Rapid Detection of AAV8 Binding Antibodies in Gene Therapy Candidates: Development of a Point-of-Care Approach

Rapid Detection of AAV8 Binding Antibodies in Gene Therapy Candidates: Development of a Point-of-Care Approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rapid Detection of AAV8 Binding Antibodies in Gene Therapy Candidates: Development of a Point-of-Care Approach Angelo GUnasekera, Alex Kozikowski, Qing Wang, Cheng Yang, Neil Gordon, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6297887/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Aug, 2025 Read the published version in Gene Therapy → Version 1 posted 11 You are reading this latest preprint version Abstract Preexisting anti-AAV antibodies pose a significant challenge to the success of Adeno-associated Virus (AAV) mediated gene therapies, as they can diminish therapeutic effectiveness, restrict patient eligibility for treatment, and cause serious health issues during treatment. This study introduces the first point-of-care (POC) test for the rapid, quantitative detection of AAV8 binding antibodies in patients’ plasma, serum, and blood, leveraging Chembio’s Dual Path Platform (DPP) technology. The DPP AAV8 Total Antibody (TAb) assay delivers results within 20 minutes, with a dynamic range of 0–32 µg/ml when evaluated with purified human polyclonal antibodies that bind to AAV8, with reasonable specificity and sensitivity relative to the AAV8 TAb ELISA (R² = 0.90). Moreover, the assay demonstrated strong correlations with the AAV8 neutralizing antibody (NAb) ELISA and cell-based NAb assays (R² = 0.97 in plasma). This rapid and reliable test can facilitate screening potential gene therapy patients for preexisting AAV8 binding antibodies and assess their suitability for AAV8-mediated gene therapy. Biological sciences/Biological techniques/Immunological techniques Health sciences/Biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Adeno-associated virus (AAV)-mediated gene delivery is emerging as a leading approach for treating genetic diseases. Since the FDA approved the first AAV-mediated gene therapy in 2017( 1 ), AAV product development has surged, with over 200 clinical trials conducted to date( 2 , 3 ). However, as more AAV therapies advance through clinical stages, mild to severe adverse events have occurred that may be attributed to immune responses against the AAV vectors used( 4 ), highlighting the importance of measuring pre-existing anti-AAV antibody levels before AAV treatments. These antibodies may stem from natural exposure to wild-type AAVs or prior exposure to recombinant AAV vectors( 5 , 6 ). Studies in murine and nonhuman primate models suggest that preexisting anti-AAV antibodies can significantly reduce transduction efficiency and transgene expression, thereby limiting the therapeutic effectiveness of AAV gene therapies( 7 , 8 ). Additionally, these antibodies pose safety risks, often disqualifying affected individuals from AAV-based clinical trials and therapies( 4 , 9 ). AAV8 vectors, frequently investigated in clinical trials targeting hemoglobinopathies and hemophilia( 10 ), face a significant limitation: up to 50% of treatment candidates carry preexisting anti-AAV8 antibodies( 11 ), which excludes them from AAV8-based therapies. Antibodies against AAVs consist of neutralizing antibodies (NAbs) and non-neutralizing antibodies (nNAbs); NAbs can interfere with treatment via the occlusion of functionally important viral domains, blocking transduction of genetic material, while nNAbs may also impair transduction by promoting opsonization, leading to vector clearance and potentially causing other severe reactions in patients( 5 , 12 , 13 ). Total anti-AAV antibodies (TAbs) can currently be measured in clinical or research labs within a few hours, enabling the identification of patients with negative or sufficiently low titers for treatment eligibility( 14 , 15 ). NAbs can be assessed using a cell-based in vitro method by detecting a reporter gene following transduction with an AAV sample pre-incubated with the patient’s serum or plasma( 16 ). However, several factors may influence the reproducibility of this method, potentially resulting in inaccurate NAb titer measurements. These factors include using serum or plasma instead of whole blood, interference from other neutralizing elements within the assay, and variability in culture conditions across sites and days. Since TAbs include both NAbs and nibs, this can negatively impact treatment. This rapid, quantitative method also utilizes fingerstick blood as a matrix for TAb measurement and could be a valuable tool for physicians in identifying suitable treatment candidates. Strategies to address the presence of preexisting antibodies have been explored in both nonclinical and clinical studies. These include plasmapheresis to reduce antibody levels( 17 , 18 ), drugs to modulate immune responses by inhibiting T-cell activation, B-cell signaling, or antigen presentation to B-cells( 19 ), and AAV capsid engineering to lower immunogenicity( 4 ). Unfortunately, antibodies removed from a patient through plasmapheresis can regenerate rapidly, often reaching unacceptable levels before they can be measured using current methods like ELISA (which takes several hours) or live cell-based neutralizing antibody (NAb) assays (which takes several days). This prolonged timeframe significantly limits the utility of these assays for monitoring antibody titers post-treatment, particularly in assessing the effectiveness of gene therapy interventions. The short window between plasmapheresis and treatment application underscores the need for a rapid and reliable method to measure anti-AAV antibody levels in patients. Also, such a rapid test can monitor the patient after vector injection for any increase in anti-AAV antibody titer. Chembio has developed a novel lateral-flow immunoassay for the rapid and quantitative detection of AAV8 binding antibodies, which utilizes its proprietary Dual Path Platform (DPP®( 20 )) technology. Employing AAV8 capsids as the capture agent on the test line, the DPP AAV8 TAb Assay delivers quantitative results within 20 minutes, offering a fast and reliable point-of-care method for measuring the levels of antibodies that would bind to AAV8 capsids and interfere with AAV8-mediated gene therapies. Here, we present the development of the DPP AAV8 TAb assay and the results from preliminary clinical studies, highlighting the assay’s potential to empower doctors and improve patients' quality of life. Materials and Methods Materials The study materials included AAV8 viral capsid particles obtained from Takeda (Lexington, MA) and the recombinant monoclonal anti-AAV8 antibody AbD25496ia human IgG1, which was fully characterized and confirmed by Takeda as a neutralizing antibody( 21 ). The ADK8 mouse anti-AAV8 monoclonal antibody (mAb) was obtained from Progen (Heidelberg, Germany). The anti-AAV8 mouse mAb, HI16, was sourced from Takeda (Lexington, MA) and Bio-Rad (Hercules, CA). For cross-reactivity and sample characterization studies, AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, and AAV9 capsids were purchased from Virovek (Malvern, PA). MAPIA Protran BA83 membranes were purchased from Whatman (Little Chalfont, Buckinghamshire, UK) or Protran 10600044 membranes from GE Amersham (Chicago, IL). Chembio (Medford, NY) prepared red gold nanoparticles for conjugation, and Protein A was obtained from Thermo Fisher (Carlsbad, CA). Protein A-Sepharose, HRP substrate, AP substrate, and Protein A/G horseradish peroxidase (HRP) conjugate, Rabbit anti-Human Antibody-Alkaline Phosphatase (AP) conjugate were purchased from Bio-Rad (Hercules, CA). Additional reagents included Biotin-HRP conjugates, EZ-Link Sulfo-NHS-LC-Biotin, Streptavidin, and Streptavidin-HRP conjugates, all from Thermo Fisher (Carlsbad, CA). Confirmed AAV8 antibody negative and positive characterized plasma and serum samples were acquired from Takeda (Lexington, MA), and the TMB substrate, stop solution, and Human IgG ELISA kit were obtained from Sigma-Aldrich (St. Louis, MO). In addition, twenty non-characterized plasma and serum samples were obtained from BioIVT (Westbury, NY). For cell-based assays, HEK 293 cells, 293 FreeStyle Expression Medium (Cat# 12338018), and Firefly Luciferase Glow Assay Kit (cat #16176) were purchased from Thermofisher (Carlsbad, CA), and the AAV8-Luciferase (Cat# SL101432) was purchased from SignaGen (Fredrick, MD) Multi antigen Print Immunoassay (MAPIA) MAPIA is a Chembio-proprietary immunoassay that employs nitrocellulose membranes imprinted with multiple antigens at different concentrations, allowing for quick detection of protein binding specificity and cross-reactivity( 22 ). This assay validated the reagents selected through ELISA and confirmed that AAV8 and other AAV capsids can be immobilized on nitrocellulose membranes, establishing a foundation for the DPP assay. MAPIA uses printed serial dilutions of AAV8 and other AAV capsid subtypes on a membrane to help identify the best serum or plasma samples and detector reagents, such as anti-human-antibody-HRP or AP conjugates for various immunoassay applications. Nitrocellulose membranes printed with AAV subtypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV9, and AAV8) and control proteins are cut into 4 mm strips. For sample analysis, these strips are hydrated in PBS, blocked with 1% nonfat milk, and then incubated with diluted serum or antibody samples. After washing with PBST, the strips are treated with an anti-human antibody HRP (or AP) conjugate and washed again. The strips are then developed with the appropriate substrates according to the manufacturer’s instructions, dried, and scanned to measure line intensities. Dual-Path Platform (DPP) AAV8 Assay The DPP AAV8 TAb assay is a rapid immunochromatographic assay developed by Chembio Diagnostic Systems (Medford, NY), and its key features and components are shown in Fig. 1 . First, the patient sample or anti-AAV8 antibody standard is diluted 1:25 in running buffer and applied to the Sample Pad, which migrates toward the Test-Path. The sample flows across the Test Line, which contains 1.2x10 13 cp/ml buffer-exchanged AAV8 viral capsid to capture the analyte of interest, and the Control Line, containing 0.75 mg/ml immobilized Rabbit anti-human IgG. After a 10-minute incubation, the buffer is added to the Running Buffer Pad to release the red gold-conjugated Protein A. The conjugate migrates along the Test-Path with the buffer front, pushing any remaining sample over the Test Line for capture and clearing the path for the conjugate to bind to the analyte captured on the Test Line. After another 10-minute incubation, the DPP assay is inserted into the Micro Reader to obtain the assay results. The presence of AAV8 binding antibodies results in an increased test signal intensity, quantified by the reader. Purification of Polyclonal Human Anti-AAV8 Antibodies Large amounts of polyclonal human anti-AAV8 antibodies were purified from 300 mL of positive patient plasma samples following a two-step protocol using Fast Protein Liquid Chromatography (FPLC); these antibodies were used as one of the anti-AAV8 antibody standards. In the first step, total IgG was purified using a 25 mL home-packed Protein A Sepharose column. In the second step, anti-AAV8 antibodies were isolated from the total IgG using a custom-made AAV8 affinity column designed to mimic the treatment approach proposed for removing anti-AAV8 antibodies in patients undergoing AAV8-based gene therapies( 23 ). This affinity column was prepared by crosslinking 4×10 13 vg of AAV8 capsid to 25 mL NHS-Sepharose; 2.5 mg/mL BSA and 200 mM NaCl were added in the elution buffer to stabilize the purified antibodies. The antibodies were eluted from the affinity column using 0.1 M glycine at pH 2.5, with fractions collected directly into vials containing 1/10th the fraction volume of 2 M Tris at pH 8 to neutralize. This two-step purification method provides a high yield of AAV8 IgG with minimal contamination. Purifying plasma directly using the AAV8-Sepharose column resulted in low yields and significant contamination from plasma proteins like transferrin and albumin, likely due to their competitive binding to the column. However, when a two-step purification was used, including isolating total IgG with Protein A-Sepharose followed by isolating anti-AAV8 antibodies on the AAV8-Sepharose column, the eluates primarily contained anti-AAV8 IgG with minimal amounts of contaminants. (data not shown). The fractions were analyzed by SDS-PAGE, and those containing the eluted antibodies were pooled and dialyzed overnight at 4°C in PBS. The concentration of the purified human polyclonal anti-AAV8 antibody was then determined using a Human IgG ELISA Kit. The concentration was measured using a Human IgG ELISA kit at 33.3 µg/mL. In-house AAV8 TAb ELISA An in-house AAV8 TAb ELISA was developed to characterize and validate plasma and serum samples, serving as a screening tool for reagents and samples provided by Takeda and as a complementary reference test for the Chembio DPP® AAV8 TAb assay. The AAV8 TAb ELISA was developed using AAV8 capsid as the anti-AAV8 antibody-binding agent and Protein A/G horseradish peroxidase (HRP) as the detection agent. The wells were coated overnight at 2–8°C with 100 µl/well of a 0.4 M Na 2 CO 3 solution (pH 9.5) containing the AAV8 capsid at a 2.9 x 10 10 cp/ml. Following the incubation, wells were washed three times for two minutes each time with 200 µl/well of phosphate-buffered saline (PBS) solution containing 0.05% Tween 20 (PBST), then blocked for 2 hours at room temperature with 200 µl/well of 1% BSA in PBS. After repeating the washing step, 100 µl/well of plasma sample or anti-AAV8 antibody standards were diluted 1:100 in blocking buffer and incubated at room temperature for 1 hour. Wells were washed as before, and 100 µl/well of Protein A/G HRP diluted 1:10,000 in blocking buffer was added and incubated at room temperature for 1 hr. Following another washing step, the colorimetric reaction was developed by adding 100 µl/well of TMB substrate, then stopped with 100 µl/well of stop solution. Reactivity was determined by measuring the optical density (OD) of the well solutions at 450 nm using the Thermoscientific Biomate 160 spectrophotometer. Calibration curves were generated using the mouse HI16 mAb antibody and purified human anti-AAV8 polyclonal antibody to determine the samples' total anti-AAV8 antibody concentrations. While the specific antibody concentration values for defining positive and negative samples in patient samples are still to be established, several Limit of Blank (LoB) cut-off values have been set using negative samples in the in-house ELISA. These calculated antibody concentrations range between 2 and 6 standard deviations above the mean of the negative plasma samples (cut-off = mean + 2–6 x standard deviation). These cut-off concentrations were determined using the mouse monoclonal anti-AAV8 antibody (clone HI16) standard (Table 1). ROC curves for the DPP-AAV8 assay were generated using these cutoff values for 132 patient samples. In-house AAV8 NAb ELISA As depicted in Fig. 2 , the AAV8 NAb ELISA was developed to revalidate characterized samples received from Takeda using AAV8 viral capsids as the capture agent, Biotinylated anti-AAV8 NAb AbD25496ia as the competitor antibody, and Streptavidin- HRP as the detection agent. The wells were coated overnight at 2–8°C with 100 µl/well of a 0.4 M Na 2 CO 3 solution (pH 9.5) that contained AAV8 capsid at a concentration of 2.9 x 10 10 cp/ml. Following the incubation, wells were washed three times for two minutes each with 200 µl/well of PBST, then blocked for 2 hours at room temperature with 300 µl/well of 1% BSA in PBS. The washing step was repeated, this time with 300 µl washing buffer. Sample and antibody AbD25496ia solutions were created by mixing 120 µl of the sample with 120 µl of Biotin-AbD25496ia diluted in blocking buffer to a concentration of 0.008 µg/ml; 200 µl of the resulting mixture was added to each well and allowed to incubate at room temperature for 1 hour. Wells were washed with 300 µl washing buffer, then 100 µl/well of Streptavidin-HRP diluted 1:20,000 in blocking buffer was added and incubated at room temperature for 1 hr. Following another wash step with 200 µl of washing buffer, the colorimetric reaction was developed via the addition of 100 µl/well of TMB substrate for 10 minutes, then stopped with 100 µl/well of stop solution (Cat# S5814, Sigma-Aldrich, St. Louis, MO). Reactivity was determined by measuring the optical density (OD) of the well solutions at 450 nm using the Thermoscientific Biomate 160 spectrophotometer. The presence of anti-AAV NAb led to a reduced test signal intensity of the competitor Biotin-AbD25496ia, compared to assay wells with Biotin-AbD25496ia alone. Cell-Based AAV8 NAb Assay The initial development of a cell-based AAV8 NAb assay followed a three-day protocol. On day one, HEK293 cells are seeded into a 96-well plate at 100µL per well in 293 FreeStyle Expression Medium at 3 x 10 5 cells/mL. The plate is then incubated overnight at 37°C with 5% CO 2 . On the second day, the medium is replaced by 100 µL/well of human serum samples diluted 1:10 in a cell culture medium containing AAV8-Luciferase at 1 x 10 10 vp/mL. The plates are then incubated overnight. The cells are lysed on the third and final day, and the results are developed using the Pierce Firefly Luciferase Glow Assay Kit. The wells are aspirated, and 100 µL/well of 1x lysis buffer from the kit is added and allowed to shake for 30 minutes at room temperature; 25 µL of each resulting lysate is taken and added to the wells of an opaque white 96-well plate, followed by 50 µL of kit working solution (contains D-Luciferin, Assay Buffer, and Signal Enhancer). The resulting luminescence was measured using the Perkin Elmer Ensight plate reader. The presence of anti-AAV neutralizing antibodies led to a low signal in the cell-based AAV8 NAb assay, as these antibodies diminish the transduction efficiency of AAV-Luciferase into cells, resulting in a decreased luminescence signal. Results MAPIA and The Purification of Human Anti-AAV8 Antibodies Twenty plasma samples, previously characterized by the AAV8 TAb ELISA (Table 2) and two anti-AAV8 antibodies, were analyzed using MAPIA. Results showed that AAV8-positive samples displayed a reasonable signal intensity on capsid AAV8-printed lines, while negative samples showed no signal. Additionally, some samples responded positively to other AAV subtypes, including AAV2, AAV5, AAV6, and AAV9, suggesting that either AAV8 shares immunoreactive epitopes with these subtypes or that these plasma samples contain antibodies to other AAV serotypes; monoclonal anti-AAV8 antibodies on strips 21 and 22 tested negative when exposed to other AAV serotype viral capsids (Fig. 3 ). As observed in MAPIA, the two anti-AAV8 mouse monoclonal antibodies (HI16 and ADK8) demonstrated specificity for the AAV8 subtype, with ADK8 showing partial reactivity to the AAV3 subtype. This suggests that some plasma samples tested may have prior exposure to other AAV subtypes. MAPIA testing identified serum samples suitable for purifying Human Anti-AAV8 polyclonal antibodies (pAb), although most samples displayed varying degrees of cross-reactivity with other AAV subtypes. MAPIA testing was beneficial throughout the purification process in determining the most efficient method to purify Human Anti-AAV8 pAb. Plasma samples positive for AAV8 displayed distinct IgG bands at 25 kDa and 50–55 kDa on SDS-PAGE. An AAV8-Sepharose immunoaffinity column was developed in-house for targeted capture of anti-AAV8 antibodies. Directly purifying plasma through this column resulted in low yields and significant contamination from plasma proteins like transferrin and albumin (Fig. 4 a, and eluate B in Figure c & d). However, when a two-step purification was used, including isolating total IgG with Protein G-Sepharose followed by isolating anti AAV8 antibodies on the AAV8-Sepharose column, the eluates contained mainly anti-AAV8 IgG with minimal amounts of contaminants (Fig. 4 b, and eluate E in Figure c & d) Further testing demonstrated that the two-step method achieved greater specificity and enhanced purity of the isolated anti-AAV8 antibodies, as confirmed by MAPIA and SDS-PAGE analysis. AAV8-positive samples subjected to the two-step purification process (protein G-Sepharose followed by AAV8-sepharose columns) exhibited strong AAV8 signals in the purified protein fractions, with minimal to no antibodies remaining in the pass-through, indicating the high binding efficiency of the AAV8 column (Fig. 4 ). The two-step approach effectively eliminated competing plasma proteins, yielding purified anti-AAV8 antibodies suitable for large-scale applications. A scaled-up purification using 300 mL of Serum #16 was performed to obtain high-purity Human Anti-AAV8 pAb. The concentration was measured using a Human IgG ELISA kit and found to be 33.3 µg/mL. Detection of Mouse and Human Anti-AAV8 TAbs using DPP AAV8 TAb Assay The DPP AAV8 TAb assay successfully detected and quantified antibodies that would bind to AAV8 using two standard curves: one generated with a negative plasma spiked with mouse monoclonal antibody (clone HI16) and the other with a negative plasma spiked with the human anti-AAV8 polyclonal antibodies purified from patient plasma. The spiked plasma samples were also used to assess key assay parameters, including dynamic range, limit of detection (LOD), and assay precision. The evaluation followed CLSI guidelines to characterize the DPP assay for detecting anti-AAV8 antibodies( 24 ). Notably, the assay detects all antibodies capable of binding to the AAV8 capsid, including specific anti-AAV8 antibodies and antibodies against other AAV serotypes with significant immunogenic homology to AAV8. Five replicates of each antibody standard were independently and serially diluted in negative plasma for the dynamic range assessment and evaluated using the DPP AAV8 TAb assay. The HI16 antibody demonstrated a direct response between 0 and 50 µg/ml, with an R² above 0.98, indicating robust detection. The human polyclonal antibody displayed a nonlinear response between 0 and 32 µg/ml, with an R² above 0.98, confirming consistent performance across the two antibody types (Fig. 5 & Table 3). Both assays exhibited low variability, ensuring reliable detection within their respective dynamic ranges. The Limit of Blank (LOB) was determined by analyzing fifty replicates of a negative plasma sample, with the 48th-ranked signal used as the LOB( 24 , 25 ). While sample limitations prevented the study from using 50 different confirmed negative samples for greater accuracy, it provided a robust LOB. To establish the limit of detection (LOD), the same two antibody standards used in the dynamic range measurements were employed. In the DPP AAV8 TAb assay, three concentrations (5, 2.5, and 1 µg/ml) of the mouse monoclonal HI16 Ab were tested, yielding signals very close to the Limit of Blank (LOB). Each concentration was evaluated with twenty replicates, concluding that the LOD for the HI16 Ab was 2.5 µg/ml, as at least 19 out of 20 replicates produced signals above the LOB. The LOD for the human anti-AAV8 polyclonal antibody was also established at 1.5 µg/ml (Table 2) using the same method. The precision of the DPP AAV8 TAb assay was assessed using the same two anti-AAV8 antibodies: HI16, tested in five replicates at concentrations of 50, 37.5, 25, and 12.5 µg/ml, and the human polyclonal antibody, also in five replicates at 32, 24, 16, and 8 µg/ml. Results showed an average %CV of less than 11% over five days for all HI16 antibody concentrations and below 10% for the human polyclonal anti-AAV8 antibody, demonstrating the DPP AAV8 TAb assay’s strong precision and accuracy (see Fig. 6 ). Sixty blinded venous blood samples and their corresponding matched plasma were tested. Fresh blood samples yielded consistent results with plasma, while older samples exhibited lower signal intensities (Fig. 7 a). The assay performance was compared to the ELISA for anti-AAV8 antibody concentration in 60 plasma samples. The DPP assay and ELISA showed uniform results, except for samples with concentrations above 12 µg/ml. DPP estimated higher values due to its broader dynamic range (0–32 µg/ml) compared to ELISA (0–12 µg/ml; Fig. 7 b) The preliminary stability study of the DPP AAV8 TAb assay was assessed at 25°C, 37°C, and 45°C over 105 days. Testing was conducted at intervals of 0, 7, 22, 52, and 98 days using the HI16 antibody, two positive plasma samples, one negative plasma sample, and a buffer control. Each sample was tested in triplicate at each time point. Results showed that the assay remained remarkably stable at 25°C, while stability decreased by 2%-19% at 37°C and 9%-47% at 45°C over the 105 days; which translates into roughly 2 years and 7 months at room temperature( 26 ) Specificity, Sensitivity, and Performance of DPP-AAV8 Assay in Correlation to AAV8 TAb ELISA To assess the specificity and sensitivity of the DPP AAV8 TAb assay in detecting human anti-AAV8 antibodies, 132 plasma samples from patients were tested alongside the AAV8 TAb ELISA, which served as the reference assay. Various cut-off values for the AAV8 TAb ELISA were established, ranging from 2 to 6 standard deviations above the average of negative plasma samples (see Table 1). Figure 8 illustrates the correlation between the concentration of AAV8 TAb and the DPP assay for each sample. This analysis indicates a strong correlation, yielding an R² value of 0.9046 across all 132 patient samples. Receiver Operating Characteristic (ROC) curves were also generated for each of the cut-off values, yielding the following Area Under the Curve (AUC) results: an AUC greater than 0.95 when the cut-off was set at negative average + 2x standard deviation and an AUC greater than 0.99 when the cut-off was set at negative average + 5 x standard deviation (Table 1 & Fig. 9 A and 9 B). Strong Correlation of DPP AAV8 TAb Assay to AAV8 NAb ELISA and Cell-Based AAV8 NAb Assay The DPP AAV8 TAb assay was evaluated for binding AAV8 TAbs. Its performance was then compared with the AAV8 NAb ELISA and the cell-based AAV8 NAb assay. The correlation between these methods was assessed using plasma and serum samples from 20 patients. Plasma results showed a strong correlation with DPP AAV8 TAb, with an R² of 0.97, indicating high consistency between the two assays. In contrast, three outliers weakened the correlation using serum samples, likely due to false positives in the AAV8 NAb ELISA or false negatives in the DPP assay. When these outliers were excluded, the R² improved from 0.16 to 0.73, indicating a good correlation for the remaining serum results. Additionally, seven negative and 7 positive plasma samples, classified by the cell-based NAb assays, were used to evaluate the correlation between the cell-based AAV8 NAb assay and the AAV8 TAb ELISA and DPP AAV8 TAb assays. The results demonstrated a high correlation, as shown in Table 4. The AAV8 NAb ELISA identified one more positive result, probably due to a false positive. Discussion To ensure the safety and effectiveness of gene therapies, it is crucial to measure pre-existing anti-AAV antibodies in patients before treatment. These antibodies are categorized as neutralizing (NAb) or non-neutralizing (nNAb). NAbs inhibit transduction by preventing AAVs from attaching to the target cell receptors, thereby diminishing the efficacy of gene therapy(8). While nNAbs do not block transduction, they can still bind to AAVs, influencing and impacting the success of gene therapy through mechanisms such as opsonization, inflammation, and complement activation(27). Total binding antibodies encompass both NAbs and nNAbs, and their combined effects are considered when assessing the potential impact on the efficacy of gene therapy. Cell-based assays for NAbs and immunoassays such as ELISA for TAbs have been commonly used in clinical trials. (28, 29). However, these methods are hindered by long turnaround times. Furthermore, cell-based assays necessitate specialized skills and expertise, making it challenging to develop them into commercially viable test kits suitable for point-of-care use. In this study, we developed a rapid, lateral-flow-based immunoassay using Chembio's Dual Path Platform (DPP) technology to detect AAV8 binding antibodies in patient samples. Unlike traditional lateral flow assays, Chembio’s DPP system uses two distinct flow paths, enhancing sensitivity, minimizing non-specific interactions, and enabling quantitative and multiplexed testing(22, 30, 31). The DPP AAV8 TAb Assay, the first point-of-care (POC) test for AAV8 binding antibodies, uses serum, plasma, and fingerstick blood samples, delivers results in just 20 minutes, and requires minimal user training. Its clinical range was calibrated using anti-AAV8 antibody standards, and results are interpreted using the DPP Micro Reader II, which processes data via integrated algorithms and assay-specific calibration curves included in the RFID card for accurate, reproducible measurements (Figure 1). Although the DPP AAV8 TAb assay does not exclusively detect NAbs due to the design limitations of the lateral flow-based assay, recent studies(32) and our experimental data reveal a strong correlation between NAbs and TAbs in patients' plasma. Specifically, the DPP AAV8 TAb assay results strongly correlated with the cell-based AAV8 NAb assay results and the AAV8 NAb ELISA. Measuring NAbs directly could be challenging because they comprise only a small portion of the total AAV8 IgG antibodies. Therefore, assessing total AAV8 IgG provides a more reliable indicator of a sample's potential presence of NAbs. After plasmapheresis removes AAV8 antibodies (including NAbs) from a patient's blood, detecting NAbs becomes even more difficult. In such cases, measuring the remaining total AAV8 IgG antibody levels is a practical alternative for monitoring the presence of NAbs. Another important consideration is the intrinsic nature of AAV serotypes, as some share high amino acid sequence homology with others. For instance, it has been reported that AAV8 shares 83% sequence homology with AAV2(33). This high homology can lead to cross-reactivity, where antibodies generated against other AAV serotypes are detected in assays designed for AAV8. This phenomenon warrants further investigation to understand its implications better. Moreover, this homology may contribute to reduced transduction efficiency of AAV8-mediated gene therapy products, as pre-existing antibodies against other AAV serotypes could interfere with AAV8 function. This interference, which the AAV8 assay could also detect, highlights the need for deeper exploration of cross-serotype antibody interactions in the context of AAV-based therapies. In conclusion, the DPP AAV8 TAb assay is a rapid, reliable, and efficient point-of-care diagnostic tool for detecting pre-existing AAV8 binding antibodies in patient plasma, serum, or fingerstick blood. It provides quantitative results within 20 minutes, making it highly suitable for testing patients at various points in the treatment; these include the initial assessment of AAV8 binding antibodies, leading to the need for plasmapheresis if the patient shows high antibody levels, measuring remaining antibodies after plasmapheresis and before vector administration, and monitoring antibody levels during treatment to follow antibody response and allow control and prevention of immunotoxicity-related episodes. The strong correlation between the DPP AAV8 TAb assay and the comparator ELISA Assays highlights its value in screening and monitoring patients undergoing AAV8-mediated gene therapies. The DPP AAV8 TAb assay addresses the limitations of the current methods and provides a practical tool to physicians to enhance the management and success of gene therapy treatments. Declarations Data Availability The original contributions presented in the study can be provided to the inquiries directed to the corresponding author. Further information and requests for additional information should be directed and will be fulfilled by the corresponding author AG ( [email protected] ) ACKNOWLEDGEMENTS The authors thank BioAgilytix EU for their valuable contributions to assay validation and sample analysis. They also thank Luying Pan (Takeda) and Konstantin Lyashenko (Chembio) for their careful review and thoughtful guidance. AUTHor contributions The Assays were designed by AG, who also played a key role in interpreting the results along with JE, CY and TP. Laboratory work was primarily conducted by QW and AK, with NG primarily handling tissue culture and cell-based studies. CV was responsible for all printing, reagent spraying, and assembly of test devices, while AY helped with the invitro assays. KC performed reagent characterization and all laboratory work at Takeda. AG drafted the manuscript, with all authors contributing to its revision and editing. The final version was reviewed, and approved by CY, TP, PL, AG, and JE. FUNDING This study was funded by the Takeda Pharmaceutical Company Limited, 650E Kendall Square, Cambridge, MA, USA DECLARATION OF INTERESTS AK, QW, NG, AY, CV, PL, JE, and AG are current or past employees of Chembio Diagnostics Inc. and have no financial interests or personal relationships that could have influenced the work reported in this paper. CY, KC, and TP are current or past employees of Takeda. Takeda provided financial support. The Takeda authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. References Rodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM. Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye. Pharm Res. 2018;36(2):29. Bougioukli S, Chateau M, Morales H, Vakhshori V, Sugiyama O, Oakes D, et al. Correction: Limited potential of AAV-mediated gene therapy in transducing human mesenchymal stem cells for bone repair applications. Gene Ther. 2024;31(9–10):527. Bulcha JT, Wang Y, Ma H, Tai PW, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target therapy. 2021;6(1):53. Yang T-y, Braun M, Lembke W, McBlane F, Kamerud J, DeWall S, et al. Immunogenicity assessment of AAV-based gene therapies: an IQ consortium industry white paper. Mol Therapy-Methods Clin Dev. 2022;26:471–94. Calcedo R, Wilson JM. Humoral Immune Response to AAV. Front Immunol. 2013;4:341. Kuranda K, Jean-Alphonse P, Leborgne C, Hardet R, Collaud F, Marmier S, et al. Exposure to wild-type AAV drives distinct capsid immunity profiles in humans. J Clin Invest. 2018;128(12):5267–79. Davidoff AM, Gray JT, Ng CY, Zhang Y, Zhou J, Spence Y, et al. Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models. Mol Ther. 2005;11(6):875–88. Wang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL, Wilson JM. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther. 2011;22(11):1389–401. Issa SS, Shaimardanova AA, Solovyeva VV, Rizvanov AA. Various AAV serotypes and their applications in gene therapy: an overview. Cells. 2023;12(5):785. Naso MF, Tomkowicz B, Perry WL III, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31(4):317–34. Rajavel K, Ayash-Rashkovsky M, Tang Y, Gangadharan B, de la Rosa M, Ewenstein B. Co-prevalence of pre-existing immunity to different serotypes of adeno-associated virus (AAV) in adults with hemophilia. Blood. 2019;134:3349. Boutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF, Masurier C. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther. 2010;21(6):704–12. Nidetz NF, McGee MC, Tse LV, Li C, Cong L, Li Y, Huang W. Adeno-associated viral vector-mediated immune responses: Understanding barriers to gene delivery. Pharmacol Ther. 2020;207:107453. Falese L, Sandza K, Yates B, Triffault S, Gangar S, Long B, et al. Strategy to detect pre-existing immunity to AAV gene therapy. Gene Ther. 2017;24(12):768–78. Stanford S, Pink R, Creagh D, Clark A, Lowe G, Curry N, et al. Adenovirus-associated antibodies in UK cohort of hemophilia patients: A seroprevalence study of the presence of adenovirus-associated virus vector-serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A. Res Pract Thromb Haemost. 2019;3(2):261–7. Calcedo R, Morizono H, Wang L, McCarter R, He J, Jones D, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol. 2011;18(9):1586–8. Chicoine LG, Montgomery C, Bremer W, Shontz K, Griffin D, Heller K, et al. Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery. Mol Ther. 2014;22(2):338–47. Monteilhet V, Saheb S, Boutin S, Leborgne C, Veron P, Montus MF, et al. A 10 Patient Case Report on the Impact of Plasmapheresis Upon Neutralizing Factors Against Adeno-associated Virus (AAV) Types 1, 2, 6, and 8. Mol Ther. 2011;19(11):2084–91. Ertl HCJ. Mitigating Serious Adverse Events in Gene Therapy with AAV Vectors: Vector Dose and Immunosuppression. Drugs. 2023;83(4):287–98. Esfandiari J. Dual path immunoassay device. US Patent. 2017;US9784734B2. Pabinger I, Ayash-Rashkovsky M, Escobar M, Konkle BA, Mingot-Castellano ME, Mullins ES, et al. Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia. Gene Ther. 2024;31(5–6):273–84. Boadella M, Lyashchenko K, Greenwald R, Esfandiari J, Jaroso R, Carta T, et al. Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa). J Vet Diagn Invest. 2011;23(1):77–83. Kruzik A, Raim R, Voelkel D, Weiller M, Hoellriegl W, Scheiflinger F, et al. AAV8-specific immune adsorption column: A treatment option for patients with pre-existing anti-AAV8 neutralizing antibodies. Blood. 2019;134:5922. Clinical, Institute LS. Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline. Clinical and Laboratory Standards Institute Wayne, PA; 2012. Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49–52. Institute CaLS. Evaluation of Stability of In Vitro Medical Laboratory Test Reagents. second ed2023. Mendell JR, Connolly AM, Lehman KJ, Griffin DA, Khan SZ, Dharia SD, et al. Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations. Mol Therapy-Methods Clin Dev. 2022;25:74–83. Gorovits B, Azadeh M, Buchlis G, Harrison T, Havert M, Jawa V, et al. Evaluation of the humoral response to adeno-associated virus-based gene therapy modalities using total antibody assays. AAPS J. 2021;23:1–17. Gorovits B, Fiscella M, Havert M, Koren E, Long B, Milton M, Purushothama S. Recommendations for the development of cell-based anti-viral vector neutralizing antibody assays. AAPS J. 2020;22:1–10. Iregbu KC, Esfandiari J, Nnorom J, Sonibare SA, Uwaezuoke SN, Eze SO, et al. Dual path platform HIV 1/2 assay: evaluation of a novel rapid test using oral fluids for HIV screening at the National Hospital in Abuja, Nigeria. Diagn Microbiol Infect Dis. 2011;69(4):405–9. Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AH, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012;6(11):e1878. Elkins S. Identification of Patient Specific Neutralizing Epitopes on the AAV8 Virus for Personalized Gene Therapy. Masters Thesis, Digital Access to Scholarship at Harvard: Harvard; 2020. Nam HJ, Lane MD, Padron E, Gurda B, McKenna R, Kohlbrenner E, et al. Structure of adeno-associated virus serotype 8, a gene therapy vector. J Virol. 2007;81(22):12260–71. Tables Tables 1 to 4 are available in the Supplementary Files section Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Table1.xlsx Table 1 Table2.xlsx Table 2 Table3.xlsx Table 3 Table4.xlsx Table 4 Cite Share Download PDF Status: Published Journal Publication published 21 Aug, 2025 Read the published version in Gene Therapy → Version 1 posted Editorial decision: revise 06 May, 2025 Review # 3 received at journal 22 Apr, 2025 Review # 2 received at journal 17 Apr, 2025 Reviewer # 3 agreed at journal 09 Apr, 2025 Review # 1 received at journal 30 Mar, 2025 Reviewer # 2 agreed at journal 28 Mar, 2025 Reviewer # 1 agreed at journal 27 Mar, 2025 Reviewers invited by journal 27 Mar, 2025 Editor assigned by journal 25 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6297887","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":434892208,"identity":"21ca22fa-f1e7-4358-aec4-f17e6a33fde9","order_by":0,"name":"Angelo GUnasekera","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBADAyBmfMDYAGQxk6CF2YBkLWwSYC2ElPL3rzF78OOPjTG/9OFnFT932ESbszMfYPhRsQ2nFokbb8wNe9vSzCT70sxu9p5Jy93ZzJbA2HPmNm5rbpwxk+BtOGxjcIbB7DZj2+HcDYd5DJgZ23BrkQdqkfzz57+N/Rn2b8WMbf8JazE432MmzcN2wMyAh8cMqPIAYS2GN9jKpGXbko0lzvAUS/a2JYP9chCfX+TOH94m+eaPnWF/D/vGDz/b7HK38x8++OBHBR7vSyRgETyAWz0Q8OOXHgWjYBSMglHAwAAAhNdXwUD7PToAAAAASUVORK5CYII=","orcid":"","institution":"Chembio Diagnostics Inc","correspondingAuthor":true,"prefix":"","firstName":"Angelo","middleName":"","lastName":"GUnasekera","suffix":""},{"id":434892209,"identity":"c8e07e4e-2d5d-4244-acb7-c21c2a7e19f7","order_by":1,"name":"Alex Kozikowski","email":"","orcid":"","institution":"Chembio Diagnostics 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18:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6297887/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6297887/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41434-025-00559-0","type":"published","date":"2025-08-21T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80811666,"identity":"25ec8c05-3210-4666-8476-167dba73871b","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170662,"visible":true,"origin":"","legend":"\u003cp\u003eDPP® AAV8 TAb Assay Test Cassette (a), open test cassette revealing internal assay components (b), and the Chembio Micro Reader II with AAV8 Test RFID card (c) (not shown to scale).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/e2d30b10ac53ad497654b30b.png"},{"id":80811668,"identity":"307fa570-7b01-42a9-9295-8629d235c743","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":78731,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of AAV8-NAb ELISA Assay.\u003c/p\u003e\n\u003cp\u003eThe illustration depicts the competitive binding between anti-AAV8 Nabs present in the sample and a biotin-conjugated mouse anti-AAV8 NAb antibody (AbD23596ia). This competition affects the absorbance measured at 450 nm, which is inversely proportional to the concentration of anti-AAV8 NAbs in the sample.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/acc0afb21492c8e295f11590.png"},{"id":80811677,"identity":"c91ec03b-c6bb-4812-9a69-9aab3424c83e","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":296894,"visible":true,"origin":"","legend":"\u003cp\u003eMAPIA results were generated by screening plasma samples characterized by ELISA, including samples 1-20 (Table 2), antibody HI16 (sample 21), and the mouse monoclonal ADK8 antibody (sample 22). The AAV subtypes were printed at two concentrations 25 \u0026amp; 12.5 mg/ml) and protein A (1mg/mL) was used as the positive control, and BSA (1mg/ml) was used as the negative control\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/350c7099fc822eda0e08fb87.png"},{"id":80813899,"identity":"a19688b4-afdb-4158-9e0b-11e297d51b3b","added_by":"auto","created_at":"2025-04-17 10:44:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":572083,"visible":true,"origin":"","legend":"\u003cp\u003ePurification and Characterization of Human Anti-AAV8 Polyclonal Antibodies (X) Column fractionation indicating the fractions A-E, (Y) Characterization of Eluates by MAPIA and (Z) Coomassie-stained SDS-PAGE of Eluate B \u0026amp; E for Serum samples 4 \u0026amp; 16\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/f66a676c0f2b09e2ad318937.png"},{"id":80814603,"identity":"c08682c7-0aca-406e-9206-ff264290d31d","added_by":"auto","created_at":"2025-04-17 10:52:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94524,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the Dynamic range of DPP® AAV8 TAb Assay using HI16 (a) and Human anti-AAV8 pAb (b)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/3b1f411f0539e212ee2b52d7.png"},{"id":80812258,"identity":"3644bcd1-b858-47b6-9dcc-fc8093e26af4","added_by":"auto","created_at":"2025-04-17 10:36:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":157727,"visible":true,"origin":"","legend":"\u003cp\u003ePrecision Testing of DPP® AAV8 Total IgG Assay using HI16 (a) and Human polyclonal Antibody (b)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/c4c0fc9c663fa208e175864c.png"},{"id":80812256,"identity":"57b7e9c6-9e50-4c5a-a6cb-c99192cb79e1","added_by":"auto","created_at":"2025-04-17 10:36:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":86781,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of 60 Serum Samples compared to matched Whole Blood on the DPP AAV8 Assay (a), and comparison of the same 60 Serum Samples on the DPP AAV8 Assay versus AAV8 ELISA\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/3b2907d8c20bda1040bbcc07.png"},{"id":80813903,"identity":"3c4f83da-df86-4f8b-a4ca-7846766acf05","added_by":"auto","created_at":"2025-04-17 10:44:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":58866,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between DPP AAV8 TAb Assay and AAV8 TAb ELISA results.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/9651747064b373db146f5ab9.png"},{"id":80811678,"identity":"195982c4-073d-4574-ac96-6654d03fc656","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":107667,"visible":true,"origin":"","legend":"\u003cp\u003eROC curves of DPP AAV8 TAb Assay with cutoff of 6.09 mg/ml determined via 2x the SD (A), and 12.02 mg/ml determined via 5x the SD (B).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/03f9daff70214fa38639a387.png"},{"id":89717252,"identity":"640d32fe-71f1-460d-abe9-28d2c412f108","added_by":"auto","created_at":"2025-08-23 07:15:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2261826,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/6df1da4b-b8b3-434d-99f4-c5823df835e2.pdf"},{"id":80811664,"identity":"5213f0d6-7b42-4557-8142-1baef27b271a","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10639,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/d05573ed9bbccd716ad6b60b.xlsx"},{"id":80811671,"identity":"a3436bf6-9404-411b-bc4a-8de5ed1edfc5","added_by":"auto","created_at":"2025-04-17 10:28:53","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13222,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/1e29955f5a2547f97d787688.xlsx"},{"id":80814602,"identity":"d702f0b3-254d-4461-9a6f-580dac76ccdc","added_by":"auto","created_at":"2025-04-17 10:52:53","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10880,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3\u003c/p\u003e","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/b172ac5a4f2b537806d87ada.xlsx"},{"id":80812252,"identity":"d0c85062-7e3c-40bd-ad30-fc40bd2162f2","added_by":"auto","created_at":"2025-04-17 10:36:53","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11889,"visible":true,"origin":"","legend":"\u003cp\u003eTable 4\u003c/p\u003e","description":"","filename":"Table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6297887/v1/44be690d72d9e4e70b42eb75.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Rapid Detection of AAV8 Binding Antibodies in Gene Therapy Candidates: Development of a Point-of-Care Approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdeno-associated virus (AAV)-mediated gene delivery is emerging as a leading approach for treating genetic diseases. Since the FDA approved the first AAV-mediated gene therapy in 2017(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), AAV product development has surged, with over 200 clinical trials conducted to date(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, as more AAV therapies advance through clinical stages, mild to severe adverse events have occurred that may be attributed to immune responses against the AAV vectors used(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), highlighting the importance of measuring pre-existing anti-AAV antibody levels before AAV treatments.\u003c/p\u003e \u003cp\u003eThese antibodies may stem from natural exposure to wild-type AAVs or prior exposure to recombinant AAV vectors(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Studies in murine and nonhuman primate models suggest that preexisting anti-AAV antibodies can significantly reduce transduction efficiency and transgene expression, thereby limiting the therapeutic effectiveness of AAV gene therapies(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Additionally, these antibodies pose safety risks, often disqualifying affected individuals from AAV-based clinical trials and therapies(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAAV8 vectors, frequently investigated in clinical trials targeting hemoglobinopathies and hemophilia(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), face a significant limitation: up to 50% of treatment candidates carry preexisting anti-AAV8 antibodies(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), which excludes them from AAV8-based therapies. Antibodies against AAVs consist of neutralizing antibodies (NAbs) and non-neutralizing antibodies (nNAbs); NAbs can interfere with treatment via the occlusion of functionally important viral domains, blocking transduction of genetic material, while nNAbs may also impair transduction by promoting opsonization, leading to vector clearance and potentially causing other severe reactions in patients(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Total anti-AAV antibodies (TAbs) can currently be measured in clinical or research labs within a few hours, enabling the identification of patients with negative or sufficiently low titers for treatment eligibility(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). NAbs can be assessed using a cell-based in vitro method by detecting a reporter gene following transduction with an AAV sample pre-incubated with the patient\u0026rsquo;s serum or plasma(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, several factors may influence the reproducibility of this method, potentially resulting in inaccurate NAb titer measurements. These factors include using serum or plasma instead of whole blood, interference from other neutralizing elements within the assay, and variability in culture conditions across sites and days. Since TAbs include both NAbs and nibs, this can negatively impact treatment. This rapid, quantitative method also utilizes fingerstick blood as a matrix for TAb measurement and could be a valuable tool for physicians in identifying suitable treatment candidates.\u003c/p\u003e \u003cp\u003eStrategies to address the presence of preexisting antibodies have been explored in both nonclinical and clinical studies. These include plasmapheresis to reduce antibody levels(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), drugs to modulate immune responses by inhibiting T-cell activation, B-cell signaling, or antigen presentation to B-cells(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), and AAV capsid engineering to lower immunogenicity(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Unfortunately, antibodies removed from a patient through plasmapheresis can regenerate rapidly, often reaching unacceptable levels before they can be measured using current methods like ELISA (which takes several hours) or live cell-based neutralizing antibody (NAb) assays (which takes several days). This prolonged timeframe significantly limits the utility of these assays for monitoring antibody titers post-treatment, particularly in assessing the effectiveness of gene therapy interventions. The short window between plasmapheresis and treatment application underscores the need for a rapid and reliable method to measure anti-AAV antibody levels in patients. Also, such a rapid test can monitor the patient after vector injection for any increase in anti-AAV antibody titer.\u003c/p\u003e \u003cp\u003eChembio has developed a novel lateral-flow immunoassay for the rapid and quantitative detection of AAV8 binding antibodies, which utilizes its proprietary Dual Path Platform (DPP\u0026reg;(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)) technology. Employing AAV8 capsids as the capture agent on the test line, the DPP AAV8 TAb Assay delivers quantitative results within 20 minutes, offering a fast and reliable point-of-care method for measuring the levels of antibodies that would bind to AAV8 capsids and interfere with AAV8-mediated gene therapies. Here, we present the development of the DPP AAV8 TAb assay and the results from preliminary clinical studies, highlighting the assay\u0026rsquo;s potential to empower doctors and improve patients' quality of life.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe study materials included AAV8 viral capsid particles obtained from Takeda (Lexington, MA) and the recombinant monoclonal anti-AAV8 antibody AbD25496ia human IgG1, which was fully characterized and confirmed by Takeda as a neutralizing antibody(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The ADK8 mouse anti-AAV8 monoclonal antibody (mAb) was obtained from Progen (Heidelberg, Germany). The anti-AAV8 mouse mAb, HI16, was sourced from Takeda (Lexington, MA) and Bio-Rad (Hercules, CA). For cross-reactivity and sample characterization studies, AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, and AAV9 capsids were purchased from Virovek (Malvern, PA). MAPIA Protran BA83 membranes were purchased from Whatman (Little Chalfont, Buckinghamshire, UK) or Protran 10600044 membranes from GE Amersham (Chicago, IL). Chembio (Medford, NY) prepared red gold nanoparticles for conjugation, and Protein A was obtained from Thermo Fisher (Carlsbad, CA). Protein A-Sepharose, HRP substrate, AP substrate, and Protein A/G horseradish peroxidase (HRP) conjugate, Rabbit anti-Human Antibody-Alkaline Phosphatase (AP) conjugate were purchased from Bio-Rad (Hercules, CA). Additional reagents included Biotin-HRP conjugates, EZ-Link Sulfo-NHS-LC-Biotin, Streptavidin, and Streptavidin-HRP conjugates, all from Thermo Fisher (Carlsbad, CA). Confirmed AAV8 antibody negative and positive characterized plasma and serum samples were acquired from Takeda (Lexington, MA), and the TMB substrate, stop solution, and Human IgG ELISA kit were obtained from Sigma-Aldrich (St. Louis, MO). In addition, twenty non-characterized plasma and serum samples were obtained from BioIVT (Westbury, NY). For cell-based assays, HEK 293 cells, 293 FreeStyle Expression Medium (Cat# 12338018), and Firefly Luciferase Glow Assay Kit (cat #16176) were purchased from Thermofisher (Carlsbad, CA), and the AAV8-Luciferase (Cat# SL101432) was purchased from SignaGen (Fredrick, MD)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMulti antigen Print Immunoassay (MAPIA)\u003c/h3\u003e\n\u003cp\u003eMAPIA is a Chembio-proprietary immunoassay that employs nitrocellulose membranes imprinted with multiple antigens at different concentrations, allowing for quick detection of protein binding specificity and cross-reactivity(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This assay validated the reagents selected through ELISA and confirmed that AAV8 and other AAV capsids can be immobilized on nitrocellulose membranes, establishing a foundation for the DPP assay. MAPIA uses printed serial dilutions of AAV8 and other AAV capsid subtypes on a membrane to help identify the best serum or plasma samples and detector reagents, such as anti-human-antibody-HRP or AP conjugates for various immunoassay applications. Nitrocellulose membranes printed with AAV subtypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV9, and AAV8) and control proteins are cut into 4 mm strips. For sample analysis, these strips are hydrated in PBS, blocked with 1% nonfat milk, and then incubated with diluted serum or antibody samples. After washing with PBST, the strips are treated with an anti-human antibody HRP (or AP) conjugate and washed again. The strips are then developed with the appropriate substrates according to the manufacturer\u0026rsquo;s instructions, dried, and scanned to measure line intensities.\u003c/p\u003e\n\u003ch3\u003eDual-Path Platform (DPP) AAV8 Assay\u003c/h3\u003e\n\u003cp\u003eThe DPP AAV8 TAb assay is a rapid immunochromatographic assay developed by Chembio Diagnostic Systems (Medford, NY), and its key features and components are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. First, the patient sample or anti-AAV8 antibody standard is diluted 1:25 in running buffer and applied to the Sample Pad, which migrates toward the Test-Path. The sample flows across the Test Line, which contains 1.2x10\u003csup\u003e13\u003c/sup\u003e cp/ml buffer-exchanged AAV8 viral capsid to capture the analyte of interest, and the Control Line, containing 0.75 mg/ml immobilized Rabbit anti-human IgG. After a 10-minute incubation, the buffer is added to the Running Buffer Pad to release the red gold-conjugated Protein A. The conjugate migrates along the Test-Path with the buffer front, pushing any remaining sample over the Test Line for capture and clearing the path for the conjugate to bind to the analyte captured on the Test Line. After another 10-minute incubation, the DPP assay is inserted into the Micro Reader to obtain the assay results. The presence of AAV8 binding antibodies results in an increased test signal intensity, quantified by the reader.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePurification of Polyclonal Human Anti-AAV8 Antibodies\u003c/h3\u003e\n\u003cp\u003eLarge amounts of polyclonal human anti-AAV8 antibodies were purified from 300 mL of positive patient plasma samples following a two-step protocol using Fast Protein Liquid Chromatography (FPLC); these antibodies were used as one of the anti-AAV8 antibody standards. In the first step, total IgG was purified using a 25 mL home-packed Protein A Sepharose column. In the second step, anti-AAV8 antibodies were isolated from the total IgG using a custom-made AAV8 affinity column designed to mimic the treatment approach proposed for removing anti-AAV8 antibodies in patients undergoing AAV8-based gene therapies(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This affinity column was prepared by crosslinking 4\u0026times;10\u003csup\u003e13\u003c/sup\u003e vg of AAV8 capsid to 25 mL NHS-Sepharose; 2.5 mg/mL BSA and 200 mM NaCl were added in the elution buffer to stabilize the purified antibodies. The antibodies were eluted from the affinity column using 0.1 M glycine at pH 2.5, with fractions collected directly into vials containing 1/10th the fraction volume of 2 M Tris at pH 8 to neutralize.\u003c/p\u003e \u003cp\u003eThis two-step purification method provides a high yield of AAV8 IgG with minimal contamination. Purifying plasma directly using the AAV8-Sepharose column resulted in low yields and significant contamination from plasma proteins like transferrin and albumin, likely due to their competitive binding to the column. However, when a two-step purification was used, including isolating total IgG with Protein A-Sepharose followed by isolating anti-AAV8 antibodies on the AAV8-Sepharose column, the eluates primarily contained anti-AAV8 IgG with minimal amounts of contaminants. (data not shown).\u003c/p\u003e \u003cp\u003eThe fractions were analyzed by SDS-PAGE, and those containing the eluted antibodies were pooled and dialyzed overnight at 4\u0026deg;C in PBS. The concentration of the purified human polyclonal anti-AAV8 antibody was then determined using a Human IgG ELISA Kit. The concentration was measured using a Human IgG ELISA kit at 33.3 \u0026micro;g/mL.\u003c/p\u003e\n\u003ch3\u003eIn-house AAV8 TAb ELISA\u003c/h3\u003e\n\u003cp\u003eAn in-house AAV8 TAb ELISA was developed to characterize and validate plasma and serum samples, serving as a screening tool for reagents and samples provided by Takeda and as a complementary reference test for the Chembio DPP\u0026reg; AAV8 TAb assay. The AAV8 TAb ELISA was developed using AAV8 capsid as the anti-AAV8 antibody-binding agent and Protein A/G horseradish peroxidase (HRP) as the detection agent. The wells were coated overnight at 2\u0026ndash;8\u0026deg;C with 100 \u0026micro;l/well of a 0.4 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution (pH 9.5) containing the AAV8 capsid at a 2.9 x 10\u003csup\u003e10\u003c/sup\u003e cp/ml. Following the incubation, wells were washed three times for two minutes each time with 200 \u0026micro;l/well of phosphate-buffered saline (PBS) solution containing 0.05% Tween 20 (PBST), then blocked for 2 hours at room temperature with 200 \u0026micro;l/well of 1% BSA in PBS. After repeating the washing step, 100 \u0026micro;l/well of plasma sample or anti-AAV8 antibody standards were diluted 1:100 in blocking buffer and incubated at room temperature for 1 hour. Wells were washed as before, and 100 \u0026micro;l/well of Protein A/G HRP diluted 1:10,000 in blocking buffer was added and incubated at room temperature for 1 hr. Following another washing step, the colorimetric reaction was developed by adding 100 \u0026micro;l/well of TMB substrate, then stopped with 100 \u0026micro;l/well of stop solution. Reactivity was determined by measuring the optical density (OD) of the well solutions at 450 nm using the Thermoscientific Biomate 160 spectrophotometer. Calibration curves were generated using the mouse HI16 mAb antibody and purified human anti-AAV8 polyclonal antibody to determine the samples' total anti-AAV8 antibody concentrations.\u003c/p\u003e \u003cp\u003eWhile the specific antibody concentration values for defining positive and negative samples in patient samples are still to be established, several Limit of Blank (LoB) cut-off values have been set using negative samples in the in-house ELISA. These calculated antibody concentrations range between 2 and 6 standard deviations above the mean of the negative plasma samples (cut-off =\u0026thinsp;mean\u0026thinsp;+\u0026thinsp;2\u0026ndash;6 x standard deviation). These cut-off concentrations were determined using the mouse monoclonal anti-AAV8 antibody (clone HI16) standard (Table\u0026nbsp;1). ROC curves for the DPP-AAV8 assay were generated using these cutoff values for 132 patient samples.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn-house AAV8 NAb ELISA\u003c/h2\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the AAV8 NAb ELISA was developed to revalidate characterized samples received from Takeda using AAV8 viral capsids as the capture agent, Biotinylated anti-AAV8 NAb AbD25496ia as the competitor antibody, and Streptavidin- HRP as the detection agent. The wells were coated overnight at 2\u0026ndash;8\u0026deg;C with 100 \u0026micro;l/well of a 0.4 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution (pH 9.5) that contained AAV8 capsid at a concentration of 2.9 x 10\u003csup\u003e10\u003c/sup\u003e cp/ml. Following the incubation, wells were washed three times for two minutes each with 200 \u0026micro;l/well of PBST, then blocked for 2 hours at room temperature with 300 \u0026micro;l/well of 1% BSA in PBS. The washing step was repeated, this time with 300 \u0026micro;l washing buffer. Sample and antibody AbD25496ia solutions were created by mixing 120 \u0026micro;l of the sample with 120 \u0026micro;l of Biotin-AbD25496ia diluted in blocking buffer to a concentration of 0.008 \u0026micro;g/ml; 200 \u0026micro;l of the resulting mixture was added to each well and allowed to incubate at room temperature for 1 hour. Wells were washed with 300 \u0026micro;l washing buffer, then 100 \u0026micro;l/well of Streptavidin-HRP diluted 1:20,000 in blocking buffer was added and incubated at room temperature for 1 hr. Following another wash step with 200 \u0026micro;l of washing buffer, the colorimetric reaction was developed via the addition of 100 \u0026micro;l/well of TMB substrate for 10 minutes, then stopped with 100 \u0026micro;l/well of stop solution (Cat# S5814, Sigma-Aldrich, St. Louis, MO). Reactivity was determined by measuring the optical density (OD) of the well solutions at 450 nm using the Thermoscientific Biomate 160 spectrophotometer. The presence of anti-AAV NAb led to a reduced test signal intensity of the competitor Biotin-AbD25496ia, compared to assay wells with Biotin-AbD25496ia alone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell-Based AAV8 NAb Assay\u003c/h3\u003e\n\u003cp\u003eThe initial development of a cell-based AAV8 NAb assay followed a three-day protocol. On day one, HEK293 cells are seeded into a 96-well plate at 100\u0026micro;L per well in 293 FreeStyle Expression Medium at 3 x 10\u003csup\u003e5\u003c/sup\u003e cells/mL. The plate is then incubated overnight at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. On the second day, the medium is replaced by 100 \u0026micro;L/well of human serum samples diluted 1:10 in a cell culture medium containing AAV8-Luciferase at 1 x 10\u003csup\u003e10\u003c/sup\u003evp/mL.\u003c/p\u003e \u003cp\u003eThe plates are then incubated overnight. The cells are lysed on the third and final day, and the results are developed using the Pierce Firefly Luciferase Glow Assay Kit. The wells are aspirated, and 100 \u0026micro;L/well of 1x lysis buffer from the kit is added and allowed to shake for 30 minutes at room temperature; 25 \u0026micro;L of each resulting lysate is taken and added to the wells of an opaque white 96-well plate, followed by 50 \u0026micro;L of kit working solution (contains D-Luciferin, Assay Buffer, and Signal Enhancer). The resulting luminescence was measured using the Perkin Elmer Ensight plate reader. The presence of anti-AAV neutralizing antibodies led to a low signal in the cell-based AAV8 NAb assay, as these antibodies diminish the transduction efficiency of AAV-Luciferase into cells, resulting in a decreased luminescence signal.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMAPIA and The Purification of Human Anti-AAV8 Antibodies\u003c/h2\u003e \u003cp\u003eTwenty plasma samples, previously characterized by the AAV8 TAb ELISA (Table\u0026nbsp;2) and two anti-AAV8 antibodies, were analyzed using MAPIA. Results showed that AAV8-positive samples displayed a reasonable signal intensity on capsid AAV8-printed lines, while negative samples showed no signal. Additionally, some samples responded positively to other AAV subtypes, including AAV2, AAV5, AAV6, and AAV9, suggesting that either AAV8 shares immunoreactive epitopes with these subtypes or that these plasma samples contain antibodies to other AAV serotypes; monoclonal anti-AAV8 antibodies on strips 21 and 22 tested negative when exposed to other AAV serotype viral capsids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As observed in MAPIA, the two anti-AAV8 mouse monoclonal antibodies (HI16 and ADK8) demonstrated specificity for the AAV8 subtype, with ADK8 showing partial reactivity to the AAV3 subtype. This suggests that some plasma samples tested may have prior exposure to other AAV subtypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMAPIA testing identified serum samples suitable for purifying Human Anti-AAV8 polyclonal antibodies (pAb), although most samples displayed varying degrees of cross-reactivity with other AAV subtypes. MAPIA testing was beneficial throughout the purification process in determining the most efficient method to purify Human Anti-AAV8 pAb. Plasma samples positive for AAV8 displayed distinct IgG bands at 25 kDa and 50\u0026ndash;55 kDa on SDS-PAGE. An AAV8-Sepharose immunoaffinity column was developed in-house for targeted capture of anti-AAV8 antibodies. Directly purifying plasma through this column resulted in low yields and significant contamination from plasma proteins like transferrin and albumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, and eluate B in Figure c \u0026amp; d). However, when a two-step purification was used, including isolating total IgG with Protein G-Sepharose followed by isolating anti AAV8 antibodies on the AAV8-Sepharose column, the eluates contained mainly anti-AAV8 IgG with minimal amounts of contaminants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, and eluate E in Figure c \u0026amp; d)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther testing demonstrated that the two-step method achieved greater specificity and enhanced purity of the isolated anti-AAV8 antibodies, as confirmed by MAPIA and SDS-PAGE analysis. AAV8-positive samples subjected to the two-step purification process (protein G-Sepharose followed by AAV8-sepharose columns) exhibited strong AAV8 signals in the purified protein fractions, with minimal to no antibodies remaining in the pass-through, indicating the high binding efficiency of the AAV8 column (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The two-step approach effectively eliminated competing plasma proteins, yielding purified anti-AAV8 antibodies suitable for large-scale applications. A scaled-up purification using 300 mL of Serum #16 was performed to obtain high-purity Human Anti-AAV8 pAb. The concentration was measured using a Human IgG ELISA kit and found to be 33.3 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Mouse and Human Anti-AAV8 TAbs using DPP AAV8 TAb Assay\u003c/h2\u003e \u003cp\u003eThe DPP AAV8 TAb assay successfully detected and quantified antibodies that would bind to AAV8 using two standard curves: one generated with a negative plasma spiked with mouse monoclonal antibody (clone HI16) and the other with a negative plasma spiked with the human anti-AAV8 polyclonal antibodies purified from patient plasma. The spiked plasma samples were also used to assess key assay parameters, including dynamic range, limit of detection (LOD), and assay precision. The evaluation followed CLSI guidelines to characterize the DPP assay for detecting anti-AAV8 antibodies(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Notably, the assay detects all antibodies capable of binding to the AAV8 capsid, including specific anti-AAV8 antibodies and antibodies against other AAV serotypes with significant immunogenic homology to AAV8.\u003c/p\u003e \u003cp\u003eFive replicates of each antibody standard were independently and serially diluted in negative plasma for the dynamic range assessment and evaluated using the DPP AAV8 TAb assay. The HI16 antibody demonstrated a direct response between 0 and 50 \u0026micro;g/ml, with an R\u0026sup2; above 0.98, indicating robust detection. The human polyclonal antibody displayed a nonlinear response between 0 and 32 \u0026micro;g/ml, with an R\u0026sup2; above 0.98, confirming consistent performance across the two antibody types (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026amp; Table\u0026nbsp;3). Both assays exhibited low variability, ensuring reliable detection within their respective dynamic ranges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Limit of Blank (LOB) was determined by analyzing fifty replicates of a negative plasma sample, with the 48th-ranked signal used as the LOB(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). While sample limitations prevented the study from using 50 different confirmed negative samples for greater accuracy, it provided a robust LOB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo establish the limit of detection (LOD), the same two antibody standards used in the dynamic range measurements were employed. In the DPP AAV8 TAb assay, three concentrations (5, 2.5, and 1 \u0026micro;g/ml) of the mouse monoclonal HI16 Ab were tested, yielding signals very close to the Limit of Blank (LOB). Each concentration was evaluated with twenty replicates, concluding that the LOD for the HI16 Ab was 2.5 \u0026micro;g/ml, as at least 19 out of 20 replicates produced signals above the LOB. The LOD for the human anti-AAV8 polyclonal antibody was also established at 1.5 \u0026micro;g/ml (Table\u0026nbsp;2) using the same method.\u003c/p\u003e \u003cp\u003eThe precision of the DPP AAV8 TAb assay was assessed using the same two anti-AAV8 antibodies: HI16, tested in five replicates at concentrations of 50, 37.5, 25, and 12.5 \u0026micro;g/ml, and the human polyclonal antibody, also in five replicates at 32, 24, 16, and 8 \u0026micro;g/ml. Results showed an average %CV of less than 11% over five days for all HI16 antibody concentrations and below 10% for the human polyclonal anti-AAV8 antibody, demonstrating the DPP AAV8 TAb assay\u0026rsquo;s strong precision and accuracy (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSixty blinded venous blood samples and their corresponding matched plasma were tested. Fresh blood samples yielded consistent results with plasma, while older samples exhibited lower signal intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The assay performance was compared to the ELISA for anti-AAV8 antibody concentration in 60 plasma samples. The DPP assay and ELISA showed uniform results, except for samples with concentrations above 12 \u0026micro;g/ml. DPP estimated higher values due to its broader dynamic range (0\u0026ndash;32 \u0026micro;g/ml) compared to ELISA (0\u0026ndash;12 \u0026micro;g/ml; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe preliminary stability study of the DPP AAV8 TAb assay was assessed at 25\u0026deg;C, 37\u0026deg;C, and 45\u0026deg;C over 105 days. Testing was conducted at intervals of 0, 7, 22, 52, and 98 days using the HI16 antibody, two positive plasma samples, one negative plasma sample, and a buffer control. Each sample was tested in triplicate at each time point. Results showed that the assay remained remarkably stable at 25\u0026deg;C, while stability decreased by 2%-19% at 37\u0026deg;C and 9%-47% at 45\u0026deg;C over the 105 days; which translates into roughly 2 years and 7 months at room temperature(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSpecificity, Sensitivity, and Performance of DPP-AAV8 Assay in Correlation to AAV8 TAb ELISA\u003c/h2\u003e \u003cp\u003eTo assess the specificity and sensitivity of the DPP AAV8 TAb assay in detecting human anti-AAV8 antibodies, 132 plasma samples from patients were tested alongside the AAV8 TAb ELISA, which served as the reference assay. Various cut-off values for the AAV8 TAb ELISA were established, ranging from 2 to 6 standard deviations above the average of negative plasma samples (see Table\u0026nbsp;1). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e illustrates the correlation between the concentration of AAV8 TAb and the DPP assay for each sample. This analysis indicates a strong correlation, yielding an R\u0026sup2; value of 0.9046 across all 132 patient samples. Receiver Operating Characteristic (ROC) curves were also generated for each of the cut-off values, yielding the following Area Under the Curve (AUC) results: an AUC greater than 0.95 when the cut-off was set at negative average\u0026thinsp;+\u0026thinsp;2x standard deviation and an AUC greater than 0.99 when the cut-off was set at negative average\u0026thinsp;+\u0026thinsp;5 x standard deviation (Table\u0026nbsp;1 \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStrong Correlation of DPP AAV8 TAb Assay to AAV8 NAb ELISA and Cell-Based AAV8 NAb Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe DPP AAV8 TAb assay was evaluated for binding AAV8 TAbs. Its performance was then compared with the AAV8 NAb ELISA and the cell-based AAV8 NAb assay. The correlation between these methods was assessed using plasma and serum samples from 20 patients. Plasma results showed a strong correlation with DPP AAV8 TAb, with an R\u0026sup2; of 0.97, indicating high consistency between the two assays. In contrast, three outliers weakened the correlation using serum samples, likely due to false positives in the AAV8 NAb ELISA or false negatives in the DPP assay. When these outliers were excluded, the R\u0026sup2; improved from 0.16 to 0.73, indicating a good correlation for the remaining serum results.\u003c/p\u003e \u003cp\u003eAdditionally, seven negative and 7 positive plasma samples, classified by the cell-based NAb assays, were used to evaluate the correlation between the cell-based AAV8 NAb assay and the AAV8 TAb ELISA and DPP AAV8 TAb assays. The results demonstrated a high correlation, as shown in Table\u0026nbsp;4. The AAV8 NAb ELISA identified one more positive result, probably due to a false positive.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo ensure the safety and effectiveness of gene therapies, it is crucial to measure pre-existing anti-AAV antibodies in patients before treatment. These antibodies are categorized as neutralizing (NAb) or non-neutralizing (nNAb). NAbs inhibit transduction by preventing AAVs from attaching to the target cell receptors, thereby diminishing the efficacy of gene therapy(8). While nNAbs do not block transduction, they can still bind to AAVs, influencing and impacting the success of gene therapy through mechanisms such as opsonization, inflammation, and complement activation(27). Total binding antibodies encompass both NAbs and nNAbs, and their combined effects are considered when assessing the potential impact on the efficacy of gene therapy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCell-based assays for NAbs and immunoassays such as ELISA for TAbs have been commonly used in clinical trials. (28, 29). However, these methods are hindered by long turnaround times. Furthermore, cell-based assays necessitate specialized skills and expertise, making it challenging to develop them into commercially viable test kits suitable for point-of-care use.\u003c/p\u003e\n\u003cp\u003eIn this study, we developed a rapid, lateral-flow-based immunoassay using Chembio's Dual Path Platform (DPP) technology to detect AAV8 binding antibodies in patient samples. Unlike traditional lateral flow assays, Chembio’s DPP system uses two distinct flow paths, enhancing sensitivity, minimizing non-specific interactions, and enabling quantitative and multiplexed testing(22, 30, 31). The DPP AAV8 TAb Assay, the first point-of-care (POC) test for AAV8 binding antibodies, uses serum, plasma, and fingerstick blood samples, delivers results in just 20 minutes, and requires minimal user training. Its clinical range was calibrated using anti-AAV8 antibody standards, and results are interpreted using the DPP Micro Reader II, which processes data via integrated algorithms and assay-specific calibration curves included in the RFID card for accurate, reproducible measurements (Figure 1).\u003c/p\u003e\n\u003cp\u003eAlthough the DPP AAV8 TAb assay does not exclusively detect NAbs due to the design limitations of the lateral flow-based assay, recent studies(32) and our experimental data reveal a strong correlation between NAbs and TAbs in patients' plasma. Specifically, the DPP AAV8 TAb assay results strongly correlated with the cell-based AAV8 NAb assay results and the AAV8 NAb ELISA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeasuring NAbs directly could be challenging because they comprise only a small portion of the total AAV8 IgG antibodies. Therefore, assessing total AAV8 IgG provides a more reliable indicator of a sample's potential presence of NAbs. After plasmapheresis removes AAV8 antibodies (including NAbs) from a patient's blood, detecting NAbs becomes even more difficult. In such cases, measuring the remaining total AAV8 IgG antibody levels is a practical alternative for monitoring the presence of NAbs.\u003c/p\u003e\n\u003cp\u003eAnother important consideration is the intrinsic nature of AAV serotypes, as some share high amino acid sequence homology with others. For instance, it has been reported that AAV8 shares 83% sequence homology with AAV2(33). This high homology can lead to cross-reactivity, where antibodies generated against other AAV serotypes are detected in assays designed for AAV8. This phenomenon warrants further investigation to understand its implications better. Moreover, this homology may contribute to reduced transduction efficiency of AAV8-mediated gene therapy products, as pre-existing antibodies against other AAV serotypes could interfere with AAV8 function. This interference, which the AAV8 assay could also detect, highlights the need for deeper exploration of cross-serotype antibody interactions in the context of AAV-based therapies.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the DPP AAV8 TAb assay is a rapid, reliable, and efficient point-of-care diagnostic tool for detecting pre-existing AAV8 binding antibodies in patient plasma, serum, or fingerstick blood. It provides quantitative results within 20 minutes, making it highly suitable for testing patients at various points in the treatment; these include the initial assessment of AAV8 binding antibodies, leading to the need for plasmapheresis if the patient shows high antibody levels, measuring remaining antibodies after plasmapheresis and before vector administration, and monitoring antibody levels during treatment to follow antibody response and allow control and prevention of immunotoxicity-related episodes. The strong correlation between the DPP AAV8 TAb assay and the comparator ELISA Assays highlights its value in screening and monitoring patients undergoing AAV8-mediated gene therapies. The DPP AAV8 TAb assay addresses the limitations of the current methods and provides a practical tool to physicians to enhance the management and success of gene therapy treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study can be provided to the inquiries directed to the corresponding author. \u0026nbsp;Further information and requests for additional information should be directed and will be fulfilled by the corresponding author AG ([email protected])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank BioAgilytix EU for their valuable contributions to assay validation and sample analysis. They also thank Luying Pan (Takeda) and Konstantin Lyashenko (Chembio) for their careful review and thoughtful guidance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Assays were designed by AG, who also played a key role in interpreting the results along with JE, CY and TP. Laboratory work was primarily conducted by QW and AK, with NG primarily handling tissue culture and cell-based studies. CV was responsible for all printing, reagent spraying, and assembly of test devices, while AY helped with the invitro assays. KC performed reagent characterization and all laboratory work at Takeda. AG drafted the manuscript, with all authors contributing to its revision and editing. The final version was reviewed, and approved by CY, TP, PL, AG, and JE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Takeda Pharmaceutical Company Limited, 650E Kendall Square, Cambridge, MA, USA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAK, QW, NG, AY, CV, PL, JE, and AG are current or past employees of Chembio Diagnostics Inc. and have no financial interests or personal relationships that could have influenced the work reported in this paper. CY, KC, and TP are current or past employees of Takeda. Takeda provided financial support. The Takeda authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM. Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye. Pharm Res. 2018;36(2):29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBougioukli S, Chateau M, Morales H, Vakhshori V, Sugiyama O, Oakes D, et al. Correction: Limited potential of AAV-mediated gene therapy in transducing human mesenchymal stem cells for bone repair applications. Gene Ther. 2024;31(9\u0026ndash;10):527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulcha JT, Wang Y, Ma H, Tai PW, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target therapy. 2021;6(1):53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang T-y, Braun M, Lembke W, McBlane F, Kamerud J, DeWall S, et al. Immunogenicity assessment of AAV-based gene therapies: an IQ consortium industry white paper. Mol Therapy-Methods Clin Dev. 2022;26:471\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalcedo R, Wilson JM. Humoral Immune Response to AAV. Front Immunol. 2013;4:341.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuranda K, Jean-Alphonse P, Leborgne C, Hardet R, Collaud F, Marmier S, et al. Exposure to wild-type AAV drives distinct capsid immunity profiles in humans. J Clin Invest. 2018;128(12):5267\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavidoff AM, Gray JT, Ng CY, Zhang Y, Zhou J, Spence Y, et al. Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models. Mol Ther. 2005;11(6):875\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL, Wilson JM. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther. 2011;22(11):1389\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIssa SS, Shaimardanova AA, Solovyeva VV, Rizvanov AA. Various AAV serotypes and their applications in gene therapy: an overview. Cells. 2023;12(5):785.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaso MF, Tomkowicz B, Perry WL III, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31(4):317\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajavel K, Ayash-Rashkovsky M, Tang Y, Gangadharan B, de la Rosa M, Ewenstein B. Co-prevalence of pre-existing immunity to different serotypes of adeno-associated virus (AAV) in adults with hemophilia. Blood. 2019;134:3349.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoutin S, Monteilhet V, Veron P, Leborgne C, Benveniste O, Montus MF, Masurier C. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther. 2010;21(6):704\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNidetz NF, McGee MC, Tse LV, Li C, Cong L, Li Y, Huang W. Adeno-associated viral vector-mediated immune responses: Understanding barriers to gene delivery. Pharmacol Ther. 2020;207:107453.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalese L, Sandza K, Yates B, Triffault S, Gangar S, Long B, et al. Strategy to detect pre-existing immunity to AAV gene therapy. Gene Ther. 2017;24(12):768\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanford S, Pink R, Creagh D, Clark A, Lowe G, Curry N, et al. Adenovirus-associated antibodies in UK cohort of hemophilia patients: A seroprevalence study of the presence of adenovirus-associated virus vector-serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A. Res Pract Thromb Haemost. 2019;3(2):261\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalcedo R, Morizono H, Wang L, McCarter R, He J, Jones D, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol. 2011;18(9):1586\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChicoine LG, Montgomery C, Bremer W, Shontz K, Griffin D, Heller K, et al. Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery. Mol Ther. 2014;22(2):338\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonteilhet V, Saheb S, Boutin S, Leborgne C, Veron P, Montus MF, et al. A 10 Patient Case Report on the Impact of Plasmapheresis Upon Neutralizing Factors Against Adeno-associated Virus (AAV) Types 1, 2, 6, and 8. Mol Ther. 2011;19(11):2084\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErtl HCJ. Mitigating Serious Adverse Events in Gene Therapy with AAV Vectors: Vector Dose and Immunosuppression. Drugs. 2023;83(4):287\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsfandiari J. Dual path immunoassay device. US Patent. 2017;US9784734B2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePabinger I, Ayash-Rashkovsky M, Escobar M, Konkle BA, Mingot-Castellano ME, Mullins ES, et al. Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia. Gene Ther. 2024;31(5\u0026ndash;6):273\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoadella M, Lyashchenko K, Greenwald R, Esfandiari J, Jaroso R, Carta T, et al. Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa). J Vet Diagn Invest. 2011;23(1):77\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKruzik A, Raim R, Voelkel D, Weiller M, Hoellriegl W, Scheiflinger F, et al. AAV8-specific immune adsorption column: A treatment option for patients with pre-existing anti-AAV8 neutralizing antibodies. Blood. 2019;134:5922.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinical, Institute LS. Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline. Clinical and Laboratory Standards Institute Wayne, PA; 2012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008;29(Suppl 1):S49\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInstitute CaLS. Evaluation of Stability of In Vitro Medical Laboratory Test Reagents. second ed2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendell JR, Connolly AM, Lehman KJ, Griffin DA, Khan SZ, Dharia SD, et al. Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations. Mol Therapy-Methods Clin Dev. 2022;25:74\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorovits B, Azadeh M, Buchlis G, Harrison T, Havert M, Jawa V, et al. Evaluation of the humoral response to adeno-associated virus-based gene therapy modalities using total antibody assays. AAPS J. 2021;23:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorovits B, Fiscella M, Havert M, Koren E, Long B, Milton M, Purushothama S. Recommendations for the development of cell-based anti-viral vector neutralizing antibody assays. AAPS J. 2020;22:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIregbu KC, Esfandiari J, Nnorom J, Sonibare SA, Uwaezuoke SN, Eze SO, et al. Dual path platform HIV 1/2 assay: evaluation of a novel rapid test using oral fluids for HIV screening at the National Hospital in Abuja, Nigeria. Diagn Microbiol Infect Dis. 2011;69(4):405\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Dami\u0026atilde;o AO, Gon\u0026ccedil;alves AH, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012;6(11):e1878.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElkins S. Identification of Patient Specific Neutralizing Epitopes on the AAV8 Virus for Personalized Gene Therapy. Masters Thesis, Digital Access to Scholarship at Harvard: Harvard; 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNam HJ, Lane MD, Padron E, Gurda B, McKenna R, Kohlbrenner E, et al. Structure of adeno-associated virus serotype 8, a gene therapy vector. J Virol. 2007;81(22):12260\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6297887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6297887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePreexisting anti-AAV antibodies pose a significant challenge to the success of Adeno-associated Virus (AAV) mediated gene therapies, as they can diminish therapeutic effectiveness, restrict patient eligibility for treatment, and cause serious health issues during treatment. This study introduces the first point-of-care (POC) test for the rapid, quantitative detection of AAV8 binding antibodies in patients\u0026rsquo; plasma, serum, and blood, leveraging Chembio\u0026rsquo;s Dual Path Platform (DPP) technology. The DPP AAV8 Total Antibody (TAb) assay delivers results within 20 minutes, with a dynamic range of 0\u0026ndash;32 \u0026micro;g/ml when evaluated with purified human polyclonal antibodies that bind to AAV8, with reasonable specificity and sensitivity relative to the AAV8 TAb ELISA (R\u0026sup2; = 0.90). Moreover, the assay demonstrated strong correlations with the AAV8 neutralizing antibody (NAb) ELISA and cell-based NAb assays (R\u0026sup2; = 0.97 in plasma). This rapid and reliable test can facilitate screening potential gene therapy patients for preexisting AAV8 binding antibodies and assess their suitability for AAV8-mediated gene therapy.\u003c/p\u003e","manuscriptTitle":"Rapid Detection of AAV8 Binding Antibodies in Gene Therapy Candidates: Development of a Point-of-Care Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:28:48","doi":"10.21203/rs.3.rs-6297887/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-05-06T09:37:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-22T17:24:33+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-17T13:12:33+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-09T11:27:54+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-03-30T06:25:52+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-28T10:53:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-27T15:31:07+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-27T14:23:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T12:17:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T12:17:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Gene Therapy","date":"2025-03-24T18:50:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"gene-therapy","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"gt","sideBox":"Learn more about [Gene Therapy](http://www.nature.com/gt/)","snPcode":"41434","submissionUrl":"https://mts-gt.nature.com/cgi-bin/main.plex","title":"Gene Therapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9d47a90a-599b-4f90-a077-9cd60d39150d","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46307943,"name":"Biological sciences/Biological techniques/Immunological techniques"},{"id":46307944,"name":"Health sciences/Biomarkers"}],"tags":[],"updatedAt":"2025-08-23T07:15:11+00:00","versionOfRecord":{"articleIdentity":"rs-6297887","link":"https://doi.org/10.1038/s41434-025-00559-0","journal":{"identity":"gene-therapy","isVorOnly":false,"title":"Gene Therapy"},"publishedOn":"2025-08-21 04:00:00","publishedOnDateReadable":"August 21st, 2025"},"versionCreatedAt":"2025-04-17 10:28:48","video":"","vorDoi":"10.1038/s41434-025-00559-0","vorDoiUrl":"https://doi.org/10.1038/s41434-025-00559-0","workflowStages":[]},"version":"v1","identity":"rs-6297887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6297887","identity":"rs-6297887","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}