Progress 09/01/18 to 12/31/19

Outputs
Target Audience:The majority of the work done on this project was technical, but there were several outreach efforts that reached the target audience. First, to ascertain whether this product would be of interest to shellfish aquaculture, several aquaculture facilities were contacted. Many of these facilities were interested. Second, over 10 regulators around the US were contacted to determine whether the product would be of interest and also whether shellfish samples containing toxins could be obtained during a Phase II award. The response from these regulators was overwhelmingly positive and there is significant interest in the product and in sharing shellfish samples during a Phase II award. Third, this research was included in several MBio presentations during the award period. In particular, this research was included in the keynote address at the Colorado Lakes and Rivers Management Association annual luncheon. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunities for training an professional development. The PI, Dr. Bickman, has significantly benefited from this opportunity to improve her project managment and supervisory skills. The research assistant who worked on this project has benefited from this project enormously. Prior to working on this project, the research assistant had not developed bioassays, and now is comfortable performing a wide range of the necessary laboratory tasks necessary for this work. How have the results been disseminated to communities of interest?MBio has contacted over 10 potential customers to better understand their needs for this product. During these discussions, MBio has shared the results of the Phase I research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this Phase I award, all of the objectives were successfully met. As described in the proposal, there were two primary technical objectives that demonstrated proof-of-concept for this system for measuring toxins generated by harmful algal blooms and concentrated in shellfish. These objectives were: Deliver one-step, 10-minute assays forsaxitoxin, domoicacid, and okadaic acid tuned to regulatory limits for shellfish meat. Demonstrate a rapid, simple, and cost-effective sample preparation and extraction protocol that is suitable for use at the sample collection site and is compatible with the assay developed under objective 1. ?Task 1.1 Two Step Assay Development. This task was to perform 2-step competitive immunoassays to determine the maximum assay sensitivity. These assays were performed with toxin-protein conjugates bound to the surface of the waveguides used in cartridges for the MBio LightDeck technology system. Anti-toxin antibodies were added to the cartridge to bind to the microarray spots containing toxin-protein conjugates when toxin is not present in the sample. When significant toxin concentrations are present in the sample, the antibody binds to the toxin in the sample instead of the microarray spot. A secondary anti-species antibody conjugated to a fluorescent dye was added to the cartridge to be used for fluorescence detection. Using this 2-step method, assay sensitivities were measured. For saxitoxin (STX) the 50% inhibition concentration (IC50) was 2 ug/L, for domoic acid (DA) the IC50 was 0.9 ug/L, and for okadaic acid (OA) the IC50 was 1 ug/L. These sensitivities are sufficient for measuring these three toxins near the regulatory limit in shellfish. Furthermore, all of these sensitivities are high enough to be of interest for measuring the toxins directly in seawater, which is an objective in Phase II. Task 1.2 One-Step Assay Development. This task was to simplify the assay by conjugating the anti-toxin antibodies with a fluorescent dye thereby simplifying the assay protocol from 2-steps down to 1-step. To accomplish this task, all three anti-toxin antibodies were conjugated to the fluorescent dye with at least three different ratios of dye to antibody. The optimal degree of labeling for the antibody was determined and one-step assays were performed. The STX and OA assays had similar sensitivities for the 1-step and 2-step assays. As expected, the 1-step DA assay had an increase in IC50 after dye conjugation from 1 ug/L to 6 ug/L. This decrease in sensitivity for the DA assay due to conjugation to a dye has been observed before both at MBio and by collaborators. Even with a 6 ug/L IC50, the 1-step DA assay still has 10x better sensitivity than is required for testing shellfish samples. Therefore, the decrease in sensitivity due to conjugation of this antibody to a fluorescent dye, is not a problem. The DA assay will be further detuned in Phase II to better match the regulatory limits for DA in shellfish. Task 1.3 Single-plex Assay Demonstration. All three assays met or exceeded the goals for the single-plex one-step assay demonstration. The IC50 of the STX assay was 6 ug/L, which corresponds to 0.5 mg/kg in shellfish according to the protocol developed under objective 2. Since the regulatory limit for STX is 0.8 mg/kg, this sensitivity provides good accuracy near the regulatory limit and sensitivity well below the regulatory limit. Similarly, the IC50 of the DA assay was 6 ug/L, which corresponds to 0.5 mg/kg, which is well below the regulatory limit of 20 mg/kg. The sensitivity of this assay will be detuned in Phase II to better match the regulatory limit by changing antibody concentrations or switching to a less sensitive antibody clone. The IC50 of the OA assay was 1.5 ug/L which corresponds to 0.12 mg/kg and the regulatory limit is 0.16 mg/kg. Assay reproducibility for all three assays was good. For each assay, 5 replicates were measured at each of 7 different concentrations. For STX and OA these concentrations were 0, 0.316, 1, 3.16, 10, 31.6, and 100 ug/L. For DA these concentrations were 0, 3.16, 10, 31.6, 100, 316, and 1000 ug/L. For the STX assay, the % coefficient of variation (%CVs) far exceeded the stretch goal of <10%. For the 6 lowest concentrations the %CV for the STX assay ranged from 3.2%-9.5% with an average of 6.8%. At the IC50, the %CV was 3.2% for STX. The DA assay also met the goals for reproducibility with a %CV of 8.5% at the IC50. The OA assay also met %CV goals with an average %CV of 12.5% and a %CV of 14.1% at the IC50. Task 2.1 Homogenization Method. Several methods were tested to effectively homogenize shellfish in the field. A commercial tissue homogenizer (Omni International P/N TH115) was purchased and compared to a low-cost battery-operated blender and a prototype made by putting a blade onto MBio's MQ Algae Lyse machine, which was originally designed to lyse cyanobacteria. The commercial tissue homogenizer provided excellent shellfish homogenates, but could only homogenize small quantities of tissue at a time. The battery-operated blender was suitable for large samples and is field portable, but the homogenate is not as well-ground as with the other options. The blade attached to the MQ Algae Lyse was found to be an excellent option for homogenization. It produced a well-homogenized sample, is designed to be portable, and is expected to lyse seawater samples as well as shellfish samples. The vision for the product is to sell the MQ Algae Lyse with either a whisk for cell lysis, or with a blade for shellfish homogenization. Therefore, the same instrument can be used for different attachments for field measurements of toxins in shellfish or in seawater. In Phase II, MBio anticipates further development of this method of shellfish homogenization in the field. Task 2.2 Extraction Method Optimization. Based on a literature search, MBio decided to extract toxins using a 50% methanol 50% water mixture. This mixture will extract hydrophilic and lipophilic toxins simultaneously. The extraction protocol that MBio developed was to combine 1 g of shellfish homogenate with 4 mL of the methanol/water mixture and to shake for 10 minutes based on the protocol in reference1. The resulting solution is withdrawn, diluted 1:10 in a dilution buffer, and added to the assay. This procedure was demonstrated to be compatible with the assay while still extracting toxin. Task 2.3 Demonstration of Sample Preparation Method. The sample preparation method was demonstrated by measuring certified negative mussel tissue, certified positive mussel tissue for domoic acid, and spikes of all three toxins into clam samples purchased commercially. Clam samples were spiked with either 0.8 mg/mL of STX, 20 mg/kg of DA, or 0.16 mg/mL of OA and measured on both the MBio assay and Neogen Reveal 2.0. All of the spiked samples had excellent reproducibility exceeding the 15% goal. The %CVs were 14.7% for STX, 12.4% for DA, and 12.1% for OA. For the DA assay, there is good agreement between the spiked value of 20 mg/kg and the measured value of 19 mg/kg. For the STX assay, the spiked value was 0.8 mg/kg and the measured value was 0.4 mg/kg. Similarly, for the OA assay the spike was 0.16 mg/kg, but the measured value was 0.4 mg/kg. The discrepancies in the measured STX and OA concentrations is under investigation. In summary, the objectives of the Phase I award were met and the project is on track to meet all objectives proposed for Phase II. (1) Campbell, K.; McNamee, S. E.; Huet, A. C.; Delahaut, P.; Vilarino, N.; Botana, L. M.; Poli, M.; Elliott, C. T. Anal Bioanal Chem 2014, 406, 6867-6881.

Publications

Progress 09/01/18 to 08/31/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has been instrumental in the professional development and training of the two people primarily working on the project. The PI, Sarah Bickman, has used this project to learn about gathering customer opinions, writing market analyses, and commercialization plans. The research assistant performing most of the experiments has learned many new laboratory techniques and protocols to fluorophore-label the antibodies and to perform two-step assays. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?There are several tasks planned to meet the project goals in the next reporting period. These tasks include: Goal 1: Task 1.1 Two step assay development on the OA assay will begin this week and is completed for the other two assays. Task 1.2: The 1-step STX and DA assays will be optimized and the OA assay will be tested and optimized. Task 1.3: Reproducibility studies of 1-step STX, DA, and OA assays: Standard curves for all three assays in a 1-step assay format will be generated with higher replicate numbers to demonstrate assay sensitivity and reproducibility. Goal 2: Task 2.1: Complete Task 2.2: The extraction method will be optimized with negative clam homogenate once task 1.2 is complete. Task 2.3: Spiked Shellfish Tissue: Extractions of toxin from spiked shellfish samples will be tested on the MBio assay and in reference to the Neogen Reveal 2.0 tests. Next, extractions of toxin from a certified reference sample of mussel tissue containing domoic acid will be tested on the MBio assay and on the Neogen Reveal 2.0 tests.

Impacts
What was accomplished under these goals? During the first half of this Phase I SBIR award, MBio has made significant progress towards the project goals. The project is on-track for time and budget and no significant problems have been encountered. Shellfish aquaculture is a significant economic sector and domestic food source with more than $240M produced in the United States annually. Expansion of this food source is desirable, since shellfish are an ideal aquaculture product; they grow readily, are environmentally friendly, are easy to seed, and are immobile. However, shellfish are filter feeders and can accumulate high concentrations of toxins generated by harmful algal blooms (HABs). HABs are increasing in frequency and severity and cannot always be visually detected. All shellfish sold in the United States, European Union, and 18 other countries have regulatory limits for three common toxins found in shellfish meat. Each species of shellfish harvested in an area must be tested, since different types of shellfish accumulate and depurate these toxins at different rates. Therefore, there is a sizable market for tests for HAB toxins in shellfish. Current testing requires performing three different tests for the three different common toxins for paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP) and diarrhetic shellfish poisoning (DSP). Performing these tests burdens aquaculture, since one toxin requires a laboratory test, which potentially has days of delay between sample shipment and receiving results. Even the two toxins which have approved rapid tests can be burdensome to aquaculture, since they have different protocols and must be purchased from different manufacturers. Therefore, testing adds significant delays and costs associated with bringing shellfish to market. In addition to the required regulatory testing, better testing would allow growers and regulators to better manage shellfish harvests. Shellfish that have been harvested that contain significant toxin levels must be disposed of appropriately, but toxin-containing shellfish that are still growing can be allowed to continue to grow while depurating. Therefore, significant costs and waste can be avoided by knowing whether shellfish contain toxins prior to harvesting. MBio Diagnostics is developing a transformative platform technology that will enable users in the field to perform cost-effective, multiplexed, rapid, laboratory-quality HAB Toxin testing. Under this award, MBio is developing tests for saxitoxin (STX) which causes paralytic shellfish poisoning (PSP), domoic acid (DA) which causes amnesic shellfish poisoning (ASP) and okadaic acid (OA) which causes diarrheitc shellfish poisoning (DSP). In Phase II, these tests will be combined into a single cartridge with a single protocol that will allow users to simultaneously test for all three toxins. This technology will protect the safety of the nation's food supply while enabling expansion of aquaculture by reducing the time and cost necessary to bring shellfish to market. There are two primary technical goals to demonstrate proof-of-concept: Deliver one-step, 10-minute assays forsaxitoxin, domoicacid, and okadaic acid tuned to regulatory limits for shellfish meat. Demonstrate a rapid, simple, and cost-effective sample preparation and extraction protocol that is suitable for use at the sample collection site and is compatible with the assay developed under goal 1. During the first half of this award, MBio has made significant progress towards both of these goals: Task 1.1 Two Step Assay Development: All reagents have been received. Significant progress has been made on the STX and DA two-step assays since these reagents in November. The conditions for printing spots on the microarray in the MBio cartridge have been optimized for both assays. Third, two-step assays for both the STX and DA targets have been performed demonstrating the necessary assay sensitivity and reproducibility. In these two-step assays, first antibody combined with a sample is added to the cartridge. Then, a secondary fluorophore-labeled detection antibody is added to the cartridge. Reagents for OA arrived in mid-January and development of this assay is now underway. Task 1.2 One-Step Assay Development: To simplify the workflow, MBio is working on 1-step assays for both STX and DA. These one-step assays require that the STX and DA antibodies be labeled with fluorophores, and the labeling conditions for both assays have been optimized. A one-step standard curve has been measured on the STX assay demonstrating sensitivity 3 orders of magnitude better than is necessary. The STX assay will be optimized for lower sensitivity to match the necessary sensitivity for the regulatory limits for STX in shellfish. The one-step DA assay has demonstrated sensitivities 8x greater than necessary. Both the STX and DA assay sensitivities will be appropriately tuned once the OA assay sensitivity has been demonstrated. Since the Phase II goal is to have all three assays measured simultaneously measured, the sensitivity of each assay will be tuned to the appropriate regulatory limits with the appropriate dilution from the sample preparation protocol. Task 1.3 Single-plex assay demonstration: This task cannot be performed until Task 1.2 is complete, but data from task 1.1 indicates that MBio will be able to meet the goals for this demonstration. Task 2.1 Homogenization Method: MBio has successfully completed this task. MBio has tested four different methods of homogenizing shellfish using live clams and demonstrated proof of concept for homogenization. These four methods include a laboratory grade tissue homogenizer, a bead beating method, a low-cost battery-operated blender, and a blade attached to MBio's system for lysing cyanobacteria (MQ Algae Lyse). Of these four methods, the battery-operated blender and the blade attached to the MBio MQ Algae Lyse were most effective for the sample sizes necessary for these assays. In Phase II one of these options will be productized. Task 2.2 Extraction Method Optimization: A protocol for extracting toxins from the shellfish homogenate has been identified and tested for assay compatibility with clam homogenate that does not contain toxins. This protocol was chosen because it should be a universal extraction protocol that will extract both lipophilic and hydrophilic toxins using a 50% methanol/50% water solution. A literature search identified this protocol (Campbell 2014), which may be refined in the second half of this grant and in Phase II. In particular, MBio is interested in whether a more environmentally-friendly protocol can be used. Katrina Campbell, Sara McNamee, Anne-Catherine Huet, Phillippe Delahaut, Natalia Vilarino, Luis Botana, Mark Poli, Christopher Elliott, Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish. Anal Bioanal Chem (2014) 406:6867-6881.

Publications

Progress 09/01/18 to 04/30/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has been instrumental in the professional development and training of the two people primarily working on the project. The PI, Sarah Bickman, has used this project to learn about gathering customer opinions, writing market analyses, and commercialization plans. The research assistant performing most of the experiments has learned many new laboratory techniques and protocols to fluorophore-label the antibodies and to perform two-step assays. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?There are several tasks planned to meet the project goals in the next reporting period. These tasks include: Goal 1: Task 1.1 Two step assay development on the OA assay will begin this week and is completed for the other two assays. Task 1.2: The 1-step STX and DA assays will be optimized and the OA assay will be tested and optimized. Task 1.3: Reproducibility studies of 1-step STX, DA, and OA assays: Standard curves for all three assays in a 1-step assay format will be generated with higher replicate numbers to demonstrate assay sensitivity and reproducibility. Goal 2: Task 2.1: Complete Task 2.2: The extraction method will be optimized with negative clam homogenate once task 1.2 is complete. Task 2.3: Spiked Shellfish Tissue: Extractions of toxin from spiked shellfish samples will be tested on the MBio assay and in reference to the Neogen Reveal 2.0 tests. Next, extractions of toxin from a certified reference sample of mussel tissue containing domoic acid will be tested on the MBio assay and on the Neogen Reveal 2.0 tests.

Impacts
What was accomplished under these goals? During the first half of this Phase I SBIR award, MBio has made significant progress towards the project goals. The project is on-track for time and budget and no significant problems have been encountered. Shellfish aquaculture is a significant economic sector and domestic food source with more than $240M produced in the United States annually. Expansion of this food source is desirable, since shellfish are an ideal aquaculture product; they grow readily, are environmentally friendly, are easy to seed, and are immobile. However, shellfish are filter feeders and can accumulate high concentrations of toxins generated by harmful algal blooms (HABs). HABs are increasing in frequency and severity and cannot always be visually detected. All shellfish sold in the United States, European Union, and 18 other countries have regulatory limits for three common toxins found in shellfish meat. Each species of shellfish harvested in an area must be tested, since different types of shellfish accumulate and depurate these toxins at different rates. Therefore, there is a sizable market for tests for HAB toxins in shellfish. Current testing requires performing three different tests for the three different common toxins for paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP) and diarrhetic shellfish poisoning (DSP). Performing these tests burdens aquaculture, since one toxin requires a laboratory test, which potentially has days of delay between sample shipment and receiving results. Even the two toxins which have approved rapid tests can be burdensome to aquaculture, since they have different protocols and must be purchased from different manufacturers. Therefore, testing adds significant delays and costs associated with bringing shellfish to market. In addition to the required regulatory testing, better testing would allow growers and regulators to better manage shellfish harvests. Shellfish that have been harvested that contain significant toxin levels must be disposed of appropriately, but toxin-containing shellfish that are still growing can be allowed to continue to grow while depurating. Therefore, significant costs and waste can be avoided by knowing whether shellfish contain toxins prior to harvesting. MBio Diagnostics is developing a transformative platform technology that will enable users in the field to perform cost-effective, multiplexed, rapid, laboratory-quality HAB Toxin testing. Under this award, MBio is developing tests for saxitoxin (STX) which causes paralytic shellfish poisoning (PSP), domoic acid (DA) which causes amnesic shellfish poisoning (ASP) and okadaic acid (OA) which causes diarrheitc shellfish poisoning (DSP). In Phase II, these tests will be combined into a single cartridge with a single protocol that will allow users to simultaneously test for all three toxins. This technology will protect the safety of the nation's food supply while enabling expansion of aquaculture by reducing the time and cost necessary to bring shellfish to market. There are two primary technical goals to demonstrate proof-of-concept: Deliver one-step, 10-minute assays forsaxitoxin, domoicacid, and okadaic acid tuned to regulatory limits for shellfish meat. Demonstrate a rapid, simple, and cost-effective sample preparation and extraction protocol that is suitable for use at the sample collection site and is compatible with the assay developed under goal 1. During the first half of this award, MBio has made significant progress towards both of these goals: Task 1.1 Two Step Assay Development: All reagents have been received. Significant progress has been made on the STX and DA two-step assays since these reagents in November. The conditions for printing spots on the microarray in the MBio cartridge have been optimized for both assays. Third, two-step assays for both the STX and DA targets have been performed demonstrating the necessary assay sensitivity and reproducibility. In these two-step assays, first antibody combined with a sample is added to the cartridge. Then, a secondary fluorophore-labeled detection antibody is added to the cartridge. Reagents for OA arrived in mid-January and development of this assay is now underway. Task 1.2 One-Step Assay Development: To simplify the workflow, MBio is working on 1-step assays for both STX and DA. These one-step assays require that the STX and DA antibodies be labeled with fluorophores, and the labeling conditions for both assays have been optimized. A one-step standard curve has been measured on the STX assay demonstrating sensitivity 3 orders of magnitude better than is necessary. The STX assay will be optimized for lower sensitivity to match the necessary sensitivity for the regulatory limits for STX in shellfish. The one-step DA assay has demonstrated sensitivities 8x greater than necessary. Both the STX and DA assay sensitivities will be appropriately tuned once the OA assay sensitivity has been demonstrated. Since the Phase II goal is to have all three assays measured simultaneously measured, the sensitivity of each assay will be tuned to the appropriate regulatory limits with the appropriate dilution from the sample preparation protocol. Task 1.3 Single-plex assay demonstration: This task cannot be performed until Task 1.2 is complete, but data from task 1.1 indicates that MBio will be able to meet the goals for this demonstration. Task 2.1 Homogenization Method: MBio has successfully completed this task. MBio has tested four different methods of homogenizing shellfish using live clams and demonstrated proof of concept for homogenization. These four methods include a laboratory grade tissue homogenizer, a bead beating method, a low-cost battery-operated blender, and a blade attached to MBio's system for lysing cyanobacteria (MQ Algae Lyse). Of these four methods, the battery-operated blender and the blade attached to the MBio MQ Algae Lyse were most effective for the sample sizes necessary for these assays. In Phase II one of these options will be productized. Task 2.2 Extraction Method Optimization: A protocol for extracting toxins from the shellfish homogenate has been identified and tested for assay compatibility with clam homogenate that does not contain toxins. This protocol was chosen because it should be a universal extraction protocol that will extract both lipophilic and hydrophilic toxins using a 50% methanol/50% water solution. A literature search identified this protocol (Campbell 2014), which may be refined in the second half of this grant and in Phase II. In particular, MBio is interested in whether a more environmentally-friendly protocol can be used. Katrina Campbell, Sara McNamee, Anne-Catherine Huet, Phillippe Delahaut, Natalia Vilarino, Luis Botana, Mark Poli, Christopher Elliott, Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish. Anal Bioanal Chem (2014) 406:6867-6881.

Publications