Progress 09/01/19 to 08/31/22

Outputs
Target Audience:We report on two significant business developments during the project period related to reaching our target audience. The goal of this USDA/NIFA award is a commercial product for the detection of toxins in shellfish and seawater. First, LightDeck is primarily a technology development and manufacturing organization, and does not have significant sales and marketing resources, particularly for serving shellfish and water customers. Our business strategy has been to secure sales and distribution partners with a commercial organization that can help get our products out into the selected markets in the United States and Globally. Toward this end, LightDeck announced an exclusive agreement with Hach, a US-based, global leader in water quality testing equipment. The Hach partnership directly benefits this program by providing a well-established sales and marketing organization to push product commercialization. Hach has assisted LightDeck in presenting its technology at conferences and webinars to reach an audience of experts about HAB testing. Second, The NOAA/NCCOS PCMHAB program awarded LightDeck funding to pursue the Single Laboratory Validation Testing necessary for the Approval for Limited Use of this shellfish test by the Interstate Shellfish Sanitation Committee (ISSC). In addition to funding the laboratory work, this award allowed LightDeck to assemble a panel of shellfish experts to advise on the transition of this product to market. Many key opinion leaders in shellfish testing are on this panel and attending a meeting to discuss the product that LightDeck is developing under the USDA award. During this project period, the results of this project were presented at the 10th US Harmful Algae Symposium in Alabama. In addition, the results of this project were presented at an invited lecture at the University of Colorado at Denver. These audiences included undergraduates and graduate students. Changes/Problems:One major change in this proposal is the timeline. Originaly, it was anticipated that this award would have been completed in Year 2. However, LightDeck experienced delays due to delays in receiving funding and delays due to COVID19 precautions. Furthermore, LightDeck is pleased to announce the partnership with Hach, but experienced some research delays while launching the first product in conjuction with Hach. Ultimately, this delay will benefit this test as LightDeck has established a sales and distribution channel for this triplex shellfish test through Hach. A second major change was a shift in focus from the detection of 3 toxins,saxitoxin (STX), domoic acid (DA), and okadaic acid (OA), to only 2 toxins, STX and DA.The resulting Triplex Assay performance was shared with a Transition Action Committee (TAC) as part of the concurrent NOAA/NCCOS Award (NA20NOS4780181). As a result of significant differences in sensitivity across all three toxins, as well as additional hydrolysis steps necessary to measure total OA concentrations, it was recommended that the test be limited to a duplex DA and STX Test for future commercial activities. What opportunities for training and professional development has the project provided?This project has provided many training and professional development opportunities for the team working on the project. During the project period, four college students have been interns assisting with the project and have learned about immunoassay development and gained valuable experience on working in an industrial setting. The majority of the day-to-day effort has been performed by a Research Assistant II (RAII), who joined LightDeck immediately after graduating from college. This RAII has learned how to efficiently develop immunoassays including optimizing the reagents and conditions. In addition, this RAII has made the significant transition from an academic research environment to an industrial setting and is thriving in the fast-paced, product-focused culture of industry. During this project period, the RAII received was promoted from Research Associate I to Research Associated based on his ability to design and execute experiments, as well as a growth in data analysis and presentation. Additionally, a Research Associate II was promoted to Research Associate III based on her ability to lead projects independently, which was partially a result of work done on this award. A new Senior Scientist, PI Lewis, was hired during the project period, and he has learned management skills while supervising the RAII and RAIII on this project. The Senior Scientist transitioned to serve as the project PI near the end of this project period, taking on responsibility for overseeing multiple ongoing research grants, including this one. During the project period, PI Bickman, applied to NOAA/NCCOS for a Prevention Control and Mitigation Harmful Algal Bloom (PCMHAB) award for Interstate Shellfish Sanitation Committee (ISSC) approval for the triplex shellfish test being developed under this award. The proposal is complimentary and non-overlapping to the work performed under this award. Writing this grant allowed Bickman to expand her grant writing skills, form new partnerships and collaborations, and examine the potential business case for this product. How have the results been disseminated to communities of interest?Project PI Bickman attended the 10th US Harmful Algae Symposium and presented the results from this project there to a wide variety of algal bloom experts. During this project period, PI Bickman wrote the PCMHAB award (NA20NOS4780181), during which she was in informal communication (email, phone calls, etc.) with key opinion leaders in shellfish testing. Two collaborators, Dr. Gregory Doucette of NOAA/NCCOS and Dr. Steven Archer from Bigelow Laboratory for Ocean Sciences, along with seven members of a transition advisory committee were assembled for the PCMHAB grant. Through the PCMHAB award, multiple meetings were convened with approximately 20 participants to review LightDeck's progress on this triplex shellfish assay and to discuss validation protocols. The results of this assay development have been shared with the Interstate Shellfish Sanitation Conference (ISSC) for the purpose of developing validation protocols for the assay. The resulting congener coverage observed for this assay significantly outperforms current commercially available immunoassays, leading to excitement from TAC members for the development and validation of the assay. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? LightDeck achieved all of the objectives and demonstrated a 10-minute, triplex assay for the measurement of paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), and diarrhetic shellfish poisoning (DSP). Objective 1 is to demonstrate a triplex, 10-minute, one-step assay for saxitoxin (STX), domoic acid (DA), and okadaic acid (OA) in shellfish homogenate with sensitivities tuned to the regulatory limits. During Year 1 of this award, a proof of concept was demonstrated using prototype reagents. In Years 2 and 3, the prototype reagents were converted to production reagents and used to demonstrate final assay performance. LightDeck converted anti-DA and anti-OA antibodies from hybridoma derived antibodies to recombinant antibodies. Anti-STX antibodies were screened before selection based on assay performance (e.g., sensitivity and precision), as well as congener cross-reactivity. The best performing anti-STX antibody was licensed and subsequently converted to a recombinant antibody, through a similar process as the other two antibodies. Leveraging work performed and knowledge gained under a NOAA/NCCOS PCMHAB Award (NA20NOS4780186), LightDeck developed the capability to conjugate small molecule toxins to carrier proteins. This capability was used to produce conjugates of DA and OA with proteins. LightDeck has been unable to secure a commercial source of STX due to regulatory concerns. As such, the STX-protein conjugate has been sourced from a reliable vendor. The production quality antibodies and toxin-protein conjugates were combined in a multiplexed format on the LightDeck platform and demonstrated to achieve %CVs of 7.7% (OA), 5.1% (DA), 9.6% (STX) across a matrix-matched calibration curve within target sensitivities. Additionally, no cross-reactivity was observed between the different assays. The resulting Triplex Assay performance was shared with a Transition Action Committee (TAC) as part of the concurrent NOAA/NCCOS Award (NA20NOS4780181). As a result of significant differences in sensitivity across all three toxins, as well as additional hydrolysis steps necessary to measure total OA concentrations, it was recommended that the test be limited to a duplex DA and STX Test for future commercial activities. Objective 2 is to demonstrate a triplex, 10-minute assay for STX, DA, and OA in seawater optimized for sensitivity. Significant progress on Objective 2 was made during Year 1 of the project, with IC50s measured of 0.3 µg/L for STX, 0.8 µg/L for DA, and 1.5 µg/L for OA. Due to sensitivity differences between the three assays, STX and DA were measured using a 2-step assay, while OA was measured in a 1-step assay. Conversion to production quality materials helped to improve assay reliability, while allowing STX and DA to be measured in a 1-step assay with sensitivities of 0.7 µg/L for STX and 18 µg/L for DA. These 1-step IC50s are closer to the desired levels of sensitivity, relative to regulatory levels in shellfish. As with the testing in shellfish matrix demonstrated in Objective 1, minimal to no cross-reactivity was observed between the different assays. Objective 3 is to measure congener cross reactivity of the assay for STX and OA. At the recommendation of the TAC from the concurrent NOAA/NCCOS Award (NA20NOS4780181), congener cross-reactivity for only STX was pursued. STX congener cross-reactivity was measured in both extracted shellfish and saltwater matrices, demonstrating broad congener coverage in both matrices. Under the initial shellfish extraction conditions developed in Task 4.3, NEO and gonyautoxins (GTX 1-6) respectively demonstrated 99% and 22-483% cross-reactivity relative to the estimated toxicity of each congener, demonstrating that the assay adequately detects a wide selection of STX congeners. When measuring in saltwater matrix, differences in congener cross-reactivity were observed relative to shellfish matrix, most noticeably NEO and gonyautoxins (GTX 1-6) respectively demonstrating 221% and 44-133% cross-reactivity relative to the estimated toxicity of each congener. It is hypothesized that differences in the final matrix pH and ionic strength impact the congener cross-reactivity. Objective 4 is to demonstrate a fully portable kit for sample preparation including homogenizing shellfish, lysing cells in seawater, extracting toxin, and hydrolysis. Objective 4 was completed during this Project period, utilizing a combination of commercial and proprietary systems. A commercial blender was utilized for homogenization of shellfish, followed by a simple, instrument-free extraction using a syringe filter. The MQ Algae Lyse System utilizes bead beating lysis of seawater cells, which can then directly be measured using the developed assay cartridges. As part of an ongoing NOAA MERHAB Award (NA19NOS4780190), this lysis system has demonstrated ease-of-use and field portability. A portable, battery powered prototype system was developed that allowed for alkaline hydrolysis at 75 °C to convert OA esters to detectable forms. Due to the negative impacts of alkaline hydrolysis on the DA and STX assays, separate workflows were developed for the quantification of OA from DA and STX. For quantification of OA, the homogenized shellfish matrix is extracted using a 50% methanol and 50% water solution, followed by alkaline hydrolysis at 75°C for 40 minutes before neutralization and measurement. As the extraction of DA and STX does not require the presence of methanol, in this separate workflow, the homogenized shellfish matrix is extracted using 100% water. Objective 5 is to determine how well this test operates with spikes of toxins into shellfish, naturally incurred toxins in shellfish, and cell cultures. During this Project period, detection of toxin spikes in shellfish, naturally incurred toxic shellfish, and toxic cell culture samples has been completed. Initial work demonstrated performance of a triplex OA, DA, and STX Test with toxin spiked into commercial non-toxic shellfish samples. Recoveries of 70%, 123%, and 78% at the regulatory limits were measured, relative to expected, for OA, DA, and STX respectively using the extraction methods described in Task 4.3. As previously indicated, the hydrolysis methods required to measure total OA concentrations negatively impacted the DA and STX assays, as such further development prioritized a Duplex DA and STX Test. Using a matrix-matched calibration curve, DA and STX recoveries were improved to reach 102% and 93% at the regulatory limits. Further improvements in performance were observed when measuring naturally incurred toxin shellfish samples. Of the 33 natural samples analyzed, 6 and 16 were within the quantitative range for the DA and STX assays respectively, resulting in average recoveries of 99% and 81% respectively. For both toxins, 100% of samples (22 DA and 3 STX) below the assay range measured below the detection limit of the assay. For the samples above the assay range (5 DA and 12 STX), 60% and 58% reported as above the limit of quantification, however all samples within this set measured as toxic. Despite difficulties measuring samples above the upper limit of quantification, no false positives were reported, supporting use of this assay as a screening test. Several saltwater cell cultures are maintained at LightDeck Diagnostics, including Lyngbya, Aphanizomenon, and Planktothryx cultures, which are expected to produce toxins. None of the cell cultures at LightDeck tested positive for DA, so no culture data was analyzed for ASP. The STX producing Aphanizomenon spp. cell culture was tested using the cell lysis method described in Task 4.2, resulting in 92% recovery relative to the gold standard freeze-thaw method. The cultured Aphanizomenon spp. contained high concentrations of STX, requiring 1000-fold dilutions to fall within the working range of the assay.

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:We report on two significant business developments during the reporting period related to reaching our target audience. The goal of this USDA/NIFA award is a commercial product for the detection of toxins in shellfish and seawater. First, LightDeck is primarily a technology development and manufacturing organization, and does not have significant sales and marketing resources, particularly for serving shellfish and water customers. Our buisness strategy has been to secure sales and distribution partners with a commercial organization that can help get our products out into the selected markets in the United States and Globally. Toward this end, LightDeck announced an exclusive agreement with Hach, a US-based, global leader in water quality testing equipment. The Hach partnership directly benefits this program by providing a well-established sales and marketing organization to push product commercialization. Hach has assisted LightDeck in presenting its technology at conferences and webinars to reach an audience of experts about HAB testing. Second, The NOAA/NCCOS PCMHAB program awarded LightDeck funding to pursue the Single Laboratory Validation Testing necessary for the Approval for Limited Use of this shellfish test by the Interstate Shellfish Sanitation Committee (ISSC). In addition to funding the laboratory work, this award allowed LightDeck to assemble a panel of shellfish experts to advise on the transition of this product to market. Many key opinion leaders in shellfish testing are on this panel and attending a meeting to discuss the product that LightDeck is developing under the USDA award. Changes/Problems:The only major change in this proposal is the timeline. Originaly, it was anticipated that this award would have been completed in Year 2. However, LightDeck experienced delays due to delays in receiving funding and delays due to COVID-19 precautions. Furthermore, LightDeck is pleased to announce the partnership with Hach, but experienced some research delays while launching the first product in conjuction with Hach. Ultimately, this delay will benefit this test as LightDeck has established a sales and distribution channel for this triplex shellfish test through Hach. What opportunities for training and professional development has the project provided?This project has provided many training and professional development opportunities for the team working on the project. During the past reporting period, two college students have been interns assisting with the project and have learned about immunoassay development and gained valuable experience on working in an industrial setting. The majority of the day-to-day effort has been performed by a Research Assistant I (RAI) , who joined LightDeck immediately after graduating from college. This RAI has learned how to efficiently develop immunoassays including optimizing the reagents and conditions. In addition, this RAI has made the significant transition from an academic research environment to an industrial setting and is thriving in the fast-paced, product-focused culture of industry. During the last reporting period, a Reaserch Associate II was promoted to Research Associate III based on her ability to lead projects indepenedenly, which was partially a result of work done on this award. A new Senior Scientist was hired during the past reporting period and he is learning management skills while supervising the RAI and RAIII on this project. How have the results been disseminated to communities of interest?Through the PCMHAB award (NA20NOS4780181), a 4 hour virtual meeting was convened with approximately 20 participants to review LightDeck's progress on this triplex shellfish assay and to discuss validation protocols. What do you plan to do during the next reporting period to accomplish the goals?The majority of the objectives on this award are complete or nearly complete, and during the next reporting period, all of the objectives will be completed. Under Objective 1, the saxitoxin assay will be completed with production-quality reagents and optimized conditions and reagent concentrations will be used to finalize the triplex assay. Objective 2 is already complete. Under Objective 3, congener cross reactivity will be tested with spikes into shellfish and seawater. Under Objective 4, the extraction protocol will be finalized and hydrolysis demonstrated. The majority of the work will occur under Objective 5 to test spikes of toxins and cell cultures into shellfish and seawater as well as testing naturally incurred samples.

Impacts
What was accomplished under these goals? During this reporting period, significant progress was made on the research and development of the triplex test to simultaneously measure saxitoxins, domoic acid, and okadaic acids in shellfish. This project has been delayed due to COVID-19, funding delays, and executing on the aforementioned contract with Hach. However, with the approved 1-year, no cost extension of this award, LightDeck will be able to achieve all the objectives and demonstrate a 10-minute, triplex assay for the measurement of paralytic shellfish poisoning, amnesic shellfish poisoning, and diarrhetic shellfish poisoning. Progress on the project in the last reporting period was significant and the remaining tasks are anticipated to be accomplished in the next reporting period. Objective 1 is to demonstrate a triplex, 10-minute, one-step assay for STX, DA and OA in shellfish homogenate with sensitivities tuned to the regulatory limits. Prior to this reporting period, a proof of concept was demonstrated with prototype reagents. During this reporting period, four of the six key reagents were converted from prototype to production reagents. Conversion of the remaining two reagents is currently underway. The anti-domoic acid and anti-okadaic acid antibodies were converted from hybridoma derived antibodies to recombinant antibodies which can be quickly and reproducibly be made at production-scale quantities, without concern of the evolution of the cell line. These recombinant antibodies perform functionally equivalently to the hybridoma antibodies in the immunoassay. During this reporting period on a NOAA/NCCOS PCMHAB award (NA20NOS4780186), LightDeck developed the capability to conjugate small molecule toxins to carrier proteins such as ovalbumin and blood serum albumin. This capability was used on the USDA/NIFA award to produce conjugates of domoic acid with proteins and okadaic acid with proteins. These materials are now being printed on the cartridge waveguide surface as a capture reagent in these competitive immunoassays. These conjugates made in- house are better characterized and more reproducible than the prototype material that we had previously been receiving from an outside vendor. Significant technical improvements in the saxitoxin or paralytic shellfish poisoning (PSP) assay have been demonstrated and the procurement of production-quality reagents is underway. In the prior reporting period, the saxitoxin assay was not able to detect several key PSP congeners such as neosaxitoxin and GTX 1&4. A result, LightDeck sourced and tested a different anti-PSP antibody, obtaining significantly improved congener PSP cross reactivity that correlates well with the known toxicity of many congeners. This antibody has demonstrated excellent sensitivity with 50% inhibition concentrations tunable between 1-5 μg/L, which is the necessary range for this assay. Reproducibility has also been demonstrated with percent coefficients of variation of 8-10%. This antibody has been licensed and is being converted into a recombinant in a process similar to the process previously used for the antibodies used in the domoic acid and okadaic acid assays. The saxitoxin-protein conjugates cannot be made at LightDeck since we have not found a commercial source of saxitoxin. As a result, we are contracting with a vendor to obtain a reliable supply. All three assays have been tested multiplexed and with the shellfish matrix and no cross-reactivity between the three assays has been observed. Objective 2 is to develop a multiplexed test in seawater. The assay development performed under Objective 1 will directly improve the results in seawater, although objective 2 was fully achieved in Year 1 of this award. Objective 3 is to demonstrate congener cross reactivity. As described above, LightDeck switched to a new anti-PSP antibody, which improved congener cross reactivity. After the assay is finalized, congener cross reactivity will be repeated, but preliminary results demonstrated cross reactivity that correlates well with the known toxicity of the congeners. Preliminary congener cross reactivity is STX 100%; C1&2 18%; dcGTX2&3 4%; dcNEO 25%; dcSTX 16%; GTX1&4 98%; GTX2&3 66%; GTX5 50%; GTX6 69%; Lyngbia Wollei Toxin 1 25%; NEO 189%. This correlation between toxicity and congener cross reactivity is ideal as the resulting immunoassay will report results that are correspond to the toxicity of the sample. Objective 4 is to demonstrate sample preparation. Shellfish homogenization has been demonstrated using a commercially available, portable, battery-operated blender that efficiently homogenizes shellfish meat. For shellfish, toxins are extracted using a 50% methanol/50% water solution that has been extensively tested at LightDeck and is performing well. This method will be tested with alternative congeners at NOAA under the related, but non-overlapping NOAA/NCCOS PCMHAB award (NA20NO24780181) to validate this test. Saltwater algal cultures were purchased and are successfully being grown at LightDeck to be used in experiments for determining the efficacy of LightDeck's freshwater algal lysis methods with algae grown in saltwater. These cell cultures are being successfully grown at LightDeck and lysis efficiency experiments are currently underway. A prototype of a hydrolysis heater has been designed and components have been ordered to assemble and are being tested. Objective 5 is to determine how well this test operates with spikes of toxins into shellfish, naturally incurred toxins in shellfish, and cell cultures. Spikes of STX have resulted in average % recoveries of 100%. Nine naturally incurred STX samples were tested with percent recoveries ranging from 46%-334% with an average percent recovery of 132%. Additional measurements of spikes and naturally incurred samples are planned for the next reporting period.

Publications

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:During this reporting period, the results of this project were presented at the 10th US Harmful Algae Symposium in Alabama. In addition,the results of this project were presented at an invited lecture at the University of Colorado at Denver. The audience included undergraduates and graduate students. In addition, informal conversations were hadwith many key opinion leaders in the field of shellfish testing. During this past year, MBio applied for a PCMHAB NOAA/NCOOS award for funds to go through the single laboratory validation procedure described by the Interstate Shellfish Sanitation Conference (ISSC). This single laboratory validation procedure is important for applying to the ISSC for approval for limited use of the test. In assembling a collaboration for this application as well as a transition advisory committee, MBio spoke to approximately 10 key opinion leaders about this test. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has offered several opportunities for training and professional development. Two college interns have worked on this project and have learned how to do laboratory work related to this project. For both interns, this project has been their first experience working in a professional laboratory setting and their first experience working in industry. Two research assistants have worked on this project and have had significant professional development. Both research assistants have learend how to plan, organize, and execute experiments and synthesize data to inform future experiments. During the past year, the PI, Bickman, applied to NOAA/NCOOS for a Prevention Control and Mitigation Harmful Algal Bloom award for Interstate Shellfish Sanitation Committee approval for the triplex shellfish test being developed under this award. The proposal is complimentary and non-overlapping to the work being done under this award. Writing this grant has allowed Bickman to expand her grant writing skills, form new partnerships and collaborations, and examine the potential business case for this product. How have the results been disseminated to communities of interest?Bickman attended the 10th US HArmful Algae Symposium and presented the results from this project there to a wide variety of algal bloom experts. In the writing of the PCMHAB award, MBio has been in contact with several key opinion leaders in shellfish testing. Although the communication has been informal (emails, phone calls etc), these key opinion leaders have been made aware of MBio's progress on the USDA award. Under the PCMHAB award, two collaborators, and seven members of a transitionadvisory committe have been assembled. The collaborators are Dr. Gregory Doucette of NOAA/NCOSS and Dr. Steven Archer from Bigelow Laboratory for Ocean Sciences. What do you plan to do during the next reporting period to accomplish the goals?As discussed above, this project suffered from a 7-month delay in obtaining funding plus a 2.5 month COVID-19 delay. Due to the slow start on this project in Year 1, MBio anticipates putting extra resources on this project in Year 2 to finish the project on time. Significant progress was made on the technical objectives in Year 1, and the remainder of the technical objectives will be accomplished in Year 2.

Impacts
What was accomplished under these goals? Despite the 7-month delay in transferring funds for this project followed by a 2.5 month delay due to COVID-19, MBio has made significant progress on this project as described below. We are anticipating that in Year 2 of this award, we will devote extra resources to accomplish the goals of the project. Objective 1 is to demonstrate a triplex, 10-minute, one-step assay for STX, DA, and OA in shellfish homogenate with sensitivities tuned to the regulatory limits. MBio has nearly completed task 1.1, which is to demonstrate a multiplexed test for testing of shellfish. A triplex assay has been demonstrated, and it has been determined that there is minimal or no cross reactivity between the three different assays. In addition, the stretch goal of <10% CVs was achieved for the STX and DA assays, with an average %CV for each of these two assays at 9%. The OA assay is being optimized now to decrease %CVs. For the data shown below, the triplex assay was performed with dried reagents that were placed in the inlet port of the cartridge. This is the preferred product configuration and allows for further assay development to occur quickly as it minimizes experimental preparation time. The simultaneously measured standard curves for the triplex assay all show the appropriate sensitivity and adequate reproducibility. Work on Task 1.2 recently started and is pending further development of the extraction method. Objective 2 is to demonstrate a triplex, 10 minute assay for STX, DA, and OA in seawater optimized for sensitivity. Significant progress on task 2.1 has been made with all 3 assays optimized for sensitivity. It was determined that a two-step assay will be necessary for DA to achieve the necessary sensitivity, and that STX and OA can be done with either 1-step or 2-step assays. Standard curves measured in a 2-step assay are shown for STX and DA show high sensitivity and good reproducibility. A 1-step assay for OA also showed high sensitivity and acceptable reproducibility. Here, the IC50's are 0.3 ug/L for STX, 0.8 ug/L for DA, and 1.5 for OA. The remainder of the tasks in objective 2 will be accomplished in Year 2 of this award. ?Objective 3 is to measure congener cross reactivity of the assay for STX and OA and will be performed in Year 2 of this award. Objective 4 is to demonstrate a fully portable kit for sample preparation including homogenizing shellfish, lysing cells in seawater, extracting toxin, and hydroloysis. Significant progress has been made on this objective. The MQ Algae Lyse was completed and successfully tested on a wide range of freshwater cyanobacteria. An incubator was purchased for culturing the saltwater cell lines, and culture media materials and saltwater culture cell lines have been identified and will be purchased over the next month. The extraction protocol using 50% methanol, 50% water has been tested, and initial results are promising. During this time period, MBio has submitted a grant proposal in conjunction with Dr. Gregory Doucette, at NOAA/NCOOS to the NOAA PCMHAB program related to obtaining Interstate Shellfish Sanitation Conference approval for this triplex shellfish test. In writing this proposal, Dr. Doucette did a thorough evaluation of MBio's proposed extraction protocol and made some recommendations for improving it. Based on these recommendations, we are optimizing the extraction protocol and are in the process of obtaining shellfish with naturally incurring toxins from Maine to test this protocol on. Similarly, Dr. Jonathan Deeds at the FDA has recommended a hydrolysis protocol, and we are in the process of purchasing components to test this protocol. Objective 5 is to test natural samples including a range of shellfish species, toxins, congeners, seawater, and cell culture samples. MBio is in the process of acquiring some of these samples for testing in Year 2 of this award. As this summary demonstrates, MBio has made substantial progress on this award despite significant delays beyond our control. We intend to continue to work as quickly as possible on this award during the next year to make up for this lost time.

Publications