Progress 07/01/24 to 06/30/25

Outputs
Target Audience: Nothing Reported Changes/Problems:At the beginning of the project's second year, our team encountered unforeseen and severe challenges due to a series of back-to-back hurricanes--Debbie, Helene, and Milton--that struck Sarasota, Florida. These storms caused extensive damage to our research facilities, rendering the campus inaccessible for extended periods during each event. Approximately 50% of our main building remains uninhabitable, and many staff members are still displaced to other campuses, severely hampering our operational capacity. These disruptions have significantly impacted our ability to maintain the original project timeline, particularly for critical field testing components. Given the importance of this research and our strong commitment to completing the project objectives with scientific rigor, we respectfully requested an extension of 12 months to ensure that all project goals are met without compromising quality or impact. 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?Further investigation can be conducted to improve the sensitivity of the sandwich lateral flow assay. However, the current lateral flow methods being investigated seem unlikely to reach the 100-300 pg/mL detection limits desired. We have improved other lateral flow assays by conjugating the target to ovalbumin before binding to a lateral flow membrane, yielding competitive assays with sensitivities in the desired range. This competitive method, or other methods, may need to be pursued with the identified effective antibodies.

Impacts
What was accomplished under these goals? In aquaculture enterprises, a non-lethal method for determining the gender of young sturgeon (i.e., three years or younger) would be highly beneficial. SIM, an atypical member of the biomarker-related protein family, is reported to be distinguishable in fish gender determination, where the maturing male sturgeon exhibit lower levels of SIM, while female sturgeon show higher concentration. SIM is constituted by dimeric units consisting of a two SIM subunits giving rise to SIM A and SIM B respectively. The intended method for determining the sexual characteristics of sturgeon fish involves collecting blood from the fish, optionally separating the serum, and measuring the concentration of SIM-A or SIM-B using a rapid test. The device comprises a target-specific capture antibody on a solid phase and a rapidly developing reporter antibody that can provide a colorimetric or other indication of SIM concentration within 15 minutes. Mote Marine Laboratory has developed three monoclonal antibodies (mAbs) targeting the SIM-A subunit, as well as three mAbs targeting the SIM-B subunit. Additionally, we have identified existing commercial antibodies targeting both SIM subunits. This brief report presents findings from a lateral flow assay, a representative rapid test method that verifies and compares the SIM detection efficiency of the mAbs, laying the groundwork for a rapid sandwich assay or other antibody-based SIM detection methods. Glucose Reader/Magnetic Beads Some promising results were previously achieved using thrombin as a model for magnetic bead/glucose reader detection; however, the sensitivity and assay time did not meet the desired criteria. This method remains a possibility for further development if an excellent SIM antibody is discovered and if a more economical SIM-A for conjugation can be sourced. For the moment, lateral flow assays are being used to investigate the mAb antibodies developed by MML alongside commercially available antibodies. Direct/Competitive Lateral Flow Assay The sandwich format is the typical configuration for lateral flow assays, where the intensity of the test line increases with the target concentration, providing a facile and intuitive test reading. However, the SIM-A and SIM-B mAbs developed by MML showed a significant amount of background in a sandwich format. We therefore initiated an investigation using a competitive assay that employed only the SIM-B subunit antibody, which would measure total SIM rather than SIM-A concentration alone. However, the SIM-B mAb [131-0047 (H3)] did not conjugate well to gold, necessitating the use of an indirect competitive format. This method detected down to 5 ng/mL, but the competition may perform better without the complexity and number of components required by an indirect assay. A covalent conjugation method was therefore attempted to attach the MML-developed SIM-B mAb H3 to gold in a more stable format than the previous passive conjugation. The conjugate was more stable, but did not yield positive spots when run with the spotted target, likely due to decreased antibody loading onto the gold through covalent attachment. To stabilize the conjugate without compromising antibody loading, filler molecules were added following the passive conjugation. However, this method was unsuccessful in minimizing instability without compromising the signal. Due to the drawbacks of the existing antibodies, commercially available SIM-B antibodies were investigated. A polyclonal (pAb) and an mAb from a commercial source were both passively conjugated to gold nanoparticles. However, the mAb did not yield a signal when run with spotted SIM in a lateral flow assay. The pAb did yield a good signal, but the conjugate was highly unstable, aggregating within a day of storage. Covalent conjugation was attempted to improve stability, but similarly to the MML-developed SIM-B antibodies, covalent attachment eliminated the lateral flow signal. We then considered reversing the arrangement of the competitive assay by conjugating the SIM subunit protein to gold and spotting the pAb on the lateral flow membrane. However, the SIM subunit's formulation was high in urea, making conjugation to gold impossible. Dialysis of the SIM subunit did not generate a renatured protein. The cost of the full SIM protein made it an economically unviable alternative for conjugation to gold. The team then considered further methods to stabilize the active but non-storable SIM-B pAb gold nanoparticle conjugate. The size of the nanoparticle used was decreased, and the resulting material was lyophilized, resulting in a stable, rehydratable detection conjugate. This pAb conjugate still yielded a strong signal when run with spotted SIM, but competition was only observed at very high sample concentrations (25 μg/mL). Decreasing the amount of SIM spotted improved the competitive assay's sensitivity to 2.5 μg/mL sample SIM, which is still much higher than the required sensitivity. Sandwich Lateral Flow Assay Given the success in developing a stable pAb conjugate and the ability of pAbs to bind a target at multiple epitopes, we attempted a homologous sandwich with the pAb, both conjugated to the gold and spotted on the lateral flow membrane. This method showed minimal background but also did not show a signal below 10 μg/mL sample SIM. It was hypothesized that the new SIM-B pAb conjugate might not exhibit cross-reactivity to the SIM-A mAbs, which were previously attempted in the sandwich assay. MML-developed SIM-A mAbs 131-0051 (H11), 131-0046 (H4), and 131-0045 (H7) were therefore spotted and run in a sandwich lateral flow with pAb conjugate. For all tested configurations, a significant amount of background signal was observed, and the signal did not appear to intensify with increasing sample SIM. The commercial SIM-A mAbs were then considered as further potential pairs to the SIM-B pAb. A commercial SIM-specific antibody was attempted to be spotted on the lateral flow membrane, as well as conjugated to gold, with the SIM-B pAb either conjugated to gold or spotted to the membrane to complete the sandwich. This antibody pairing proved promising. With the spotted Abexxa mAb and pAb conjugate configuration, the lateral flow system exhibited minimal background and was further capable of detecting concentrations as low as 10 ng/mL SIM.

Publications

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.

Publications