The Journal of Immunology, 2007, 178: 4709.
Copyright © 2007 by The American Association of Immunologists, Inc.
Response to Comment on "Aberrant Regulation of Synovial T Cell Activation by Soluble Costimulatory Molecules in Rheumatoid Arthritis"
Bing Wan,
Hong Nie,
Ailian Liu and
Jingwu Z. Zhang
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
In a recent paper published in the Journal of Immunology (1), we reported a novel pathway in which the regulatory function of PD-1 in synovial T cells is interrupted by soluble PD-1 over-produced in rheumatoid synovium. An ELISA was developed to detect levels of soluble PD-1 in sera and synovial fluid in the study. However, in a letter to the editor by Nielsen et al. (2), the authors claimed that, in their hands, similar experiments failed to show the specificity. They attributed this failure, among many other possibilities, to cross-reactivity of the Abs and thus questioned whether some bias might be introduced in our ELISA results. The authors’ interpretation was that the immunogen used to generate the PD-1 Ab was a PD-1-Fc fusion protein. Thus, the Ab may recognize epitopes within the human Fc, which may result in cross-reactivity with human IgG. In analyzing the information described in the letter, we were surprised that some of the critical technical details were overlooked in reaching their interpretation. For example, the immunogen (PD-1-Fc fusion protein) was cleaved and purified to remove the Fc before immunization. The actual immunogen was a purified PD-1 protein without the Fc. This critical information was given and further confirmed by the manufacturer. As a result, there is no basis for such cross-reactivity with human IgG to occur as they claimed. In addition to the primary Ab, our secondary detection Ab was pre-absorbed against human IgG (1031-05; Southern Biotechnology Associates) before use. These critical steps largely exclude the possibility of cross-reactivity of the Abs with human IgG, which was taken into careful consideration in our experimentation. It is advisable that Nielsen and colleagues explore alternative explanations related to their own experimental conditions that are not necessarily identical or comparable to ours.
References
- Wan, B., H. Nie, A. Liu, G. Feng, D. He, R. Xu, Q. Zhang, C. Dong, J. Z. Zhang. 2006. Aberrant regulation of synovial T cell activation by soluble costimulatory molecules in rheumatoid arthritis. J. Immunol. 177: 8844-8850. [Abstract/Free Full Text]
- Nielsen, C., T. Barington, S. Hansen, S. T. Lillevang. 2007. Comment on "Aberrant regulation of synovial T cell activation by soluble costimulatory molecules in rheumatoid arthritis.". J. Immunol. 178: 4708[Free Full Text]
