Monash Antibody Technologies Facility

Transcription

1 Monash Antibody Technologies Facility The Monash Antibody Technologies Facility: A high throughput monoclonal antibody production platform for phospho-kinomics Tecan Symposium Zürich September 2007

2 What are Antibodies? They are proteins They are produced by the body to fight diseases They bind with incredible specificity to their target

3 What are Antibodies? Antibodies come in several different isotypes IgM (pentameric) IgG1 IgG2a IgG2b What are Monoclonal Antibodies?

4 Monoclonal antibodies are secreted by a single cell Antibody Molecules B-Cell

5 ..or from multiple copies of the same original cell

6 Uses of Monoclonal Antibodies Academics Functional Genomics Diagnostics Therapeutics

7 Monoclonal Antibodies are Produced by Fusing Together Two Cells.. Myeloma Cell: Does not secrete antibody Lives in culture (immortal) B-cell: Secretes antibody Does not live in culture (mortal)

8 to form a Hybrid Cell or Hybridoma Hybridoma: Lives in culture and divides Secretes monoclonal antibody

9 Mix and Fuse Myeloma Cell: 50 million B-cell: 100 million

10 to form a Hybrid Cell or Hybridoma ields 1500 Hybridomas

11 Fuse, plate-out & wait Fusion cell slurry plated into well plates in selection medium Wait 2 weeks Antibodies secreted into culture medium tested by ELISA (1920 ELISAs per fusion) Positive cells selected and grown up

12 Traditional Screening - Manual Fusion cell slurry plated into well plates in selection medium (wait 2 weeks) 8 fusions = well plates ELISAs in triplicate = 480 plates Against an average of 2 targets = 960 plates

13 EMBL Screening: The Antigen Microarray Assay Protein microarray Treat ordinary glass microscope with amino-silane slide to allow target to bind Aminosilane Ordinary Glass coat Microscope Slide

14 EMBL Screening: The Antigen Microarray Assay Protein microarray Coat the chip with the target

15 EMBL Screening: The Antigen Microarray Assay Protein microarray Print the chip with 6000 supernatants in triplicate (18,000 total)

16 The Solutions EMBL Screening: The Antigen Microarray Assay Protein microarray technology Wash, probe with fluorescently labelled, isotype-specific antibody mix

17 EMBL Screening: The Antigen Microarray Assay Protein microarray technology Wash and scan in microarray scanner

18 The Protein Chip

19 Advantages of EMBL s AMA Screening High affinity antibodies in as little as 12 weeks Screen against multiple targets simultaneously High throughput and low cost IgG isotypes guaranteed Additional subsets of monoclonals detected

20 Advantages of EMBL s AMA Screening Manual Screening: Throughput (per capita per annum) = 10 projects EMBL s AMA screening plus automation: Throughput (per capita per annum) = 100 projects

21 Monash Antibody Technologies Facility Throughput (per capita per annum): 1000 Total facility throughput 3000 to 5000 per year

22 Monash Antibody Technologies Facility Dual strategy: Massive automation Two screening technologies

23 Monash Antibody Technologies Facility Massive Automation

24 Monash Antibody Technologies Facility Massive Automation

25 Monash Antibody Technologies Facility Massive Automation Small Sample Store Reception Workstation

26 Monash Antibody Technologies Facility Massive Automation ELISA Workstation X2

27 Monash Antibody Technologies Facility Massive Automation ELISA Workstation X2

28 Monash Antibody Technologies Facility Massive Automation ELISA Workstation X2

29 Monash Antibody Technologies Facility Massive Automation Fusion Workstation

30 Automated wash/centrifuge cycles Monash Antibody Technologies Facility Massive Automation Multiple Fusions Fusion Workstation

31 Monash Antibody Technologies Facility Massive Automation AMA Workstation

32 Monash Antibody Technologies Facility Massive Automation Genetix CloneSelect Imager Cell Expansion Workstation

33 Monash Antibody Technologies Facility Massive Automation Aliquoting Workstation

34 Monash Antibody Technologies Facility Massive Automation Cryotubes Labelling Workstations 15 ml Falcons

35 Monash Antibody Technologies Facility Dual strategy: Massive automation Two screening technologies

36 In-license the Antigen Microarray Assay Protein microarray Print the chip with 6000 supernatants in triplicate (18,000 total)

37 In-license the Antigen Microarray Assay 32 channel, high speed non-contact array printer Footfall technology - matches any plate to any slide

38 In-license the EMBL Antigen Microarray Assay Low antigen amounts (< 400 µg) Differential screening Three (or more) phosphorylation states Epitope mapping at primary screen Insoluble antigens Isoform Screening

39 Genetix ClonePixFL

40 Genetix ClonePixFL

41 Genetix ClonePixFL Bulk screening High antigen amounts (> 400 µg) Single or double target screening Phospho- & Non-phospho- peptides 24 fusions per day capacity

42 Monash Antibody Technologies Facility Start Date: April/May 2008

43 Monash Antibody Technologies Facility Who we are Alan Sawyer Director Mike Spiegel Deputy Director

44 What we do Generate novel hybridoma cell lines for monoclonal antibody production using: Recombinant Proteins also truncated (100 a.a.) Insoluble Recombinant Proteins - gel bands, inclusion bodies Peptides - Phospho-specific

45 Additional Services Peptide design Coupling peptides to carrier proteins Custom screening Full subcloning to monoclonality Isotyping Bulk production Cell line banking Production of stock (e.g. tag) antibodies

46 What we need Two service levels - two sets of requirements Basic service (Clonepix screening) Premium service (AMA screening) 500 µg recombinant protein 250 µg recombinant protein down to 80 µg 1 mg synthetic peptide (we do coupling) OR 200 µg coupled peptide µg uncoupled Screen against up to 2 targets 1 mg synthetic peptide (we do coupling) OR 200 µg coupled peptide + 50 µg uncoupled Screen against up to 200 targets

47 Typical Project - Duration From receipt of sample to shipping of cell: Target 12 weeks MATF Capacity 5000 Projects per year

48 What will we do with all the capacity? Phospho-kinomics A mouse monoclonal specific for every phosphorylation site of every human kinase 5000 phospho-specific antibodies 5000 additional monoclonals against non-phospho form of same peptide 5000 peptide pairs 100,000 cell lines

49 What will we do with all the capacity? Phospho-kinomics A rat monoclonal specific for every human kinase Rat and mouse complementary pair monoclonals for every human kinase Capture and detect assays Quantitative arrays Multiplex assays (Luminex, Bioplex)

50 Acknowledgements Mike Spiegel Federico De Masi Heike Wilhelm Pieranna Chiarella Marzia Massimi Belinda Bullard Melanie Leuener Christian Fasci