Portal de Periódicos da CAPES

Epitope mapping of SCLC-cluster-2 MAbs and generation of antibodies directed against new EGP-2 epitopes

1994; Wiley; Volume: 57; Issue: S8 Linguagem: Inglês

10.1002/ijc.2910570714

1097-0215

Wijnand Helfrich, Pepijn Wittop Koning, T. Huw The, Lou de Leij,

RNA and protein synthesis mechanisms

Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide- bond -dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it difficult to generate and select antibodies against other potentially useful EGP-2 epitopes. Using PCR, we have generated mutant EGP-2 cDNA (δEGP-2) from which the coding sequences for a putative immunodominant 6-kDa intra-chain loop structure has been removed. δEGP-2 transfected COS-7 cells reacted with MM104, an antibody detecting a linear epitope on EGP-2, but were not recognized by any cluster-2 MAb. To generate new anti-EGP-2 antibodies we constructed another mutant EGP-2 protein (ΔEGP-2) from which additional domains, irrelevant for antibody generation (signal peptide, trans-membrane and cytoplasmic domains), were removed. ΔEGP-2 was introduced in a prokaryotic expression system that adds a polyhistidine affinity tag to the ΔEGP-2 N-terminus, making possible one-step purification by immobilized metal-ion-affinity chromatography (IMAC). Western blot analysis showed that sera derived from mice immunized with purified ΔEGP-2 had high-titer antibodies to reduced EGP-2 samples. We conclude that the availability of prokaryotic and eukaryotic EGP-2-expression constructs might facilitate the selection of new anti-EGP-2 MAbs otherwise difficult to obtain.