery Four (4) groundwater samples, were spiked with various levels of Saxitoxin and then assayed using the Abraxis Saxitoxin Assay. The following results were obtained: Amount of-------- Recovery ---- --- SaxitoxinMeanS.D. Recovery Added (ppb)(ppb)(ppb)( %) ____________________________ ___ 0.0400.0330.00482 0.0800.0670.00483 0.2000.1840.00892 Average 86 ____________________________ ___ Table 1. Precision and Recovery Data www.abraxiskits.com Phone: 215-357-3911 FAX: 215-357-5232 Email: info@abraxiskits.com Sample Extraction: -Remove tissue from shell by separating the adductor muscles and tissue connecting at hinge. -Collect about 120-150 mL shellfish tissue. Wash and drain thoroughly with fresh water. -Blend at high speed until pureed. -Transfer 10 mL of shellfish puree to 20 mL plastic tube. -Prepare extraction liquid by mixing 2.5 parts 70% isopropanol + 1 part 5% Acetic Acid. -Add 10 mL extraction liquid to 10 mL shellfish puree. -Filter through small pore filter (i.e coffee filter) and transfer to a clean labelled test tube. Sample is ready for dilution – 1:20,000 in pre-dispensed vials.. Saxitoxin Toxin Profile – New England Saxitoxin Ship Board Standard Curve Data Analysis SAMPLING SITE PROCEDURE DEVELOPMENT OF IMMUNOASSAY