Multiplex Immuno-PCR for Simultaneous AAV Capsid and Genome Titering
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for determining AAV capsid titer and genome copy titer are costly, time-consuming, and lack sensitivity, requiring separate assays that do not efficiently quantify both parameters simultaneously.
Innovation Solution
A novel immuno-PCR (IPCR) method using antibody-DNA oligonucleotide conjugates in a multiplex PCR reaction to simultaneously determine AAV capsid titer and genome copy titer, employing anti-AAV antibodies coupled with DNA oligonucleotides for PCR amplification, and paramagnetic beads for purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If separate ELISA and PCR assays are used to determine AAV capsid titer and genome copy titer, then both parameters can be measured, but the process is costly, time-consuming, and lacks sensitivity
Solution Approach 1:
The patent combines ELISA and PCR assays into a single immuno-PCR assay. Antibody-DNA conjugates are used to capture AAV particles via antibody binding, and simultaneously enable PCR amplification of captured genomes. This merging of capture and detection functions into one assay resolves the contradiction by achieving both parameter measurement in a single step, improving sensitivity while reducing time and cost.
Solution Approach 2:
The antibody-DNA conjugate serves multiple functions: it acts as both the capture reagent (like antibody in ELISA) and the template for PCR amplification. This multi-functional molecule enables the assay to simultaneously perform capsid detection and genome quantification, resolving the time and cost issues of separate assays while maintaining high sensitivity through PCR amplification.
2Adaptability or versatility
If separate ELISA and PCR assays are used to determine AAV capsid titer and genome copy titer, then both parameters can be measured, but the process is costly and requires multiple separate procedures
Solution Approach 1:
The patent merges two separate assay systems (ELISA for capsid, PCR for genome) into a single integrated immuno-PCR platform. The antibody-DNA conjugate bridges the immunological capture step and the molecular amplification step, allowing simultaneous quantification of both capsid and genome in one procedure, thereby improving versatility while reducing overall complexity.
Solution Approach 2:
The antibody-DNA conjugate acts as an intermediary molecule that connects the antibody-based capture mechanism with the DNA-based amplification mechanism. This intermediary enables the two previously separate assays to function together in a unified system, allowing simultaneous measurement without requiring multiple separate procedures.
3Reliability
If conventional ELISA method is used to measure AAV capsid titer, then the procedure is well-established, but it lacks sensitivity compared to PCR-based methods
Solution Approach 1:
The patent changes the detection parameter from enzyme-based colorimetric/fluorometric readout (ELISA) to PCR-based amplification readout (immuno-PCR). By replacing the signal amplification mechanism while maintaining the antibody-based capture, the assay achieves ELISA's reliability in particle capture combined with PCR's superior sensitivity and quantification capability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method provides improved sensitivity and efficiency by allowing simultaneous quantification of both titer types in a single assay, reducing costs and time, and enhancing precision through absolute quantification.
Implementation Method 1
anti-AAV antibodies coupled with DNA oligonucleotides for PCR amplification
Implementation Method 2
multiplex PCR reaction to simultaneously determine AAV capsid titer and genome copy titer
Implementation Method 3
employing anti-AAV antibodies coupled with DNA oligonucleotides for PCR amplification, and paramagnetic beads for purification
Data Source
AI summary
Disclosed is a substrate and a method for quantitative immuno-PCR (IPCR) for simultaneous determination of AAV capsid titer and AAV genome copy titer. The substrate is an antibody to the capsid of an AAV coupled to a DNA oligonucleotide. AAV bound to the AAV antibody-DNA oligonucleotide conjugate is purified from crude or purified cell lysate using paramagnetic beads. Multiplex PCR is performed with a primer and probe set for amplification of the DNA oligonucleotide of the conjugate and a second primer and probe set for amplification of the AAV genomic sequence to determine the AAV capsid titer and AAV genome copy titer in a single reaction.