I was going to order short peptides 20-30AA conjugated to KLH to immunize a rabbit for antibodies against a plant pathogen.
Fortunately i stumbled across this article which reports KLH-antibodies cross reacting with plant proteins. https://www.ncbi.nlm.nih.gov/pubmed/18992832
It also claims " Polyclonal antisera from herbivorous animals, such as rabbits and chicken, often display non-specific background
reaction towards plant antigens, while rat antisera usually exhibit minimal such cross-reactivity. "
but in our experience with rabbits this has never happened, do you believe rats are actually a better option?
Back to main issue, i do not want to perform affinity chromatography to purify my peptide specific antibodies.Would cross absorption with KLH do the trick?
Alternatively, could you recommend another carrier protein that is as good as KLH in inducing an immune response without the cross reactivity with plant antigens?
Many thanks :)
I agree with John that BSA is a very good carrier molecule for this purpose. It also has the advantage that peptide conjugation can easily monitored by MALDI-MS.
From my point of view, affinity chromatography with the peptide would be the best option in most cases, particularly in the context of complex samples. Many companies offering custom antibodies purify the antibodies this way, if the peptide is available. Cross-adsorption on KLH might be beneficial, but leaves all other cross-reacting antibodies in the serum. It only helps, if you can track down your problem definitely on the KLH issue.
I agree with John that BSA is a very good carrier molecule for this purpose. It also has the advantage that peptide conjugation can easily monitored by MALDI-MS.
From my point of view, affinity chromatography with the peptide would be the best option in most cases, particularly in the context of complex samples. Many companies offering custom antibodies purify the antibodies this way, if the peptide is available. Cross-adsorption on KLH might be beneficial, but leaves all other cross-reacting antibodies in the serum. It only helps, if you can track down your problem definitely on the KLH issue.
Michael is right about affinity purification, although you will unfortunately lose the tightest binders. If you do it, but be sure to use the native protein for your stationary phase, if possible, as you probably only want the Ab fraction that binds the native epitope.
The choice of carrier will not get rid of cross-reactivity against plant proteins. What you can do is to pre-absorb your polyclonal sera against extracts from non-infected plants to reduce the background. This is a well established method also used to reduce/eliminate cross-species reactivities for anti-IgG. To get a first feeling of how significant the cross-reactivity might be you can make experiments with unrelated rabbit sera or pre-immune controls. Based on that you may then decide whether you tackle the problem by optimization of the assay conditions or by absorption against the "negative" control, i.e. non-infected plant extract. The protocol for absorption essentially works by mixing your rabbit serum with your plant extract, incubate overnight and spin down to remove the antibody-antigen complexes. Make sure to add a preservative to inhibit bacterial growth and once your done and the sera works for your assay, aliqout and freeze. Good luck.
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