Typical Antibody Work-up

In order to aid investigators in budgeting for antibody work-ups, we have prepared an outline of the typical sequence we follow when we work up a new antibody. This outline assumes that there is a commercially available antibody available that has been used for a similar application (tissue fixative, host range, etc.) as required by the investigator.

Antibody optimizations are placed in a queue in the order they are submitted. There may be several antibody optimizations that need to be completed before we can start your project. Once started, stages A, B, and C can usually be completed within 3 weeks. The time required to run the project tissue depends on the number of tissues and antibodies. Projects are scheduled in the order they are submitted.

A. Identify potential antibody, the conditions for its use, and the appropriate controls:

Professional Time: 1 - 2 hours

B. Try antibody at recommended concentration with standard antigen retrievals:

We test 4 different antigen retrieval conditions using test antibody and the isotype, each with and without test tissue.

  • No Ag Retrieval
  • pH 6 Tris
  • EDTA
  • Proteinase K

Workprocess:

  • Make control block
  • Cut sections: 16
  • IHC: 16
  • Tech Time: 2 hours

C. Titer antibody:

In this step we test 4 different antigen concentrations, again using Ab and isotype, both with and without test tissue.

Workprocess:

  • Cut sections: 16
  • IHC: 16
  • Tech Time: 2 hours

D. Test antibody on project tissue:

Finally, the actual project tissue is tested with the Ab and isotype (including controls of + or - test tisue).

Workprocess:

  • Cut sections: 6
  • IHC: 6
  • Tech Time: 2 hours

E. Additional parameters that may require testing (not limited to the following):

  • Blocking
  • Detection systems
  • Other antigen retrieval methods