The Anti-AAV9 ELISA is used as an analytical tool for qualitative determination of antibodies to AAV9 in serum.
Contents of Kit
No.
Components
Size
Storage Conditions
1
AAV9 Capsid Coated Microtiter Plate
12 × 8 wells
2-8°C
2
Negative Control, ready to use
1 × 1 ml
2-8°C
3
Anti-AAV9 Positive Control, ready to use
1 × 1 ml
2-8°C
4
Cut-off Control, , ready to use
1 × 1 ml
2-8°C
5
Anti-human IgG Conjugate, ready to use
1 × 12 ml
2-8°C
6
Sample Diluent, ready to use
1 × 50 ml
2-8°C
7
Wash Buffer, 20 × concentrate
1 × 50 ml
2-8°C
8
TMB Substrate, ready to use
2 × 6 ml
2-8°C
9
Stop Solution, ready to use
1 × 7 ml
2-8°C
10
Instruction Manual
1
Storage
1. All reagents should be stored at 2°C to 8°C for stability. 2. All reagents and wash buffer should be used within 12 months from manufacturing date. 3. Before using, bring all components to room temperature (18-25°C). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions. 4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.
Performance Characteristics
Function verification Non-scaled Venn diagram depicting the relationship between total (tAb) and specific confirmed total antibodies in samples. The results are highly consistent, indicating that ELISA has high accuracy in detecting AAV antibodies.
Precision
General Description
Researchers have used AAV-based vectors in pre-clinical research and in clinical trials in which AAV-based vectors have demonstrated a good safety profile. AAVs have also demonstrated lasting therapeutic gene expression following a single treatment in preclinical and clinical studies.Scientists today have used the AAV9 variant, or serotype, of vector in potential product candidates in hemophilia B and Huntington's disease. Research suggests that a greater portion of patients can be effectively treated with AAV9 gene therapy, compared to other AAV-based gene therapies.AAV9 gene therapies present a favorable immunogenicity profile. To date, insect-cell manufactured AAV9 gene therapies have been administered to 22 patients across three clinical studies by a leading gene therapy company without any evidence of AAV9 capsid-specific cellular immune responses or long-term safety complications. One of the major challenges in AAV-based gene therapy is the presence of circulating anti-AAV neutralizing antibodies, which can pre-exist in patients and may prevent successful gene transfer. High levels of circulating anti-AAV neutralizing antibodies can develop after a single administration of gene therapy and can prevent successful gene transfer in patients.
Citations
Publication ()
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Transabdominal ultrasound guided AAV9-GFP delivery in fetal pigs: a translational and minimally invasive model for in utero fetal gene therapy
Di Donfrancesco A, Adelizzi A, Giri A, Duchi R, Boito S, Barandalla M, Massaro G, Santanatoglia C, Cappellozza E, Perota A, Di Meo I, Tiranti V, Bottani E, Galli C, Persico N, Brunetti D.
Applications: ELISA Reactive species: Pig
"Abstract: In utero fetal gene therapy (IUFGT) has the potential to correct severe monogenic disorders before irreversible damage occurs. Despite promising results in small and large animal models, its translation to clinical practice remains limited by technical challenges, safety concerns, and the lack of standardized protocols in relevant disease models species. We established and validated a minimally invasive, ultrasound-guided approach for systemic gene delivery in fetal pigs using a self-complementary AAV9 vector encoding GFP under a CAG promoter. Injections were performed at different gestational ages (GA 80 and GA 108) via intracardiac or umbilical venous routes. Postnatal outcomes were monitored, and transgene biodistribution and expression were assessed by qPCR, ddPCR, immunofluorescence, and Western blotting. Inflammatory response, toxicity, and maternal safety were evaluated through cytokine profiling and histological analyses. The procedure was well tolerated, with no significant maternal morbidity or adverse obstetric outcomes beyond one preterm delivery. Biodistribution analysis revealed widespread vector presence in peripheral tissues, with robust GFP expression in liver and heart. Importantly, there was no evidence of significant tissue toxicity, necrosis, or fibrosis in any of the organs analyzed. Mild increases in pro-inflammatory cytokines (GM-CSF, GRO-α, IFN-γ) were observed but were not associated with histopathological changes. No anti-AAV9 capsid antibodies were detected in sera from piglets or sows, suggesting a minimal immune response to the vector. These findings demonstrate the safety, feasibility, and efficacy of ultrasound-guided IUFGT in pigs, supporting its potential as a translational platform for therapeutic gene delivery in fetuses affected by severe congenital diseases. This model offers a valuable framework for further preclinical development of prenatal interventions, particularly for disorders with early onset, such as mitochondrial diseases." Article snippet: The presence of binding Abs reactive against the AAV vector capsid was determined by semiquantitative indirect enzyme-linked immune-sorbent assay (ELISA) technology, following manufacturer’s instructions (Creative Diagnostics, United States # DEIASL348). AAV9 capsid protein was precoated onto 96- well plates. Absorbance at 450/620 nm was read in duplicate experiments using an ELISA Microplate reader.
Figure 1. Evaluation of immune response to fetal scAAV9 delivery.
Background
Adeno-associated virus (AAV), which belongs to the parvovirus family, is the simplest single-stranded DNA virus without envelope found so far. AAV has become one of the most important gene carriers in the field of gene therapy because it cannot self-replicate, does not cause any human diseases, long-lasting gene expression and has high tissue specificity, providing a new possibility for the treatment of a variety of genetic diseases. Currently, there are 13 serotypes of AAV based on capsid protein composition (AAV1-AAV13), which differ in their proclivity and transduction efficiency in different tissues, of which AAV9 was found to have the highest proclivity to the central nervous system.
AAV9, which can bypass the blood-brain barrier and enter the central nervous system without the need to use other drugs or physical assistance, exhibits high transgenic expression and extensive central nervous system transduction in neonatal and adult mice, and is the most widely studied intravenous delivery vector. AAV9 is typically used to repair defective genes, such as lysosomal storage disease and spinal muscular atrophy type 1; to suppress dominant genetic mutations such as Huntington's disease; or to introduce a disease-modifying gene that may alleviate neuropathology, as in the case of Alzheimer's disease. In addition, due to the high efficiency of AAV9 transduction of spinal cord MN, many preclinical studies have focused on motor neuron diseases, especially spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS).
While AAV vectors are characteristically low in immunogenicity, the body's immune system can still recognize and respond to them, about 30%-80% of people have pre-existing anti-AAV antibodies, which have the possibility of affecting the effectiveness of gene therapy. Therefore, the detection of immune response to AAV vector in AAV9 therapy is very important to evaluate the safety and success rate of gene therapy. Creative Diagnostics has developed an ELISA kit for the detection of AAV9 antibodies, which can efficiently detect circulating AAV9 antibodies in human serum and ensure the efficacy and safety of gene therapy as much as possible.
Figure 1. schematic diagram of anti-AAV neutralizing antibody inhibiting transgenic expression (Source: Schulz M, et al. 2023)
1. Saraiva J, et al. Gene therapy for the CNS using AAVs: The impact of systemic delivery by AAV9. J Control Release. 2016 Nov 10;241:94-109.
2. Schulz M, et al. Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy. Mol Ther. 2023 Mar 1;31(3):616-630.
Q: I would need to know the specific antibodies that do the detection. Is this available?
A: Human anti-AAV9 Capsids IgG.
Q: Do we have to pre-dilute our samples?
A: Dilution: 1:50
Q: Are the negative / positive / cut-off controls serum, plasma or buffer?
A: All three of our controls contained antibody buffers and no serum components
Q: How did you determine the reference limit? Was it according to CLSI EP28-A3c guidelines?
A: Our method is similar to CLSI EP28-A3c guidelines, both of which use statistical methods. But we don't refer to this file
Q: How many samples did you use?
A: AAV9(118 samples)
Q: Which samples (sera, plasma, etc.) did you use to determine the cut-off?
A: Serum
Q: How did you determine the cut-off point?
A: We used our kit and a confirmed assay method in the literature to determine the cut-off
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References
Intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 shows safety and efficacy in preclinical studies
J Clin Invest
Authors: Chen X, Dong T, Hu Y, De Pace R, Mattera R, Eberhardt K, Ziegler M, Pirovolakis T, Sahin M, Bonifacino JS, Ebrahimi-Fakhari D, Gray SJ.
Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay, and subsequent intellectual disability, secondary microcephaly, and epilepsy. We preformed preclinical studies evaluating an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50 and describe in vitro studies that demonstrate transduction of patient-derived fibroblasts with AAV2/AP4M1, resulting in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1-KO mice were intrathecally (i.t.) injected with 5 × 1011, 2.5 × 1011, or 1.25 × 1011 vector genome (vg) doses of AAV9/AP4M1 at P7-P10 or P90. Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in WT mice, rats, and nonhuman primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1 × 1015 vg. Of note, similar degrees of minimal-to-mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future i.t. AAV studies. These preclinical results identify an acceptably safe and efficacious dose of i.t.-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50
Endothelial Retargeting of AAV9 In Vivo
Adv Sci (Weinh)
Authors: Bozoglu T, Lee S, Ziegler T, Jurisch V, Maas S, Baehr A, Hinkel R, Hoenig A, Hariharan A, Kim CI, Decker S, Sami H, Koppara T, Oellinger R, Müller OJ, Frank D, Megens R, Nelson P, Weber C, Schnieke A, Sperandio M, Santamaria G, Rad R, Moretti A, Laugwitz KL, Soehnlein O, Ogris M, Kupatt C.
Adeno-associated viruses (AAVs) are frequently used for gene transfer and gene editing in vivo, except for endothelial cells, which are remarkably resistant to unmodified AAV-transduction. AAVs are retargeted here toward endothelial cells by coating with second-generation polyamidoamine dendrimers (G2) linked to endothelial-affine peptides (CNN). G2CNN AAV9-Cre (encoding Cre recombinase) are injected into mTmG-mice or mTmG-pigs, cell-specifically converting red to green fluorescence upon Cre-activity. Three endothelial-specific functions are assessed: in vivo quantification of adherent leukocytes after systemic injection of - G2CNN AAV9 encoding 1) an artificial adhesion molecule (S1FG) in wildtype mice (day 10) or 2) anti-inflammatory Annexin A1 (Anxa1) in ApoE-/- mice (day 28). Moreover, 3) in Cas9-transgenic mice, blood pressure is monitored till day 56 after systemic application of G2CNN AAV9-gRNAs, targeting exons 6-10 of endothelial nitric oxide synthase (eNOS), a vasodilatory enzyme. G2CNN AAV9-Cre transduces microvascular endothelial cells in mTmG-mice or mTmG-pigs. Functionally, G2CNN AAV9-S1FG mediates S1FG-leukocyte adhesion, whereas G2CNN AAV9-Anxa1-application reduces long-term leukocyte recruitment. Moreover, blood pressure increases in Cas9-expressing mice subjected to G2CNN AAV9-gRNAeNOS . Therefore, G2CNN AAV9 may enable gene transfer in vascular and atherosclerosis models.
COA
Publication 
Figure 1. schematic diagram of anti-AAV neutralizing antibody inhibiting transgenic expression
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