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)LJXUH:HVWHUQEORWVKRZLQJWKHUHDFWLYLW\RISXULILHGP*3,P$%WRUHFRPELQDQWPRXVH*3,OHIWDQG KXPDQ*3,ULJKW Primary antibody used (A) GPI mAB 11H3.C10. (B) GPI mAB 1E3. (C) GPI mAB 46H9
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Three GPI reactive antibodies from K/BxN mice were purified. Now it was needed to know whether the three GPI hybridoma clones 1E3, 11H3.C10 and 46H9 recognized the same epitope or different epitope on GPI. Antibodies that recognized different epitopes on the GPI antigen would allow the formation of immune complexes. Transfer of a combination of antibodies should induce arthritis in mice. The classical ways of identifying whether two or more antibodies bind the same epitope region or to different epitopes on a antigen is by competition ELISA or more recently, by surface plasmon resonance technique. In both these methods the competition by the antibodies for epitope binding on the antigen are studied. A novel and simple method called peptide fingerprint western blot technique was developed for similar purpose. The principle of this method is based on a fact that on probing of SDS PAGE resolved protease cleaved antigenic peptides blot with antibody a unique western blot peptide pattern is obtained. This western blot peptide pattern will be different for other antibodies if these bind different epitope regions. Briefly the purified recombinant antigen GPI was partially digested with proteolytic enzymes Trypsin or Glu-c and the peptides were separated by SDS PAGE and blotted. The blots were screened by western blot assays with the GPI antibodies in question. By this method it could be clearly seen that the GPI mAb 11H3.C10 and 1E3 have similar western blot peptide patterns and 46H9 different with trypsin digested GPI (figure 21). This could be confirmed with Glu-c digested GPI peptide blot (figure 22).
Thus it could be concluded that 1E3 and 11H3.C10 GPI mAb recognized the same epitope on GPI, while 46H9 had a different pattern and thus recognized an epitope on GPI different from 11H3.C10 and 1E3. K/BxN sera had the additive pattern from 11H3.C10/1E3 and 46H9. The higher intensity of 11H3.C10/1E3 type over the 46H9 seen very prominently in Glu-c digest
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for K/BxN sera (figure 22) meant that in K/BxN mice the titers of antibodies of 11H3.C10/1E3 are higher than antibodies of 46H9 type. Also to be noted is 1E3 and 11H3.C10 were cloned from different K/BxN mice and yet both recognized the same epitope.
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)LJXUH3HSWLGHILQJHUSULQWZHVWHUQEORWHSLWRSHPDSSLQJRI*OX&SDUWLDOGLJHVWRIPRXVH*3,E\P*3, P$%DQWLERGLHV(A) Marker. (B) Ponceau stained lane of Glu-C digested GPI. Lanes with Glu-C digested GPI screened with primary Abs (C) K/BxN sera. (D) 46H9 Ab. (E) 11H3.C10 Ab. (F) 1E3 Ab.
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In order to elucidate the epitopes recognized by the GPI monoclonal antibodies 11H3.C10, 1E3 and 46H9, use of epitope extraction in combination with mass spectrometry was made.
For Mass spectrometric epitope mapping (Macht, Fiedler et al. 1996; Parker, Papac et al.
1996), the purified GPI mAbs and non-specific mouse IgG (for control) were cross-linked to epoxy activated paramagnetic DynabeadsR. The purified recombinant antigen GPI was
