FAQ on Antibodies (part II)

Can nanobodies be used in place of Fab fragments? 

Yes. Nanobodies are also monovalent.

Nanobodies, also known as VHH antibodies or single-domain antibodies, are the “Fab fragment” equivalent of antibodies from Alpacas or Illama. 

How do nanobodies compare to Fab fragments? Why did we prefer Fab fragments?

Fab fragments produce better signal amplification, and with OmniStain the small size of nanobodies is only marginally beneficial.

Usually, only 1-3 fluorophores can be conjugated per nanobody molecule due to their small size, while a Fab fragment can carry 9-12 fluorophores. Furthermore, because nanobodies are monoclonal, they can only bind to two sites on the primary antibody IgG molecules. Fab fragments are polyclonal and on average 3 of them bind to each primary antibody molecules. Hence, for each antigen:

• With nanobodies, max signal amplification is 2 x 3 = 6-fold.
• With Fab fragments, max signal amplification is 3 x 12 = 36-fold.

One can double the nanobody-based signal by using a mixture of nanobodies targeting different sites on the IgG molecules, but these are not widely available with doubled costs.

Nanobodies have a smaller size (~14 kDa), and theoretically, they can achieve deeper penetration. However, in the context of 3D immunostaining, the penetration depth and uniformity are limited not due to slow diffusion, but due to the depletion of antibody concentration gradients by the superficially located tissue antigens. Hence, without OmniStain, nanobodies will not enhance the penetration depth. 

With OmniStain, nanobodies do enhance penetration depth by approximately 2-3x more. Nonetheless, this is minor compared to the 10-20x penetration depth enhancement by OmniStain while sacrificing signal intensities. 

Fc-specific Fab fragments

Fab fragments can be selected for targeting specific subclasses of Fc regions of IgGs. 

Mouse and rat monoclonal antibodies for immunostaining can be of IgG1, IgG2a, IgG2b, and IgG3 subclass. They differ in the Fc regions and can be distinguished by Fc-specific Fab fragments. This is useful to expand the multiplexing capabilities of indirect immunofluorescence.

For example, anti-IgG1 Fc-specific Fab fragments will not bind to IgG2a antibodies, conversely, anti-IgG2a Fc-specific Fab fragments will not bind to IgG1 antibodies. So their signals will not be confused with one another by Fab fragment crosstalk.

As an illustration, you can have the following primary antibodies:

• Mouse monoclonal antibody, IgG1, anti-p53
• Mouse monoclonal antibody, IgG2a, anti-Ki67
• Mouse monoclonal antibody, IgG2b, anti-keratin 7
• Mouse monoclonal antibody, IgG3, anti-somatostatin

If you add Alexa Fluor 647-conjugated anti-mouse Fab fragments, all 4 antigens will show in the 647 channel. But if you use the following secondary antibody Fab fragments:

• Goat anti-Mouse IgG1 Fc-specific Fab fragments, Alexa Fluor 488-conjugated
• Goat anti-Mouse IgG2a Fc-specific Fab fragments, Alexa Fluor 584-conjugated
• Goat anti-Mouse IgG2b Fc-specific Fab fragments, Alexa Fluor 647-conjugated
• Goat anti-Mouse IgG3 Fc-specific Fab fragments, Alexa Fluor 680-conjugated

Then you will have 

• p53 in the 488 channel
• Ki67 in the 594 channel
• Keratin 7 in the 647 channel
• Somatostatin in the 680 channel

What is the maximal amount of primary IgG-secondary Fab pair that can be applied with OmniStain? 

Theoretically only limited by the compatible primary antibodies available, for example, here is one 8-plex experiment we performed with primary antibodies:

• DAPI / INSIGHT buffer C pre-mixed nuclear stain
• Mouse IgG1 anti-NeuN antibody
• Mouse IgG2a anti-MAP2 antibody
• Rat IgG1 anti-neurofilament antibody
• Rat IgG2a anti-GFAP antibody
• Rabbit anti-ALDH1L1 antibody
• Guinea pig anti-TMEM119 antibody
• Chicken anti-CNP1 antibody
• Goat anti-CD31 antibody

And the secondary Fab fragments are

• Goat anti-Mouse IgG1 Fc-specific Fab fragment, Atto 430LS-conjugated
• Goat anti-Mouse IgG2a Fc-specific Fab fragment, Atto 490LS-conjugated
• Goat anti-Rat IgG1 Fc-specific Fab fragment, Alexa Fluor 488-conjugated
• Goat anti-Rat IgG2a Fc-specific Fab fragment, BODIPY 581/591-conjugated
• Donkey anti-Rabbit Fab fragment, BODIPY-TMR-conjugated
• Goat anti-Guinea pig Fab fragment, Alexa Fluor 610-conjugated 
• Goat anti-Chicken Fab fragment, Alexa Fluor 647-conjugated
• Bovine anti-Goat Fc-specific Fab fragment, Alexa Fluor 680-conjugated

The anti-Goat Fab fragment must be Fc-specific. Otherwise, it will cross-react with the other secondary Goat Fab fragments.