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Antibody Microarray Limitations

Protein and Antibody Microarrays

Problems and Possible Solutions for Antibody Microarrays

 

Availability of Antibodies
            Another challenge to antibody arrays is to obtain antibodies against the ≥ 100,000 proteins that comprise the human proteome.  There are currently a very small fraction of antibodies available of the proteome.  In addition, the specificity of many of these antibodies is poorly documented and additional antibodies may be required to
permit the detection of post-translational modifications.  The detection of proteins by antibody–antigen interactions is also characterized by a broad range of specificity and affinity.  Also, it is difficult to obtain a large set of highly specific antibody
molecules. Therefore, most antibody arrays are limited in their use and contain a few well-defined capture agents directed at a particular class of protein markers (9,17) 
-to not act similar glycosylation

Other Problems Facing Antibody Microarrays and Possible Solutions
Classical strategies of antibody generation by animal immunization seem to be impractical. Recombinant antibody display libraries are more promising (59-61). Recently, many recombinant methods of generating antibodies have been investigated.
These include phage antibody-display, ribosome display, SELEX (systematic evolution of ligands by exponential enrichment), mRNA display, and affibody display, all of which have been developed to advance the production of antibodies and/or antibody mimics (16,54,56,62).  These methods all involve the construction of large repertoires of viable regions with potential binding activity, which are then selected by multiple rounds of affinity purifications. Candidate clones can be further selected using maturation selection which improves binding affinities.  However, an ideal selection system, which is fast, robust, sensitive, low cost, automated, and minimized, is yet to be fully developed (16,54).  These smaller antibody fragments usually have decreased cross-reactivity and similar properties upon attachment (7,8,9,27).
Also because recombinant antibodies have a potentially high affinity, high specificity, and their smaller molecular weight, which is usually about 30 kDa while non-recombinant antibodies are about 150 kDa, dense and oriented attachment to support surfaces is facilitated (63,64).
            Antibodies are not the only molecules that may bind proteins.  Aptamers are short oligonucleotids that can bind and crosslink covalent target proteins.  This facilitates
higher stringency wash conditions which promotes detection of specific binding.  Furthermore, these molecules are easily selected, arrayed, and synthesized.  Purified protein target is a requirement for their use however, and aptamers can exhibit biased binding as RNAs tend to be highly negatively charged (9). A group (Somalogic) recently used immobilized anti-human immunodeficiency virus-gp120 aptamer to detect subnanomolar concentrations of target protein in 5% human serum (65). 

Next: Protein Microarray Detection Methods and Analysis

References for Protein and Antibody Microarrays

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Introduction and Background to Protein Chips and Antibody Chips.

Types of Antibody and Protein Chips

 

Questions & Troubleshooting Forum Discussions

 

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