A microengraving method for rapid selection of single cells producing antigen-specific antibodies

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A microengraving method for rapid selection of
single cells producing antigen-specific antibodies

• J Christopher Love, Jehnna L Ronan, Gijsbert M
  Grotenberg, Annemarthe G van der Veen, Hiddie L
  Ploegh
• Presented by Raven Reddy and Omar Abudayyeh

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Monoclonal Antibodies

• Currently, dilutions of cell suspensions are
  diluted, grown, and tested for desired antibody.
  The process is repeated until monoclonality is
  achieved
• ELISA assay requires high protein concentrations,
  achieved after 7-10 days
• Impractical to screen many clones in a single
  round.

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Engraved Microwell Screening

• 1. Incubate cell suspension over microwells
• 2. Allow cells to settle, remove unbound cells
• 3. Compress against functionalized surface
• 4. Remove microwells with cells, leaving secreted
  products
• 5. Perform assays to identify desired product

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Antibody Detection and Specificity

• Secondary antibody bound to surface
• Primary antibody secreted by cells
• Incubate with labeled antigen

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Antibody Detection and Specificity

• Fluorescence correlated to wells with cells
• Low nonspecific binding in wells without cells
• Specificity of antibodies produced by individual
  cells could be determined from array

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Antibody Detection and Specificity

• Alternatively, antigen could be bound first
• Observed similar results to first method
• Could quantify number of cells based on
  fluorescence intensity

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Multiple Uses of Microwells

• Attempted to conduct two assays with same cells
• Observed similar secretion patterns, but not
  identical
• Cells secrete different amounts of protein
  depending on stage of division
• Future work aims to investigate how many
  repetitions can be conducted

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Selection of Antibody Producing Cells

• Generated polyclonal mixtures to
  H-2Kb/strepatvidin tetramers
• Selected 50 of 4300 positive cells, grew cells
  further
• 17 of 42 showed responses compared to control
  antibody
• Characterized four of their clones
• Three of them produced highly specific and unique
  antibodies against H-2kb

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Conclusions

• Earlier segregation of cells from polyclonal
  mixture than with serial dilution or ELISA
• Requires less maintenance of candidate clones
• Can screen for multiple antigens at once
• Not perfect since clones must be manually
  retrieved from wells
• Microengraving can be expanded to characterize
  other secretions from cells, such as cytokines

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Questions?
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