Source: MOTE MARINE LABORATORY submitted to
AN IN-SITU BIOSENSOR FOR RAPID SEX DETERMINATION: A CRITICAL TOOL FOR ECONOMIC GROWTH AND PROFITABILITY OF STURGEON CAVIAR PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1030763
Grant No.
2023-70007-40205
Cumulative Award Amt.
$300,000.00
Proposal No.
2022-06014
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2025
Grant Year
2023
Program Code
[AQUA]- Aquaculture Research
Project Director
Wetzel, D.
Recipient Organization
MOTE MARINE LABORATORY
1600 KEN THOMPSON PARKWAY
SARASOTA,FL 34236
Performing Department
(N/A)
Non Technical Summary
Sturgeon caviar is the most expensive aquaculture product in the world. About 25% of adult female sturgeon's weight is from eggs at harvest. A large species may produce 22kg of caviar with a value of between $70k-$175k, suggesting that relatively small farms can be highly successful. So why is caviar production not a more significant part of the American aquaculture economy? Each year, the amount of caviar produced in the US fails to meet the total consumer demand; thus, our economy is a net importer of caviar. US sturgeon aquaculture suffers from inefficiencies across the value chain. There are many opportunities for value chain R&D to have a tremendous impact on growing the American caviar industry resulting in significant profit yields. The barrier to farmers getting involved in this highly profitable fishery, where demand exceeds supply, is the long-term investment needed before sturgeon on the farm mature enough to be profitable. Females take a decade or more to reach sexual maturity and produce valuable eggs. However, juvenile sturgeon can take 5-6 years to be sexually differentiable. So, producers pay to feed and house the non-productive males, 50% of each cohort, for 5-6 years without any financial reward. Who would invest in such a venture or trust someone with their passions and promises to turn profits?1 The earlier the males can be removed from the production process, the greater the profits for this industry. Urgently needed by this artisan fishery is a rapid sex identification tool, resulting in doubling caviar capacity, dramatically improving production efficiencies, increasing profitability, and stimulating growth. When the start-up costs can be drastically reduced because the rearing population will comprise only valuable females, the door will open for new entrepreneurs to venture into the caviar production market. A field-based sex determination biosensor for sturgeon aquaculture, with potential applications for other commercial fish farms and sturgeon conservation research, can be a game-changer.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
60108101040100%
Knowledge Area
601 - Economics of Agricultural Production and Farm Management;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
1. Generate nucleic acid aptamers (chemical antibodies) to create an in-situ biosensor prototype for real-time detection and quantification of Sex identfication markerlevels.2. Validate the biosensor's efficacy utilizing MML's considerable archive of sturgeon serum and white and Russian sturgeon serum collected from farms in California and North Carolina and hatchery-sourced Atlantic sturgeon by establishing concentration gradients and threshold levels for male and female determination in each sturgeon species.3. In collaboration with sturgeon farms, optimize in-situ testing procedures for the SIM biosensor prototype, evaluating ease of use and economic feasibility.
Project Methods
Activity 1. Creation of a rigorously tested biosensor prototype for early sex determination of sturgeon. For a biosensor to be an effective tool for aquaculture, it should have real-time detection with high specificity in a very complex medium (i.e., whole blood), well-characterized binding properties, high stability, and the potential for low-cost, large-scale production. One such technology that exhibits all the above qualities is aptamers. Aptamers, single-stranded DNA or RNA, are target-recognition elements with high affinity and specificity against small molecules, protein biomarkers, cells, and tissues. Aptamers have been successfully used as an alternative platform to antibodies for diagnostic, therapeutic, and bio-industrial applications, proving their utility as the recognition element in a biosensor. DNA aptamers will be screened in vitro to bind to the MML discovered SIM. To identify DNA aptamers that will bind to the target SIM across several commercially important sturgeon species, we propose the strategic use of multiple SIM targets from various sturgeon species simultaneously in a multiplex Systematic Evolution of Ligands by EXponential enrichment (SELEX), increasing the chances of discovering aptamers with high selectivity and sturgeon species cross-reactivity. Selection will increase stringency until it has been determined that the library is sufficiently enriched with DNA binders to the SIM targets. Approximately 10-20 candidate aptamers will be synthesized and screened for the binding affinity for the SIM targets. The top candidates will be used to generate SIM biosensor prototypes for evaluation.Activity 2. Establish SIM testing strategies for minimal invasiveness while maintaining the accuracy of the test. MML scientists will travel to Marshallberg Farm, NC, Sterling Caviar, CA, farms, and the Patuxent Naval Base, Chesapeake Bay, where the Atlantic sturgeon are housed, testing the SIM biosensor prototype. Onsite testing methods will include evaluating the real-time blood sampling method, detection efficiency, and ease of use. As a more manageable, less invasive, and stressful blood collection method than caudal vein draws, blood will be collected using precut and heparinized glass microcapillary needles to collect blood from the gill. The blood will then be analyzed in real-time using the prototype device. Sampling and SIM detection will be repeated with multiple persons (farmer and personnel) under the supervision of an MML scientist to demonstrate the ease of use.Activity 3. Farmer collaboration for further evaluation of the accuracy of the biosensor to assess sex in several sturgeon species. In partnership with sturgeon farmers, further evaluation of the accuracy of the biosensor for sexing will be done. Since in-situ sex determination analysis will be done using blood, paired blood and serum samples will be collected from multiple cohorts per species for SIM analysis. Sample analyses will begin with the oldest to the youngest age classes until the SIM levels can no longer discriminate between sexes. These results will determine the minimum age that sex can be identified for each species. A subset of these sampled fish will be tagged for later gonadal histological validation of gender.Feasibility of methods and qualification of partners. MML currently has cloned sturgeon SIM recombinant proteins and several commercially available SIM recombinant proteins from different species as targets for developing the DNA aptamers. Once developed, validation testing of the DNA aptamer's SIM concentration data will be compared and correlated for linearity of responses to the data derived from the SIM antibody-based ELISA of the archived sturgeon serum samples MML has of White, Siberian, Russian, and Atlantic sturgeon. In addition to the SIM biosensor, an aptamer-based ELISA will be developed for continued validation of sex determination of farm-derived sturgeon samples. Sturgeon farm owners with years of experience in sturgeon rearing will provide the facilities and economic expertise to further evaluate the biosensor's feasibility as a tank-side sex-determining tool.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Sturgeon caviar growers in the United States who are interested in determining early sex identification of their stocks. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?Next Steps As observed in the previous assays, thrombin can be efficiently detected using the mAb immobilized MB and HRP-TMB based signal amplification system. The method can be further optimized and translated to detect Inhibin using the most effective detection mAb identified during the validation tests using lateral flow assays. This approach has the potential to be developed into a rapid field test using a portable absorbance reader.

Impacts
What was accomplished under these goals? In aquaculture enterprises, a non-lethal method for determining the gender of young sturgeon (e.g., three years or younger) would be highly beneficial. Inhibin, an atypical member of the TGF-β superfamily, is reported to be distinguishable in fish gender determination where the maturing male sturgeon exhibit lower level of inhibin, while female sturgeon shows higher concentration. Inhibin is constituted by dimeric units consisting of an alpha subunit and either a beta A or beta B subunit, giving rise to inhibin A and inhibin B, respectively. The intended method for rapid sturgeon fish sexual characteristics determination involves collecting blood from fish, optionally separating serum, measuring the concentration of Inhibin A or Inhibin B using a hand-held device. The device comprises a target-specific capture antibody on a solid-phase and a rapid-developing reporter antibody that can provide colorimetric or other indication of inhibin concentration within 15 minutes. Mote Marine Laboratory has developed three monoclonal antibodies (mAbs) targeting the Inhibin alpha subunit and three mAbs for the beta A subunit. This brief report presents findings from lateral flow assay, a representative rapid test method verifying and comparing inhibin detection efficiency of the mAbs, as well as bead-based assay development strategies, thus laying the groundwork for a rapid sandwich assay or other antibody-based inhibin detection methods. Testing detection antibody efficiency Assay format: Lateral flow immunoassay (LFA) Target: Inhibin A (InhA) Detection probe: Gold nanoparticle (AuNP) conjugated mAbs Mote InhA mAbs - Anti-alpha subunit: 131-0050 (G11); 131-0048 (G6); 131-0047 (H3) Anti-beta A subunit: 131-0051 (H11); 131-0046 (H4); 131-0045 (H7) Sandwich LFA with different mAb pair combinations: Detection H7-AuNP showed high cross-reactivity/ non-specific binding (NSB) paired with capture anti-alpha subunit mAbs. Similarly, detection H4-AuNP paired with capture G6 and G11 showed weak signal and NSB, respectively. Furthermore, BSA blocking on capture test spot and PEG blocking on mAb-AuNP did not resolve the NSB. On the other hand, detection H4-AuNP paired with capture H3 showed better detection signal-noise ratio. Note: All three anti-alpha subunit mAbs-AuNP and H11-AuNP were not achieved due to aggregation. Mote mAbs paired with Ansh mAbs (commercially purchased) showed a high level of NSB or an absence of detection signals. However, using the Ansh mAb pair alone for detection efficiently identified InhA and detected InhA positive serum samples with a good signal-to-noise ratio. When comparing the Mote and Ansh mAb pairs for detecting InhA in different species of sturgeon serum samples, the Ansh mAb pair, unlike the Mote mAbs, demonstrated better detection, corresponding to the known InhA concentrations. Sandwich LFA using different Commercial mAbs: Since functional Mote mAb pairs could not be obtained according to the findings from sandwich LFA, the assay proceeded using the following mAbs from different sources: Anti-alpha subunit: CloudClone 21 (C21); CloudClone 22 (C22); LSBio alpha (LSa) Anti-beta A subunit: Abbexa (Abx); LSBio betaA (LSb) Among the tested commercial mAb combinations, the sandwich LFA using anti-alpha mAb C21 and anti-beta A mAb Abx-AuNP demonstrated efficient cross-species serum InhA detection. It exhibited a concentration-dependent response in Russian sturgeon serum samples with a favorable signal-to-noise ratio. However, other combinations resulted in high NSB or the absence of any signal. While the C21 and Abx mAb pair showed positive detection, the signal intensity was not as robust as observed with the Ansh mAb pair. Competitive LFA detection of rInhA Most anti-alpha and anti-beta A mAb pairs exhibited high NSB or no signal. This can be due to adjacent binding sites availability, poor binding selectivity, or complexity of the matrix, causing high cross-reactivity, or steric hindrance, due to which the sandwich assay can become challenging. Consequently, the assay transitioned to a competitive format using only anti-alpha mAb, capable of detecting all inhibin rather than just inhibin A. Due to the incompatibility of anti-alpha mAbs for AuNP conjugation, an indirect assay principle was adopted using anti-IgG-AuNP. Mote and the commercial anti-alpha mAbs exhibited positive detection of the Inhibin alpha subunit in the indirect LFA without any background noise. In the indirect competitive assay, G6, H3, and C21 mAbs demonstrated InhA detection through signal inhibition in the presence of free 5 ng/mL InhA in the diluent. Further optimization can enhance signal resolution for a more effective detection dynamic range. Bead-based competitive immunoassay for rapid Inhibin detection with glucometer readout Based on mAb binding efficiency observed in the LFA analysis, a magnetic bead-based competitive assay can be developed for a two-step incubation, providing bound-free phase detection of inhibin in serum samples at sturgeon farming sites. The method involves immobilizing an anti-inhibin alpha subunit mAb on magnetic beads (MB), using a reference standard inhibin-invertase conjugate to compete with sample inhibin. The invertase enzymatic reaction generates glucose, which is readable by a glucometer--a measurement correlating to the inhibin present in the original sample. After mixing the sample with the mAb-coated MB, inhibin in the sample binds to the mAb-MB. Following incubation, the removal of the supernatant eliminates the complex matrix. Addition of the reference inhibin-invertase conjugate, which binds to the remaining binding sites on the mAb, is then followed by another incubation. Removing the supernatant for sucrose hydrolysis enzymatic reaction produces glucose that can be read by a glucometer. Higher glucose levels indicate more inhibin-invertase in the supernatant due to more inhibin in the sample, occupying most of the mAb on MB. The key benefits of this method include a competitive two-step incubation, potentially eliminating the need for a wash step, minimizing matrix complexity, and removing the necessity for an antibody pair, reducing the chance of cross-reactivity. Crucial factors for the assay development include immobilizing mAb on MB, MB-mAb target binding, protein-invertase conjugation, invertase sucrose hydrolysis reaction parameters such as time and pH and ensuring compatibility with competitive assay method. Method development To develop the method, thrombin is used as a mock target due to being a well-characterized protein, commonly available and having a similar molecular mass as Inhibin. Initially, thrombin mAb immobilization on magnetic beads (MB) is optimized using direct detection of as low as 5 ng/mL biotinylated thrombin within 15 minutes with an HRP-TMB-based signal amplification system. Reference thrombin-invertase conjugate is generated using thiol-maleimide click chemistry. Thrombin-invertase conjugate is individually validated using commercial thrombin detection lateral flow strips and sucrose hydrolysis reaction with a glucometer readout. However, using the thrombin mAb immobilized MB to detect thrombin-invertase conjugate in a direct assay does not yield positive detection, likely due to invertase (approx. MW: 270 kDa) hindering the thrombin epitope. To address this, the thiol-maleimide crosslinking is modified with a 12-PEG spacer between thrombin and invertase conjugation, which also could not be positively detected by the direct assay. Another approach uses thrombin-specific detection aptamer immobilized on agarose beads in a direct assay for thrombin-invertase conjugate. This assay detects 100 µg/mL thrombin-invertase conjugate after 30 minutes of incubation with 0.25M sucrose at 37°C, yielding a corresponding positive signal on the glucometer. While effective, this method may not be suitable for rapid and sensitive assays.

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