1. 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

2. 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.

3. 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

4. Antibody Detection and Specificity Secondary antibody bound to surface Primary antibody secreted by cells Incubate with labeled antigen

5. 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

6. 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

7. 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

8. 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

9. 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

10. Questions?