Presuming the purity of your recombinant protein is good (>98%) and it is immunogenic in the animal species you choose for polyclonal antibody production you can produce large quantities of affinity purified antibody with little problem. Here are a few tips I use routinely. You will produce two affinity matrices 1. Expression protein negative matrix. Save the supernatant from the culture you have removed the recombinant protein from or produce a culture supernatant grown in the same fashion but without the expressed protein of interest. You will use this to clean up your polyclonal antibody prior to affinity purification. 2. Produces an expressed protein (purity > 98%) positive affinity matrix. I use sodium periodate method to activate Sepharose CL4B for both matrices. Following activation of Sepharose and removal of periodate by washing add your recombinant protein negative preparation (dialyzed in sodium bicarbonate 0.2 M + NaCl 150 mM ~ ph 8.2) to an aliquot of the activated Sepharose at a ratio of 10 mg recombinant protein negative growth media / 1 gram Sepharose and adjust pH to 9,5 with sodium carbonate (0.2 M). Repeat using your second aliquot of activated Sepharose this time adding your recombinant protein (>1 mg/ml / gram Sepharose). Incubate overnight at 4C. Wash out non bound proteins and save the recombinant protein eluate for quantification. You should have > 90% purified recombinant protein attached and have saturated the Sepharose with the unwanted contaminants. Left over protein from the unwanted contaminant matrices is not significant. Reduce both Sepharose-protein conjugated gels with sodium borohydride To the Sepharose, add equal volume of FRESHLY prepared 5 gram / liter sodium borohydride (NaBH4) in 200 mM sodium acetate, NaCl 150 mM buffer pH 5 and incubate with mixing 3 - 4 hours. Wash extensively with PBS and store in phosphate buffered 1 M NaCl at 4 C.

See:http://www.google.co.nz/patents?hl=en&lr=&vid=USPAT3947352&id=gSUuAAAAEBAJ&oi=fnd&dq=sodium+periodate+activated+sepharose&printsec=abstract#v=onepage&q&f=false

Affinity purification will likely help reducing background but things can go wrong. Why not to test the polyclonal antibody and take a decision based on the results?

I have doing the same exact thing now!! I got a very strong antibody to my antigen. I then purified it with a column with Protein A and now i am in the process of making a column for affinity purification. You can use a multiplex assay!

For example, if you use a magpix assay (similar to elisa). Have your affinity purified antibody conjugated to a magnatic bead. Then mix ur antigen. Then add a secondary antibody which is sensitive to the entire protein containing the epitope. The secondary antibody is conjugated to biotin which if ur using the magpix machine, it will give u a strong signal (ie. if you are detecting a rice allergen, then ur primary antibody will be specific for that antigen but your secondary antibody will be able to recognize all the rice proteins). Similarly, you can do an sandwich elisa. Affinity purified Ab->antigen-> Secondary antibody that detects ur antigen but is not specific only to your epitope.

Polyclonal antibodies are good choice, because may have much more applications as compared with monoclonal antibodies. You will need to take especial care about purity of immunizing antigen and verify specificity of obtained antibodies (these two are critical). If the affinity purification will be performed on column with immobilized purified antigen, you can expect to obtain perfect results.

Before making a choice about peptide affinity purification, I suggest to perform some basic characterization work on the whole serum lot using the peptide antigen in a blocking step. You will most likely benefit from further affinity purification if the blocking peptide reduces you specific signal, but not the background immunoreactivity. If it turns out that the blocking peptide also reduces your background signal, then most likely you will not benefit from doing a further affinity purification step.

We had the experience where an antibody recognized nonspecific bands on Western blots, but immunoreacted very specifically in immunocytochemistry. We tried to see if the peptide antigen would block the specific signal (and NOT the nonspecific bands) on Western blots, but it turned out that it blocked both. For this reason, we decided that further affinity purification will not eliminate the nonspecific signal. You could try the same concept for your specific application ICC or IHC. Hope that helps!

Presuming the purity of your recombinant protein is good (>98%) and it is immunogenic in the animal species you choose for polyclonal antibody production you can produce large quantities of affinity purified antibody with little problem. Here are a few tips I use routinely. You will produce two affinity matrices 1. Expression protein negative matrix. Save the supernatant from the culture you have removed the recombinant protein from or produce a culture supernatant grown in the same fashion but without the expressed protein of interest. You will use this to clean up your polyclonal antibody prior to affinity purification. 2. Produces an expressed protein (purity > 98%) positive affinity matrix. I use sodium periodate method to activate Sepharose CL4B for both matrices. Following activation of Sepharose and removal of periodate by washing add your recombinant protein negative preparation (dialyzed in sodium bicarbonate 0.2 M + NaCl 150 mM ~ ph 8.2) to an aliquot of the activated Sepharose at a ratio of 10 mg recombinant protein negative growth media / 1 gram Sepharose and adjust pH to 9,5 with sodium carbonate (0.2 M). Repeat using your second aliquot of activated Sepharose this time adding your recombinant protein (>1 mg/ml / gram Sepharose). Incubate overnight at 4C. Wash out non bound proteins and save the recombinant protein eluate for quantification. You should have > 90% purified recombinant protein attached and have saturated the Sepharose with the unwanted contaminants. Left over protein from the unwanted contaminant matrices is not significant. Reduce both Sepharose-protein conjugated gels with sodium borohydride To the Sepharose, add equal volume of FRESHLY prepared 5 gram / liter sodium borohydride (NaBH4) in 200 mM sodium acetate, NaCl 150 mM buffer pH 5 and incubate with mixing 3 - 4 hours. Wash extensively with PBS and store in phosphate buffered 1 M NaCl at 4 C.

See:http://www.google.co.nz/patents?hl=en&lr=&vid=USPAT3947352&id=gSUuAAAAEBAJ&oi=fnd&dq=sodium+periodate+activated+sepharose&printsec=abstract#v=onepage&q&f=false

Can use ,but if you are trying first time It is very difficult for you to avoid background staining

I should note high quality of Frank's answer. The method described do results in immunoaffine sorbents possessing very low nonspecific binding due to the absence of ion-exchange properties. This allows to reduce ionic strength of buffers and to elute the protein of interest even by water if extreme pH values denaturate it. I successfully used Sepharose CL6B too.

Dmitry Kazansky's first answer is a full satisfaction to your question. The purity of the antigen and the purity of the antibodies raised against the antigen are the important components which will decide the results in your assay. Try the affinity purification of the antibodies with purified antigen first. lalitha kabilan

it's always a good chioce. You can combine negative (to remove possible cross reactions) and positve affinity chromatography.