Technical Tips

Technical Tips for Secondary Antibodies

Simultaneous detection of more than one antigen depends on:

Secondary antibodies that do not;

  • recognise one another
  • recognise other primary antibodies
  • recognise immunoglobulins from other species present in the assay
  • cross-react with the tissues or cells under investigation.

Probes that are well resolved (enzyme-reaction products, fluorophores, or electron-dense particles).


Minimised cross reactivity antibodies:

  • Most multiple-labelling experiments require minimised cross reactivity antibodies.
  • Tested by ELISA and/or adsorbed against the IgG and serum proteins of other species.
  • Use when immunoglobulins from other species may lead to interfering cross-reactivities.
  • Anti-Mouse IgG (min X Rat and other species) and Anti-Rat IgG (min X Mouse IgG and other species) have diminished epitope recognition.


  •  picture3Warning: Antibodies against one species may cross-react with a number of other species.
  • Warning: Bovine serum albumin (BSA) and dry milk may contain IgG which reacts with anti-bovine IgG, anti-goat IgG, anti-horse IgG, and anti-sheep IgG antibodies.

Example of the use of minimum cross reactivity secondaries

Mouse tissue antigen A

Mouse tissue antigen B

Mouse tissue antigen C

Step 1: 
4-5% Normal Donkey Serum
Step 2:
Goat Anti-Mouse antigen A
Step 3:
Probe 1-conjugated
Donkey Anti-Goat IgG
(H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Serum Proteins) 
Step 4:
4-5% Normal Donkey Serum
Step 5:
Rabbit Anti-Mouse antigen BStep 6:
Probe 2-conjugated
Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP,
Sy Hms, Hrs, Hu, MsRat, Shp Serum Proteins)
Step 7:
4-5% Normal Donkey Serum
Step 8:
Rat Anti-Mouse antigen CStep 9:
Probe 3-conjugated
Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP,
Sy Hms, Hrs, Hu, MsRb, Shp Serum Proteins)

Blocking and Double Labelling with Fab Fragments

  • Fab fragments of affinity-purified, secondary antibodies are used to sterically cover the surface of immunoglobulins for…
  • Double labelling primary antibodies from the same host species,
  • To block endogenous immunoglobulins on cell or tissue sections.
  • As a means to label primary antibodies in solution without compromising activity.

Why monovalent Fab fragments?

  • Whole IgG molecules and F(ab’)2 fragments of IgG have two antigen binding sites.
  • After binding to its primary antibody most secondary antibodies will still have one open binding site, which can capture a different second primary antibody from the same species.
  • Overlapping labelling of the two antigens will occur.
  • Not necessary when primary antibodies from the same host species are different classes of immunoglobulins, such as IgG and IgM.
  • Not necessary when primary antibodies from the same host species are different subclasses of IgG, e.g. Mouse IgG1 and Mouse IgG2a.
  • Class-specific / Subclass-specific antibodies can be used.

Possible protocols for double labelling using Fab fragments

  • Empirical optimisation will be necessary.
  • Block with appropriate 4-5% normal serum between certain steps to reduce background.
  • In experiments with multiple layers, a fixative step may be needed.


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