I'm trying to develop an experiment for measuring BSA-AntiBSA binding using polyclonal primary IgG anti-bsa antibodies. I see that it is sold purified or in serum. In regard to the serum/anti-serum, here are some questions I have:
1. Since serum is 55% albumin, is there albumin left in anti-serum? Would the BSA antibodies in anti-serum be free, or are some of them going to be bound already to BSA molecules in the serum?
2. If I took pure, primary polyclonal antibodies and mixed them with normal serum from rabbit, mouse and human, would you expect my antibodies to interact with the albumin in the serum of all of these species, or only with certain species? Since these are primary, polyclonal antibodies, I'd expect them to bind to albumin proteins in serum across species, but I'm new to this.
Thanks
I need some clarification to help you. First I'll define "antiserum" which is the blood serum polyclonal antibodies. Animals including man may be specifically immunised with a highly purified immunogen to increase the polyclonal antibody concentration in serum to the immunogen. Thus, antiserum ("sera" plural) is whole serum with increased polyclonal IgG & IgM concentration to the specific immunogen + tens of thousands of other antibody specificities. Anti BSA within a species (rabbit for example) does not react with albumin from that species but will react with epitopes on BSA and albumins from closely related species of bovine which are not epitopes on rabbit albumin. Therefor rabbit anti BSA will react with goat. sheep and other albumins from species distantly related to rabbit but closely related to bovine. When you speak of "solid purified" what do you mean? If you are speaking of affinity purified antibody to BSA then the antibody should only react with BSA and albumins from different species which share epitomes with BSA that are not on the host (rabbit as example) species. In a sensitive ELISA assay you can easily show cross reactivity to many species albumin with an affinity purified polyclonal anti BSA. To achieve a species specific antibody a well described monoclonal antibody is your best bet. Cross absorption with unrelated albumins may reduce you anti BSA activity to unusable concentrations.
Your question is a good one and has application in ELISA assay development because highly pure BSA frequently has small amounts of bovine IgG as a contaminant. Thus rabbit anti goat IgG, commonly used a a secondary antibody, will react with and be neutralised, in part, by IgG contaminated BSA when BSA is used as a diluent. Likewise when BSA is used as a blocking agent the background signal is frequently unacceptably elevated. Cross species reactivity with affinity purified anti-immunogen needs to be considered in assay development and animal species protein or serum needs to be considered in assay design to achieve specific measurements to the target of interest.
Thank you Frank that's a very helpful answer.
Just to clarify, if one introduces BSA into rabbit, the rabbit will make polyclonal anti-bsa IgG as you mentioned. Does this mean the rabbit anti-serum still has the original BSA immunogen as well? Or has that been removed and/or consumed by the rabbit?
The BSA will be eliminated by the rabbit immune system. Typically, for antibody production, one would mix an adjuvant with the BSA (immunogen) and inject the solution inter dermal. Inter muscle and other injection routs are also used. If the immunogen (foreign protein) is introduced by inter vascular route and presuming preexisting antibody to the protein (BSA) is absent the rate of clearance is related to the production of antibody (usually 4 - 10 days). Immunogen delivered IV typically forming immune complexes which, in a dose dependent fashion, can lead to a systemic Arthus reaction or serum sickness. When immunogen is mixed with adjuvant such as Freund's complete adjuvant the immunogen stays in the tissue and is processed by tissue macrophage and dendritic cells exposing immune reactive cells to immunogen related epitopes over several weeks. The half life of immunogen delivered IV varies and is dependent on immunogen size. For example bacteria (micron particles) are cleared in minutes, proteins (BSA, IgG etc are cleared over several days with the onset of antibody production, peptides Mr < 20,000 are cleared in minutes to hours by the kidney with little or no immune stimulation. The attached link provides much literature that can be educational for the novice.
http://en.wikipedia.org/wiki/Immune_system
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