Assessment Of Immune Response With Antigen Arrays

Many diagnostic approaches currently use conventional immunoassays and most popular among them is the enzyme-linked immunosorbent assay (ELISA), an outstanding multiplexed approach to assess enzymatic and binding parameters of proteins, including antigens and antibodies, in individual chambers of microtiter plates. Antigen microarrays provide an alternative to immunoassays by high-throughput monitoring of target molecules in a single assay. This is crucial for the precise comparison of binding affinity or parallel characterization of antigenic properties of numerous samples (for reviews, see Neuman de Vegvar and Robinson [58] and Sakanyan [59]). Miniaturized arrays on planar supports are also more economical in terms of the consumption of samples and reagents, the labor and time of analysis, and the cost per analyte. Moreover, the advantages of array and ELISA strategies can be combined by placing the arrays in wells [51,60-62] or by arraying antigens in separate chambers of commercial nitrocellulose-coated slides. These provide much versatility for performing binding assays simultaneously in a high-throughput and multiplexed fashion.

The feasibility of antigen microarrays was demonstrated by measuring the concentration of antibodies generated in patients against pathogen viruses and parasites as a result of a host immune defense [63-65]. Using internal calibration curves, a linear concentration-dependent response was observed with ~10% coefficient of variation between the arrayed slides, and the array data were in agreement with ELISA results [63]. The arrayed antigens of herpes simplex virus types 1 and 2, cytomegalovirus, the virus of rubella and Toxoplasma gondii, were able to detect specific IgG and IgM antibodies in the sera of patients. In another study, a panel of 430 chemically synthesized peptides and recombinant proteins was prepared to detect the immune response in rhesus monkeys immunized with genetically engineered vaccinia virus derivatives carrying antigenic determinants from a chimeric simian-human immunodeficiency virus [64]. This study indicated that an immune response was generated against immunodominant epitopes and some not yet characterized epitopes. More prolonged reactivity was monitored in immunized rather than nonimmunized animals, and surviving animals had a higher antibody titer against a wider spectrum of virus antigens. This first successful example of guiding vaccines with antigen microarrays emphasizes the utility of the high-throughput method for similar applications.

In our study, we applied microarrays to better understand the antigenic diversity of the human HIV-1 gp41 immunodominant epitope used for AIDS diagnosis [66] by evaluation of the immune response of mutant epitope sequences [65]. Mimetic peptides were selected in phage display libraries by consecutive panning with IgG from patients. Then, an arrayed panel of mimotopes was fabricated on a nitrocellulose membrane. The parallel assessment of peptides before and after "highly active anti-retroviral therapy" showed a good correlation between the binding affinity of mimotopes monitored by ELISA and array-based immunoassay with the sera of AIDS patients. However, microarray immunoassay supported by NIR fluorescence detection at 800 nm was found to reflect the binding prevalence better (i.e., the difference between reference and infected probes, which is of great value in the detection of suboptimal concentrations of antibodies in HIV-1 infected patients).

These data are encouraging in terms of the development of microarrays with a large panel of epitopes and mimotopes for the diagnosis of viral and perhaps bacterial infections that provoke levels of pathogenic agents in humans too low to be detected by convenient methods. In contrast, the generated antibodies are sufficient to provide a higher sensitivity for detection with arrayed antigens [67]. Antigen microarrays will also be useful to assess the antibody repertoire in patients infected by Epstein-Barr or hepatitis B and C viruses since acute, chronic, and convalescence forms of the corresponding infections are characterized by modulation of IgG and IgM immunoglobulin titer against particular viral antigens (reviewed in Neuman de Vegvar et al. [64] and Storch [68]).

Antigen microarrays have been employed to target other human diseases as well. Different sets of potential antigens and allergens (both peptides and proteins) were used to evaluate auto-antibodies in the sera of patients with systemic rheumatic diseases, autoimmune encephalomyelitis, or multiple sclerosis [69-71]. Recently, tumor-derived proteins have been arrayed and incubated with sera obtained from cancer patients and healthy controls [72-74]. Positive responses have been observed in prostate cancer and lung cancer patients, indicating the wider applicability of antigen microarrays to elicit specific antibodies in complex biological fluids.

The data accumulated during recent years show that antibody, antigen, and protein microarrays offer various strategies to elucidate the biological diversity and complexity of regulatory networks and biological responses to environmental actions with wide destinations for different domains of biomedical research (table 10.2).

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