Establishment and Assessment of ELISA Detecting AAV9-Binding Antibodies in Biological Samples
To establish an assay for detecting adeno-associated virus 9(AAV9)binding antibodies in biological samples.An indirect Enzyme-linked immunosorbent assay(ELISA)detecting the AAV9-binding antibodies with use of AAV9 virus particle-coating plates was exploited.The cut point,sensitivity,precision,specificity and drug tolerance are validated using the sera from healthy donors and mice antisera administrated with a rAAV9 bio-therapeutic product(BTP),GC301.The screening cut point(SCP)of this assay is 1.50,titer cut point(TCP)is 2.46,confirmation cut point(CCP)is 45.75％and its sensitivity is 75.79 ng/mL and the inter-assay precision is 7.28％.Both anti-AAV5 mAb and AAV8 mAb at 5 μg/mL showed no binding to AAV9.The anti-AAV9 mAb as the high-,moderate-,and low-quality control could tolerate 1×109 capsid particle(CP)/mL AAV9 virus particles.The serum anti-AAV9 binding Abs from 5 mice treated with GC301 was measured.Sera conversion on D28 after treatment was detected and the positive-binding was identified by confirmatory assay.The inhibition rate of the antisera at the dilution of 1∶8 000 was above the value of CCP,indicating confirmed positive.The titer of those antisera is 1∶128 000～1∶256 000.The ELISA for detection of AA V9-binding antibodies reported in this study reach the CDE requirement,as listed in"the Technical Guidelines for Immunogenicity Assessment of bio-therapeutic product".The assay for biological samples could also be utilized in pre-clinical and the ongoing clinical trials on rAAV9 BTPs.
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维普
