Protein antigens aggregation

Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96], But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97], Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98-101], Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser... 

Jiang, W., and Schwendeman, S. P. (2000), Formaldehyde-mediated aggregation of protein antigens Comparison of untreated and formalinized model antigens,Biotechnol. Bioeng., 70,507-517. 

Jiang W, Schwendeman SP. Formaldehyde-mediated aggregation of protein antigens comparison of untreated and formali-nized model antigens. Biotechnol Bioeng 2000 70 507-517. 

Like all immunoreceptor family members, FceRI lacks intrinsic tyrosine kinase activity. IgE and antigen-induced crosshnking of FceRI initiates a complex series of phosphate transfer events via the activation of non-receptor Src, Syk and Tec family protein tyrosine kinases (fig. 1). The Src family kinase Lyn, which associates with the FceRI p subunit in mast cells, transphosphorylates neighboring FceRI ITAMs after receptor aggregation [7, 26]. Once phosphorylated, the p chain ITAM binds to the SH2 domain of additional Lyn molecules, while the phosphorylated y chain ITAM recruits Syk to the receptor complex, where it is activated by both autophosphorylation and phosphorylation by Lyn [2, 7,15, 26]. 

The ELISA is an appropriate method to detect some, but not all, of these product variants. The reactivity of antibodies is not affected much by glycosylation of the antigen. An ELISA would not be the most appropriate method to analyze product variants due to differences in glycosylation. ELISA would be appropriate for analysis of variants, such as aggregates, that contain a different protein structure that can be specifically recognized by an antibody. 

The classical pathway may be activated immunologically by antigen-antibody complexes and aggregated immunoglobulins, and non-immunologically by a number of chemically diverse substances, including DNA, C-reactive protein. Staphylococcal protein A, trypsin-like enzymes and certain cellular membranes (Table III). 

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