Achievements and challenges in standardization of ELISpot

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Achievements and challenges in standardization of
ELISpot

• Dr. Guido Ferrari
• Duke University Medical Center
• gflmp_at_duke.edu

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CMI Response Assays

• Standard IVS/51Cr release assay (CRA) labor
  intensive, non-quantitative, difficult to perform
  in large scale trials, difficult to transfer to
  Southern countries, difficult to apply on
  cryopreserved samples.
• Lymphoproliferative assay (LPA) mainly used to
  detect CD4 T cell responses after 7 days Ag
  stimulation in vitro with incorporation of
  3H-Thymidine. Non-quantitative assay, can be
  performed using cryopreserved cells.
• ELISPOT assay quantitative easier to perform
  and to transfer can be performed with
  cryopreserved samples.
• ICS/CFC assay quantitative high sensitivity,
  identification of CD4 CD8 responses in same
  sample, affected by operator gating bias.

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Factors Influencing ELISpot Assay
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Assay Validation

• The assay validation identifies sources of
  potential assay variability and addresses the
  quantification of these errors in the assay
  method.
• The assay validation report follows the
  successful completion of a validation protocol,
  which defines
• the assay
• its proposed use
• lists the ranges for the sample matrix
• prospectively assigns acceptable values for the
  Assay Parameters (Accuracy, Precision, Limit of
  Detection, Limit of Quantitation, Specificity,
  Linearity, Reproducibility, Robustness and System
  Suitability).

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Assay Validation vs. Assay Optimization

• Assay optimization and pre-validation are
  experiments that determine how a range of matrix
  and sample elements, as well as assay conditions,
  effect assay parameters and assay performance.
• These data, along with scientific judgment, set
  the acceptance criteria for the assay validation.
  It is important to establish acceptance criteria
  before executing the validation protocol.

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Assay Validation vs. Assay Qualification

• Assay qualification is an experimental protocol
  which demonstrate that an accepted method will
  provide meaningful data for the specific
  conditions, matrix and samples that it will be
  used to analyze.
• Users of analytical methods described in the USP
  and NF are not required to validate accuracy and
  reliability of these methods, but merely verify
  their suitability under actual conditions of use
  CFR 211.194(a), USP 23/NF 18 1225 pp 77.

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Assay parameter definitions

• Specificity
• Accuracy
• Precision
• Detection Limit
• Limits of Quantitation
• Linearity
• Range
• Ruggedness
• Robustness

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M
MMM
TNTC (4000)
Sample ID
High-resp
SFC/million PBMC
Low/med-resp
Non-resp
VRC8
15160
VRC9
VRC5
VRC7
VRC6
BC557
BC238
VRC10
VRC2
VRC3
Median SFC to CEF pool (excludes TNTC only)
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Responses in seronegative individuals for control
reagents
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Acknowledgments