Identification of antigenic determinants on growth hormone by mass spectrometry

Background- Monoclonal antibodies (mAb) are routinely used in different immunoassays for analysis of growth hormone (GH) concentration in serum. Identification of the epitopes on GH being recognized by different antibodies may contribute to understanding the spread in quantitative results for the hormone in clinical practice. Mass spectrometry compared to other methods available, is faster and can be done with low amounts of the antigen even in complex matrix.

Method- Epitope extraction [1] has been used for characterization of the interaction between GH and mAb 10A7. The method consists of tryptic proteolysis of the antigen and subsequent identification of the fragments bound by the antibodies. 10A7 was used as immobilized to magnetic beads. The tryptic GH fragments, after release from the mAbs, were identified by mass fingerprinting using LC/MS and confirmation by LC/MS-MS.

Results/Conclusion- Out of the 20 tryptic peptides of 22 kDa GH, aa21-39 and aa147-159 were found to be recognized by mAb 10A7. These peptides correspond to two different regions on the surface of 22 kDa GH. Interestingly, aa21-39 is not present with the 20 kDa isoform. This might contribute to the known preferential recognition [2] of 22 kDa GH as compared to the 20 KDa form.