Cross-reaction of antibodies to coupling groups used in the production of anti-peptide antibodies.

A polyclonal antibody raised against a peptide conjugated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to maleic anhydride-derivatised lysozyme showed substantial cross-reactivity with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised haemocyanin. This was due to antibodies produced against maleic anhydride-derivatised groups on lysozyme that reacted with m-maleimidobenzoyl-N-hydroxysuccinimide ester-derivatised groups on haemocyanin. This observation is important because it is common practice, in the production of anti-peptide antibodies, to use two conjugates. The same peptide is coupled to two different protein carriers by two different coupling methods. One conjugate is used for immunisation and the other for testing the serum. This method assumes that the only antigen common to the two conjugates is the peptide and this was not the case here. A method is described for screening sera which involves affinity purification of the anti-peptide antibody and comparison of binding to the immunogen with that to an appropriate control conjugate. This method avoids the problem of any cross-reaction to coupling groups or proteins.