GGS LIVE - Antibody Generation

Hello BioFreakers,


Today in the GGS LIVE section we will cover how to generate rabbit polyclonal antibody:)

Method: Established methodology for antibody generation is an essential tool to study cellular functions of the protein of interest.

About: Antibodies are generated by injection of the antigen into host animal (mouse, rat, rabbit etc) and subsequent isolation of antibodies from the animal serum  (for instance from rabbit serum).

What: Generation of antibody against protein X.

There are many ways to generate antibodies but in our study case we are going to cover a protocol for generation of rabbit polyclonal antibody [monoclonal (recognise single epitope), polyclonal (recognises multiple epitopes)]. Ok, so lets see what are the main steps and tools here:

1. Antigen - design and generation - may be performed by you or company.
2. Animals - injection and animals maintanance - usually company
3. Validation of antibody - isolation and purification of antigen specific antibodies - you or company

In our study case, we will generate a polyclonal antibody against the protein of interest (size 25kDa). Because our protein is relatively small we can use its full sequence for rabbit immungenisation. In case of larger proteins (they still could be used as a whole, however purification of a big protein may be a limiting factor) a small peptide, usually from either N- or C-terminus of the protein is selected as an antigen. Please have a look at this animation showing an immune system response to antigens (Biology Animation -The Activatioof the Humoral and Cell-Mediated Pathways). This animation shows how antibodies are produced by the immune system.

Antigen generation

First we need to clone the Protein X DNA sequence into an appropriate vector that will allow us to express and purify the Protein X. In our study case we will use a GST tag for that purpose and we are going to purify it from E.coli cells. Cloning, expression and purification of tagged proteins have been previously described within different posts (just click GGS LIVE - PCR, -Cloning, -Protein tagging, -Expression and -Purification). Once we have the antigen purified (please see below):

we can contact the company that will generate the antibody for us. Before the immunogenisation we should check several pre-immune sera (serum from rabbits before the immunogenisation) to see if they are able to react with the proteins of a similar size as our protein of interest (important when antibody is going to be used primarily for immunoblotting aka Western blotting). Such a test by immunoblotting is shown below (click here for GGS LIVE - Immunoblotting tutorial):

As you can see, in our study case the test with pre-immune sera showed that all four sera react with proteins in lysates from human cells and detect a band of 75kDa. Moreover, three of them strongly recognise a band of molecular size of approximately 50kDa (sera 1, 3 and 4) and serum 2 picks up an extra band around 27kDa. Additionally, sera 3 and 4 recognise weakly two additional bands (size ~20kDa and ~30kDa for serum 3,  ~18kDa and ~35kDa for serum 4). This analysis allow us to pick up a serum (which corresponds to an animal that will be injected with the antigen) that does not recognise a band of a similar size as our protein of interest before immunogenisation. As you can see sera 1 and 4 are the only ones that do not pick up any bands in the area where our protein of interest may be migrating in the gel (between 20 and 35 kDa). Usually, the same antigen is injected into two animals to increase the chance of generating a working antibody, therefore we are going to choose rabbit 1 and 4 for our immunigenisation protocol.

After antigen generation and choosing the appropriate animals for immunogenisation, we send our antigen to the company which is going to inject it into rabbits and then provide us with immunogenised sera. This usually takes 4-12 weeks depending on the protocol used. During this time company usually provides three sera (1st, 2nd and final). These are then tested to see if they recognise the protein of interest using immunoblotting or any other immuno-technique (such as immunofluorescence or ELISA). Purified protein of interest (antigen - in our case Protein X) and cell lysates are used for these purposes. In the ideal situation the final serum (or even better 1st or 2nd one) recognises the protein of interest in the cell lysates without prior purification of the antigen specific antibodies from the serum. However, commonly the final sera recognises the antigen (a very good start and a must do in this case:)) but fail to recognise the protein of interest in the cell lysates. It also happens that the final serum is "dirty" and the immunoblots have a lot of non-specific, background signal that interferes with the detection of our protein (see below).

As you can see serum before and after the purification can easily detect the antigen but only the purified antibody can recognise the protein X in the cell lysates. This is very often due to the low concentration of the antigen specific antibodies in the serum compared to other antibodies. Therefore, the stronger the specific signal we want to obtain the stronger the background. This way it is very hard to get a strong enough signal (nice band on the right hand film above) for the protein X in the cell lysates that would be visible over the background noise. However, as you can clearly see purification of the antibody solved the problem (see here GGS LIVE - Antibody purification against the antigen).

Next step is to test if the antibody can be used in other applications such as immunofluorescence (GGS LIVE - Immunofluorescence) and immunoprecipitation (GGS LIVE - Immunoprecipitation).

I hope you enjoyed.

GGS TEAM