Antibodies antibody arrays

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

Antibody arrays are used in detection and expression profiling of e.g. cytokines (Figure 3)2 4 and allergens (www.vbc-genomics.com). Microarrays containing antigens are developed for detection of autoantibodies in rheumatoid diseases5. 

D.J. Cahill, Protein and antibody arrays and their medical applications. J. Immunol. Methods 250, 81-91 (2001). 

R.M.T. de Wildt, C.R. Mundy, B.D. Gorick, and I.M. Tomlinson, Antibody arrays for high-throughput screening of antibody-antigen interactions. Nature Biotech. 18, 989—994 (2000). 

Currently, protein microarrays can be divided into various types depending on the strategies to be chosen. For example, according to the array structure and shape, protein microarrays include 3D-surface structure [30, 31], nanowell [32], and plain chips [33-36], Meanwhile, considering the field of application, protein microarrays can be classified into five categories antibody array, antigen or reserve array, functional array, capture array, and solute array. Table 11.6 shows the differences among them. 

Antibody array Polyclonal or monoclonal antibodies are arrayed and used to detect and quantify specific proteins in a biological sample. An antibody array is effectively a parallel series of miniature immunoassays... 

Antigen or reverse array The converse of an antibody array, this chip has immobilized antigens that are used to detect and quantify antibodies in a biological sample... 

An ordered antibody array has also been assembled on the solid surface by a combination of Langmuir Blodgett (LB) film method and self-assembling method. An ordered monolayer of protein A is deposited on the solid surface by LB method, which is followed by self-assembling of antibody. Individual antigen molecules which are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). 

In addition, fabrication of antibody arrays on the solid surface by LB film technology coupled with self-assembly technology will also be presented. 

Protein A is a cell-wall protein of Staphylococcus aureus with a molecular weight of 42,000. Since protein A binds specifically to the Fc part of IgG from various animals, it has been widely used in immunoassay and affinity chromatography. We found that protein A could be spread over the water surface to form a monolayer membrane by the LB method [21]. On the basis of this finding, an antibody array on the solid surface can be obtained by the following two steps. The first step is fabrication of an ordered protein A array on the solid surface by the LB method. The second step is self assembly of antibody molecules on the protein A array by biospecific affinity between protein A and the Fc of IgG as shown in Fig.34. 

To prepare an antibody protein array, a monolayer of protein A, which was compressed at a surface pressure of 11 mN m l was transferred to a compartment containing anti-ferritin antibody in 10 mM pH 7.0 phosphate buffer. The antibody molecules were self assembled onto the protein A layer. The protein A/antibody molecular membrane was transfered to a compartment containing ultrapure water for rinsing, and was then transfered onto the surface of an HOPG plate by the horizontal method. AFM measurements were made in a pH 7.0 of 10 mM phosphate buffer solution. 

The antibody array that was self-assembled on the protein A array was also visualized in molecular alignment by AFM. The antibody array was in contact with a pH 7.0, 10 mM phosphate buffer. The AFM measurement was conducted at a controlled force of molecular size of the antibody was estimated as 7 nm in diameter. 

The antibody array was soaked in different concentrations of ferritin solutions for 1 h, and was assayed for AFM imaging in solution. Ferritin molecules were recognized and fixed by the antibody array. The ferritin concentration was 10 ng ml l. Individual ferritin molecules on the antibody array were selectivity quantitated by AFM imaging. 

Several protein assemblies have successfully been fabricated on the solid surfaces sifter the bioinformation transduction. These include the following molecular systems molecularly interfaced redox enzymes on the electrode surfaces, calmodulin / protein hybrides, and ordered antibody array on protein A. These protein assemblies find a wider application in various fields such as biosensors, bioreactors, and intelligent materials. 

Huang etal. (2002) prepared an antibody array for the simultaneous detection of 43 cytokines. They were able to verify the down-regulation of MCP-1 cytokine in transfected cells (human glioblastoma cells transfected with cx43 expression vector) relative to control cells. The antibody array is an emerging technology. In at least one study based upon the use of a commercial membrane format, the cytokine microarray failed to accurately determine cytokine levels in bacterial and lipopolysaccharide (LPS)-stimu-lated whole human blood (Copeland, 2004). 

Turtinen, L.W., Prall, D.N., Bremer, L.A., Nauss, R.E., and Hartsel, S.C., Antibody array-generated profiles of cytokine release from THP-1 leukemic monocytes exposed to different amphotericin B formulations, Antimicrob. Agents Chemother., 48, 396-403, 2004. 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

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