Griffin:Antibody Protein Coupling (F:P) Ratios

Antibodies from the same manufacturer can exhibit batch to-batch variation. In addition, some control antibodies are not marketed at the same concentration as the test antibody. Ideally the F:P ratio (fluorochrome : protein ratio) should be 1:1. Many available reagents have high F:P ratios and are better avoided if alternatives exist (over- conjugation can produce highly charged acidic species which tend to stick non-specifically to cells, especially if they are fixed). It is important to establish the correct working dilutions for every new antibody, including those from different batches, for each detection system.

3 to 8 biotin per IgG molecule (biotin MW: 244)

4 to 7 FITC per IgG molecule (FITC MW: 390)

1 to 2 HRP per IgG molecule (HRP MW: 40,000)

1 APC per IgG molecule (APC MW: 100,000)

~1 PE per IgG molecule (PE MW: 240,000)

AF405: 1-3 moles of dye: mole of IgG

AF488: 4-9 moles of dye: mole of IgG

AF647: 3-7 moles of dye: mole of IgG

1. Reilly JT. Use and evaluation of leucocyte monoclonal antibodies in the diagnostic laboratory: a review. Clin Lab Haematol. 1996 Mar;18(1):1-5. DOI:10.1111/j.1365-2257.1996.tb00728.x |