Dataset Information

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

ABSTRACT: Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

SUBMITTER: Guo P

PROVIDER: S-EPMC6317305 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

Guo Ping P Zhang Junping J Chrzanowski Matthew M Huang Jianhe J Chew Helen H Firrman Jenni A JA Sang Nianli N Diao Yong Y Xiao Weidong W

Molecular therapy. Methods & clinical development 20181129

Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the ...[more]

PMID: 30623003

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Project description:Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

Dataset Information

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

Publications

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

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