Effect Of The Glutaraldehyde-cross-linking Method On Immunogenicity Of Hemoglobin And Its Polymerized Product

HBOCs are kinds of hemoglobin chemically modified through the surface or cross-linked among of the molecules. The raw materials mainly come from human, pigs and cattle. Intermolecular cross-linking polymerization method use glutaraldehyde as crosslinker. With the advantages of non-pathogenic microbial contamination, simple composition, can be long-term preservation in vitro and without cross-matching, however, such agents also exist some potential problems, that is heterologous protein and protein modification/polymerization reaction may make such products stimulate or suppress immune system function, affecting the humoral and cell immunity, making toxic effects to body. In addition, immunogenicity is an important component of the immunotoxicity evaluations, which has been the focus of major research. In this paper, using the antibody-producing method, a preliminary study about the changes of immunogenicity of the pigs, bovine hemoglobin before and after glutaraldehyde polymerization have been made in order to know the impact of protein polymerization to immunogenicity.The experimental results showed that the antibody titer of anti-pHb or bHb was very low, but it can be enhanced after cross-linked by glutaraldehyde. There were no cross-reactions between IgG anti-bHb and its polymerized product; IgG anti-intramolecular crosslinked bpolyHb can't cross-react to the bovine hemoglobine, but IgG anti-intermolecular crosslinked bpolyHb can cross-react to the bovine hemoglobine. There were cross-reactions between IgG anti-pHb and its polymerized product, and IgG anti-ppolyHb can cross-react to the porcine hemoglobine.It can be seen from that the immunogenicity of pigs, bovine hemoglobin were low, but glutaraldehyde polymerization changed the proteins spatial structure, significantly increased the immune stimulation and stimulate the body to generate more antibodies. In addition, the different changes in the protein spatial structure had different impact on the immunogenicity. Intramolecular glutaraldehyde polymerization has not changed the structure of pig epitope, but changed the epitope of bovine hemoglobin; intermolecular polymerization did not affect bovine hemoglobin epitope. Truth of glutaraldehyde polymerization reaction can cause the two types of protein immunogen different changes showed that although the amino acid sequences of different types of hemoglobin are similarity, but the immune response of the polymerization may be totally different. This effect was closely related with the spatial structure of protein and its adjacent parts. Different hemoglobin product using the same modified method may be with different characteristics of the immunological stimulation, and the same species hemoglobin products using the different methods also may have different immunological characteristics.Therefore, it is necessary to study the immunological characteristics of different HBOCs products separately and should not inferred the characteristics of the products with the same method but the different sources of hemoglobin or the products with the same sources of hemoglobin but the different method based solely on the immunological characteristics of only one product.