An In Depth Look at Assay Development at Quansys Biosciences

Quansys Biosciences specializes in developing custom multiplex ELISA panels. Our custom development process ensures that the customer’s array is built quickly and efficiently while meeting their set quality specifications and keeping costs low. The process is flexible, and can be customized to meet the customer’s needs. Below is an outline of what a typical custom development entails, including common steps, specifications, and troubleshooting examples.

Phase I: Proof of Concept (2-3 weeks)

The purpose of Phase I is to evaluate the feasibility of building and optimizing the assay(s).

  1. Phase I Meeting: Reagent sources and target specifications are determined.
  2. Reagent Acquisition (may be commercial or provided by the customer).
  3. Basic Reagent Testing & Optimization:
    1. Functionality & Specificity: Reagents for each target are screened for functionality and intra-panel specificity, which must typically be less than 1%. If a matched antibody pair is does not capture the desired antigen or displays unacceptable cross-reactivity, we may try an alternate antibody source or type, adjust reagent concentrations, run western blot tests with the reagents to confirm functionality, or try a different assay format (i.e. competitive).
    2. Method Optimization: Testing of basic production methods begins and may include optimization of plate printing and processing, the assay protocol and calibrator ranges, diluent formulations, and reagent storage.
    3. Intra-Assay Precision: Twenty replicates of a high, mid, low, and negative control are run, %CV must typically be less than 15%. If %CVs are too high, other plate production methods or diluents may be explored.
    4. Recovery and Linearity: Recovery and linearity of dilution is investigated, and must typically be 80%-120%. If recovery or linearity for one or more analytes is poor, incubation times or diluent formulations may be adjusted.
  4. Phase I Deliverable(s): Phase I Feasibility Report.

Phase II: Assay Optimization and Characterization (8-16 weeks)

The purpose of Phase II is to create the assay protocol and fully develop the array.

  1. Phase II Meeting: Customer specifications for production are set.
  2. Assay Optimization: Production methods and assay protocols are optimized.
  3. Beta kit production and characterization testing:
    1. Calibrator Analysis: Kit calibrator concentrations are confirmed using commercially available ELISA kits or other reference standard. Percent Backfit of all calibrator curve points must typically be 80%-120%.
    2. Cross-Reactivity and Interference testing: Intra-Panel cross-reactivity must be less than 1%.
    3. Full Precision:
    4. Recovery and Linearity: Recovery and linearity of dilution is investigated, and must typically be 80%-120%. If recovery or linearity for one or more analytes is poor, incubation times or diluent formulations may be adjusted.
      1. Intra-Assay Precision: Twenty replicates of a high, mid, low, and negative control are each run on three plates, %CV must typically be less than 15%.
      2. Inter-Assay Precision: Four replicates of high, mid, low, and negative controls are tested over 20 plates from 3 different plate batches by 3 users on multiple days. % CV between plates must typically be less than 15%. If plates from a particular batch are causing the %CV to be too high, we may reexamine plate printing and processing protocols.
    5. Full Recovery and Linearity: Recovery and linearity of dilution testing is repeated, percent recovery and linearity of dilution for high, mid, and low controls spiked into biological matrix samples must typically be 80% – 120% of that spiked into the sample diluent.
    6. Assay Drift: Twenty-six replicates of a high control added 0, 10, and 20 minutes early are run. The percent difference must typically not exceed 10%.
    7. Edge Effects: A mid control is run over an entire plate. The %CV between the inner wells, outer wells, and whole plate must typically be less than 10%, the % Difference between the mean inner and outer wells must typically be less than 8%.
    8. Sample Correlation: Inter-lab and/or inter-method comparison of sample results.
    9. Stability: Real-time, accelerated, open-kit, and shipping stability tests are begun. If a reagent is found to degrade unacceptably, diluents may be re-optimized or further lyophilization testing may be performed.
    10. Software Validation: Use of kit product codes are checked in Q-View.
  4. Phase II deliverables: Phase II Characterization Report, assay protocol, and alpha kits if desired.

Phase III: Assay Performance Verification (8-10 weeks)

The purpose of this phase is to verify the characterization results of Phase II with a new lot of product produced in Phase III.

  1. Phase III Meeting: Product Specifications are finalized; design of the product is frozen.
  2. Verification testing (repeat of characterization, see above) is repeated: cross-reactivity, interference, precision (intra, inter, and lot-lot), recovery, linearity, drift, edge effects, sample correlation, stability (real-time, accelerated, open-kit, and shipping), software.
  3. Deliverables: Phase III Verification Report, technology transfer documentation, customer beta kits.

Phase IV: Pilot Production Run and QC Release (6-8 weeks)

The purpose of Phase IV is to produce the first lot of custom Q-Plex kits for customer use.

  1. Phase IV Meeting: Shipping and QC requirements, kit components.
  2. Kit document design and write up: Kit component labels, kit manuals, Certificates of Analysis.
  3. Pilot kit production of kits.
  4. Quality control testing of kits (repeat of characterization, see above), establishment of Pass/Fail criteria for all kit components.
  5. Phase IV Deliverables: Pilot lot of kits, QC Release Report, final production protocols
  6. Project Closing Meeting: Ensure Phase IV verification results match product specifications, ensure customer satisfaction.

With this flexible product development system in place, Quansys is able to take a custom multiplex ELISA from concept to product in just 24-37 weeks. We provide the multiplexing expertise to build a sensitive, reliable, low-cost product to meet your research needs.

For more Tech Tips, see http://www.quansysbio.com/tech-tips,
Contact us with questions at info@quansysbio.com or 888-782-6797.


About Us

"We are dedicated to the development of protein arrays that aid researchers and clinicians in better understanding, diagnosing, and treating disease. Our products and services provide value to customers by improving the accuracy and efficiency of their testing."

Quality

Quansys Biosciences is an ISO 9001 and ISO 13485 registered company. We pride ourselves in the quality of our product and our commitment to customer satisfaction.


Quansys Bioscience
Fax: 435-750-6869
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