Together, these studies may suggest that a second serotype could be used to boost expression if the neutralization titer is usually below a certain threshold that remains to be defined. sample sets. We thus investigated pre- vs post-vector inoculation sera samples from rhesus macaques that received AAV1 or AAV8 vector inoculations for cross-reactive anti-AAV antibodies. All 12 macaques seroconverted to the vector they received, but many also reacted to the other serotypes. Our results validate an easy-to-use ELISA for reliable detection of antibodies to individual serotypes OCTS3 of AAV. Our results also demonstrate that an antibody response post-AAV inoculation may partially cross-react with other AAV serotypes. Overall, these results suggest that either assay can be used by academic labs for prescreening samples for preexisting anti-AAV antibodies. Keywords: adeno-associated virus, AAV, capsid, serotype, ELISA, neutralization assay, gene therapy Graphical abstract Open in a separate window As more studies deploy novel AAV gene therapies, the need has grown for identifying hosts unfavorable for anti-AAV capsid antibodies. Gardner et?al. compare and show a high degree of correlation between an ELISA and neutralization assay to identify hosts unfavorable for binding and neutralizing antibodies against AAV capsids. Introduction Adeno-associated virus (AAV) vectors bring promise for treating diseases through gene therapy strategies. With the U.S. Food and Drug Administration (FDA) approval of Luxturna in 2017 and Zolgensma in 2019, the interest in using AAV vectors continues to grow. Clinical trials using AAV vectors have targeted hemophilia, alpha 1 anti-trypsin deficiency, Duchene muscular dystrophy, and Pompe disease.1 Our labs have been developing AAV vectors to express HIV-1 broadly neutralizing antibodies and antibody-like inhibitors to prevent and treat HIV-1 infection.2, 3, 4, 5, 6, 7, 8, 9 These studies have relied extensively on testing AAV vectors in rhesus macaques to evaluate their efficacy against SHIV Melanotan II and SIV contamination. Thus, we have screened hundreds of macaques for preexisting anti-AAV antibodies before conducting the studies. With the popularity of AAV vectors growing, more groups will need to evaluate their therapies in preclinical animal models. This includes the use of nonhuman primates (NHPs) such as rhesus macaques Melanotan II (neutralization assay. In the studies described here, we compared these two assays for their performance in identifying preexisting anti-AAV antibodies. For a group of 50 rhesus macaques, we observed a high degree of correlation between the two assays from serum samples tested against AAV1, AAV8, and AAV9 vectors. Correlation of the results from the two assays was also observed for these three vectors when human serum samples were tested. Additionally, both assays were able to identify cross-reactive anti-AAV antibodies after an AAV1 or AAV8 intramuscular (i.m.) vector inoculation. These data indicate that either assay could be used equivalently or together by academic labs to identify seronegative macaques for their preclinical studies. Results The goal of this study was to compare ELISA- and neutralization-based sample screening methods for preexisting antibodies against different AAV Melanotan II serotypes. ELISA plates were coated with intact virions at a concentration of 1 1? 1010 vector genomes (vg)/mL of AAV1 vectors, 5? 109 vg/mL AAV8 vectors, or 8? 109 vg/mL AAV9 vectors, and the neutralization assays used 7? 109 vg/mL of AAV1 vectors, 2? 1010 vg/mL AAV8 vectors, or 2? 1011 vg/mL AAV9 vectors. The varying amounts of vectors in the neutralization assay were used to obtain a readout of >10,000 relative light units (RLUs) from AAV transduction of HEK293T cells. Using both these assays, we assessed 50 rhesus macaque serum samples for preexisting AAV1, AAV8, and AAV9 binding and neutralizing antibodies. Binding antibodies were decided using ELISA, and neutralizing antibodies were decided using an cellular assay Melanotan II (Physique?1A). Because these methods are used to screen a large number of samples, for these studies, binding is usually defined as the absorbance value at 450?nm at 1:20 dilution, and neutralization is defined as the decrease of transduction signal (firefly luciferase) at 1:10 sample dilution. Results from the ELISA showed varying degrees of antibody binding to the three different AAV serotypes.?We observed absorbance values at 450?nm ranging from <0.1 to >1.0 (Table S1). Similar to the ELISA results, varying degrees of neutralizing.