Comparison of two different methods using overlapping synthetic peptides for localizing linear B cell epitopes in the U1 snRNP-C autoantigen.
We have compared the performances of two different approaches using overlapping synthetic peptides to identify the location of linear epitopes of the U1 snRNP-C autoantigen. The first method was based on the use of 15 overlapping peptides (16-30 residue-long) synthesized using conventional Fmoc chemistry, removed from the resin by a standard cleavage procedure, and tested by ELISA after direct coating to polyvinyl microtiter plates. The second approach used a commercial kit (SPOT) to synthesize 75 overlapping decapeptides on cellulose membrane which were assayed by a direct immunoenzymatic test. Both standard and SPOTscan methods were evaluated with antibodies raised in rabbits against synthetic peptides of U1C and sera from patients with autoimmune diseases. In addition to inherent problems linked to the SPOT synthesis (in particular the impossibility of checking the quality of peptides), a number of limitations in the SPOTscan method were identified (e.g. a certain lack of sensitivity and, in one case, the complete lack of peptide reactivity due to the removal of charged end groups at both extremities). However, we found no background with sera from autoimmune patients in the SPOTscan and the antigenic maps obtained using the two approaches generally agreed. This study shows that the SPOTscan approach represents a simple, relatively non expensive and rapid method for initial screening to identify candidate sequences that may be dominant linear epitopes in a protein. Subsequent analysis and controls should include the preparation of conventionally synthesized peptides for formal immunochemical investigations.
Halimi
H
H
Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, Strasbourg, France.
Dumortier
H
H
Briand
J P
JP
Muller
S
S
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000032:analysis*; D001402:B-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000032:analysis*
D001324:Autoantigens; D010455:Peptides; D017412:Ribonucleoprotein, U1 Small Nuclear