The exquisite sensitivity and specificity of antibody based assays is limited by their discontinuous nature. Separate samples must be withdrawn and tested to determine changes in the concentration of an analyte over time. This proposal focuses on a way to make antibody-based assays continuous, i.e., for a single probe to continually report changes in the concentration of various materials. The approach is based on a new type of stable antibody that conducts electricity. The flow of electrons through the antibody is interrupted whenever antigen is bound giving a proportional decrease in the signal seen by the transistor. An “approach-to-equilibrium” method is used to analyze the signal and obtain results in “real time.”
It has been known for over 50 years that proteins conduct electric current. Most recently proteins attached to a gold post by a sulfur bond were found to be reasonable conductors when brought close to a source of electrons. Our objective is to chemically synthesize a relatively new type of stable antibody (heavy-chain variable domain, VHH, nanobody) in which one of the?b-sheets that comprises the core of the VHH (C or F-segment) is modified by replacing the normally occurring amino acid residues with non-coded amino acid analogues that are better conductors of electricity. Residues in the relatively invariant C-segment of the antibody shown (-Met32-Gly-Trp-Phe-Arg-Gln-Ala-Pro39-) will be replaced with amino acid analogues such as p-methoxyphenylalanine. Previous research shows that both the affinity and specificity of antibodies for antigen are relatively insensitive to changes in this region. The VHH will be synthesized in solution by the excluded protecting group method. This is both rapid and cost-competitive with cloning. Analogues of the C-segment octapeptide would be inserted into the antibody using fragment synthesis, obviating the need for complete re-synthesis of the protein for every substitution.
Research will involve a VHH that is directed against the gp100 glycoprotein from the AIDS virus. Extension of this technology to other antibodies with different specificities should be straightforward since changes in the first and third hypervariable regions, where antigen binding occurs, ought not to affect the conductive core of the nanobody. Dozens, if not hundreds, of VHH having different specificities have been sequenced and can used in place of this particular antibody. Binding the gp120 glycoprotein antigen at the bottom (hypervariable region) of the antibody blocks transmission of electrons from solution through the conductive channel in the antibody to the attachment site on the transistor (anode).
Ultimately it may be possible to design and build a chip that can perform dozens if not hundreds of assays for clinically relevant materials in a sample simultaneously in real time.