Then, you are right. 

So are doing an ELISA like this:

//Ab1 GuineaPig / AG / Ab2Rabbit / antiRabbit-HRP 

so leave everything out what is between the solid phase antibody and the anti-rabbit. 

1.             //Ab1 GuineaPig / - / Ab2Rabbit / antiRabbit-HRP 

2.             //Ab1 GuineaPig / AG / - / antiRabbit-HRP 

3.             //Ab1 GuineaPig / - / - / antiRabbit-HRP 

If No 3 still highly reactive than your anti-Rabbis recognized the guinea pig antibodies.

Two opportunities to solve this issue:

1.     You can absorb the anti-rabbit (which is mostly not that successful or

2.     You should label the rabbit-antibody directly. This is done easily via biotinylation. It’s for sure that you a huge amount of any other proteins in the serum but only the specific antibodies will recognize the antigen. For later experiments, it is always good to prepare via positive affinity chromatography the specific antibodies only for labelling.

If you are inducing antibodies against the same antigen in different species, it is expectable that they are recognizing the same epitopes. Therefor you should keep in mind, what defines an epitope. It’s has to have antigenic properties, i.e. the immune system should be able to recognize this. It’s has to be on the protein surface, it’s has to be presentable by antigen presenting cells, … This is what can make the antigen recognition between different species different. Here we have related species, both are rodents. Assuming this, there will be some epitopes on your antigen, which are differently recognized by both species but the majority, will be identically recognized.

Considering this for an ELISA setup, you can expect the competition, but not for 100%.

If you are doing the same experiment with a single epitop (e.g. using a 10 -15 AA peptide), you can observe this as well. This will work even in different ELISA setups.

//AG(Epitop)  / Ab1 + Ab2-HRP / Substrate / Stop

//Ab1 / Epitop-Biotin / Strepavidin-HRP / Substrate / Stop

//Ab2 / Epitop-Biotin / Strepavidin-HRP / Substrate / Stop

You can run also the experiment with monoclonals from rat and mice. This will word as well showing a competition.

(// = solid phase; / = washing, HRP = label peroxidase, Ab1 = from Species 1, Ab2 ...)

regarding dr Kiessigs remarks: guinea pig and rabbit are not related. A rabbit = Oryctolagus cuniculi belongs to the order Lagomorpha. Whether a guinea pig is a real rodent has been disputed in the 90-ies

Thank you all for the suggestions and advice. I think using one different host might solve my problem - I will consider using a commercial antibody for this.

Hi, sometimes you may find cross-reaction between Rabbit and GP hyperimmune sera. You can use BSA-agaraose to minimize the signsl-to-noise ratio.

Sheets

Thank you Jitendra. I will try that as well.

I would suggest that you add 1ul normal guinea pig serum per ELISA well to your detection antibody. It is a good chance that this will solve your problem. 

Crossreactivity between different species is depending on the phylogenetical distance between the antigen and the antibody producing animal and the similarity between the two species that you compare. Forinstance if you make a rabbit anti-human IgG antibody, it will crossreact with other mammalian IgG e.g. pig, goat, mouse, rat, horse, dog, cat andcow IgG due to the similarities. It will not react with rabbit IgG (tolerance) or fish or chicken IgG (due to the phylogenetical difference).A chicken antibody will react with more epitopes on mammalian proteins (due to the phylogenetical difference) than a mammalian antibody. If you take insulin as an example there is 2%difference between pig and human insulin while 14% of the aminoacids differ between chicken and human insulin. In theory you will have 7 times more eptitopes that can cause immune response when you use chicken antibodies. It is a good chance that these epitopes are available on other mammalian insulin molecules leading to crossreactivity. To reduce crossreactivity, commercial antibodies may be absorbed with serum from other species. This reduces the total amount of the antibodies in the antiserum reducing the activity of the antiserum.  

The crossreactivity that you describe may be due to rheumatoid factors in your antisera. Rheumatoid factors is used as a marker for rheumatoid arthritis, but it frequently is present in response to immune stimulation. Usually we see it in response to bacterial or viral infections, but immunization is also a way of inducing immune stimulation. A classical method to reduce rehumatoid factor or HAMA interference is to add normal serum to the detection antibody. A suggestion is to add the equivalent of one microliter normal guinea pig serum per well to your diluted detection antibody. Anti-guinea pig IgG antibodies in your detection antibody will be blocked by the IgG in the guinea pig serum and it will reduce rhumatoid factor reactivity. 

If you want to block it even more efficiently you could heat-aggregate the guinea pig serum. You could look at some of the earlier publications by Bjerner J and Nustad K if you want to read more about heterophilic antibodies.

Hi Anders,

here we are talking about the induction of antisera against the same antigen in different species. It's not a cross-reaction as you assume, here we have the recognition of the same epitopes by different species. This is mainly driven by factors like antigenicity, immunogenicity that is realized via different antigen presentations in different species. 

A simple example: monoclonals from different species, recognizing the same peptide epitope compete for binding on the antigen. 

Thank you Prof Larson for the valuable explanation and advice. I am definitely going to try blocking in the way you described, it sounds like that might just solve the issue. Dr Kiessig - I am having this problem even in the absence of antigen (i.e. I get a positive signal if I coat with guinea-pig antibody, then add rabbit antibody and then add anti-rabbit conjugate, and vice versa. I even have this problem when coating with guinea pig antibody, then adding normal rabbit serum and then anti-rabbit conjugate). Could this still be because of the reasons you described?

Then, you are right. 

So are doing an ELISA like this:

//Ab1 GuineaPig / AG / Ab2Rabbit / antiRabbit-HRP 

so leave everything out what is between the solid phase antibody and the anti-rabbit. 

1.             //Ab1 GuineaPig / - / Ab2Rabbit / antiRabbit-HRP 

2.             //Ab1 GuineaPig / AG / - / antiRabbit-HRP 

3.             //Ab1 GuineaPig / - / - / antiRabbit-HRP 

If No 3 still highly reactive than your anti-Rabbis recognized the guinea pig antibodies.

Two opportunities to solve this issue:

1.     You can absorb the anti-rabbit (which is mostly not that successful or

2.     You should label the rabbit-antibody directly. This is done easily via biotinylation. It’s for sure that you a huge amount of any other proteins in the serum but only the specific antibodies will recognize the antigen. For later experiments, it is always good to prepare via positive affinity chromatography the specific antibodies only for labelling.

Thank you so much for the great advice Dr Kiessig!

This is not commonly observed, cross-reactivity between guinea pig and rabbit IgG antibodies. Responses by Dr. Kiessig are the most logical and scientific. I have nothing to add to these excellent responses, except wanted to mention my experience with no cross-reactivity between guinea pig and rabbit IgG antibodies.

in my experience anti rabbit secondary antibodies cross react with guinea primary antibodies.