The lower the homology, the higher the immune response. When working with highly conserved mammalian proteins, we recommend immunizing chickens to produce specific IgY. Non-mammalian hosts are better at generating specific polyclonal antibodies against highly conserved mammalian proteins.
For large batches of antibodies, we recommend immunizing one goat or one sheep (~1L serum per animal), several rabbits in parallel (~50ml serum each), or one hen to collect several eggs (4 eggs contains as much antibody as the serum of one rabbit).
For double-staining experiments, we recommend immunizing a combination of one mammal (eg rabbit or mouse) and one hen. Hence the anti-IgG antibodies will not cross-react with IgY, and vice versa.
The amount of antigen available influences the host selection. For example, immunizing one mouse requires 15 µg protein per injection, while goat immunization will require 200 µg per injection.
Determining the quantity of antigen to supply
If you provide the antigen for the immunization, the requested quantity depends on the immunized host species.
Indeed, a small rodent will require a smaller antigen volume than a larger animal to trigger the immune response and produce specific antibodies.
The antigen size is also to consider. Antigens with molecular weight above 18kDa will activate the immune response more efficiently than a smaller one. Accordingly, the requested quantity for a large antigen will be smaller than for an antigen with a MW below 18 kDa.
Antigen quantity and bleed volume comparison
|Min. antigen quantity / injection||Bleed volumes / animal|
< 18 kDa
> 18 kDa
|Pre-immune||Small Bleed||Large bleed||Final bleed||Comment|
|Mouse||40 µg||15 µg||40-70 µl||40-70 µl||40-70 µl||300-500 µl||Good to test antigenicity|
|Guinea pig||50 µg||30 µg||1 ml||1 ml||2-3 ml||10-15 ml||For small serum volumes|
|Rat||50 µg||30 µg||± 2 ml||± 2 ml||± 2 ml||5-7 ml||For small serum volumes|
|Chicken||200 µg||100 µg||1 egg||1 egg||± 8-10 eggs||± 8-10 eggs||For mammalian antigens and large quantities of Antibodies, 4 eggs = 1 rabbit final bleed|
|Rabbit||200 µg||100 µg||2 ml||2 ml||20-25 ml||50-70 ml||For most applications|
|Goat||400 µg||200 µg||2 ml||2 ml||250 ml||1000 ml||For large batch volumes|
|Sheep||400 µg||200 µg||2 ml||2 ml||250 ml||1000 ml||For large batch volumes|
in max 5 ml
|200 µg||2 ml||2 ml||250 ml||1000 ml||Single chain antibodies and for large batch volumes|
|Horse||400 µg||200 µg||2 ml||2 ml||250 ml||1000 ml||For large batch volumes|
|Other||Contact us for recommendations|