Affinity Binding
As part of an effort to find alternative capture and/or detector elements that might increase the sensitivity, reliability, and/or shelf-life of standard antibody-based assays, whole antibodies (Ab) to E. coli O157:H7 were compared to their (Fab'2 antibody fragments (Fab) for both their ability to capture (C) and detect (D) target. Preliminary experiments do not reveal a substantial difference in sensitivity of detection between Ab-C:Ab-D, Ab-C:Fab-D, Fab-C:Ab-D, and Fab-C:Fab-D assays.
Biosensor
One limitation of current fiber optic biosensor assays is that they have relied on only one sample point to determine whether a specific target is present in a sample, which reduces confidence in the data. To overcome this drawback, a multi-sample method is being researched that will permit statistical analysis of the data on a specific sample. This leads to an increased confidence in the results, particularly near the lower limit of sensitivity. Investigations of different methods of preventing non-specific binding have led to several improvements in biosensor assays by decreasing the variability in baselines and sample data. Experiments have demonstrated that a separate blocking step is not needed if the detector antibody is diluted with a blocking medium. PBST was found to be better than either PBS or PBS with 2% casein and 2% BSA in antibody- and antibody fragment-based assays.
Another limitation of current fiber optic biosensor assays is the inability to reduce the limits of detection below 103cells/ml. Diffusion of microorganisms from bulk flows to the capture surface is limited by laminar flows that exist within microfluidic biosensor devices. Limited diffusion of microorganisms in laminar flows occurs within the short time periods typical of biosensor assays, which limits the capture efficiency of target microorganisms. The influence of laminar flows must be overcome to increase the capture efficiencies of biosensor assays. Methods to modify flow environments are being investigated by ABL researchers that may overcome this drawback and increase assay sensitivity.
Ligands
GM1– Cholera Assay
Vibrio cholerae is the etiological agent of cholera, an acute enteric disease. The organism is ingested with contaminated food or water and colonizes inside the intestinal tract. The pathogenesis results from the production of a heterohexameric AB5 enterotoxin. In the harshest form of the disease, symptoms (watery stools, nausea and vomiting) begin to appear 12 to 72 hours after ingestion of the organism and an individual may die within three hours if left untreated. The rapid diagnosis of the causative agent of suspect intestinal distress is important for rapid patient treatment and as a preventive measure against an epidemic. Currently, definitive diagnosis depends on the isolation and identification of the organism from feces, which typically requires multiple steps: enrichment, isolation and serological and/or biochemical tests. These methods can be time consuming and result in delayed detection of the causative organism and subsequent treatment of the presenting patient.
Biosensor assays utilize a sandwich immunoassay based on the specificity of receptor-ligand binding. The b-subunits of the cholera toxin bind a glycolipid cellular receptor, ganglioside GM1, on the intestinal epithelium cells in vivo. ABL researchers have developed methods to use the natural receptor for cholera toxin, ganglioside GM1 as a capture molecule which is adsorbed to a fiber optic waveguide through hydrophobic interactions. This waveguide is exposed to a sample containing the cholera toxin-b and subsequently exposed to a polyclonal b-subunit cholera toxin detection antibody conjugated to the fluorophore cyanine 5 (Cy5). Current studies are utilizing unshelled oysters spiked with cholera toxin. This toxin is then detected and quantified using the biosensor assay. The toxin produced by Vibrio cholerae O1 and O139 causes human illness; therefore, rapid detection of the toxin represents an alternative to whole cell detection. The assay thus far has the potential to detect cholera toxin at levels of 10 ng/ml or higher in contaminated oysters.
Human Serum Albumin
Streptavidin has a high binding affinity for biotin, and biosensor capture surfaces have typically been coated with streptavidin as a starting layer for forming streptavidin-biotin bridges. This strong binding ability has been utilized in a variety of assays by biotin labeling nucleic acids, amino acids 
and antibodies to enable target capture by creation of a streptavidin-biotin bridge attached to a solid support. Capture antibody orientation is believed to be directly responsible for the proper interaction between antigen and antigen binding site. As biotin can be bound to the carbohydrate moiety found on either side of the Fc portion of the antibody or to the numerous lysine residues found on Fc and Fab portions of the antibody, biotin labeling cannot ensure proper orientation of the antibody. This indiscriminate labeling results in random orientation of the antibody when immobilized on a streptavidin coated surface. Thus, opportunities to capture target antigens may be missed due to a lack of functional orientation of the antibody. Unfortunately, this lack of orientation control to immobilize capture antibodies in biosensor assays has led to low capture efficiency of whole bacterial cells.
As an alternative, Streptococcal protein G is being investigated as it specifically binds to the constant region of the antibody heavy chain, resulting in strict orientation when immobilized on a human serum albumin (HSA) coated surface. This proper antibody orientation on the biosensor surface may lead to increased capture efficiency and sensitivity of the biosensor assay. In addition to multiple hydrogen bonds and van der Waals attractions, the bound complex remains intact in solution due to a hydrophobic area created by the interaction of side chains belonging to charged residues. This conformational binding has been observed while the molecules were in crystal form, as well as in solution, indicating that liquid flow over the complex will not denature the protein G-antibody construct. Protein G has a high amount of secondary structure and contains a hydrophobic core that makes it a heat-stable protein, which adds greater use in biosensor assays. Proper IgG orientation with paratopes exposed for antigen capture is the optimal situation for a capture antibody on a solid surface. As a possible solution to capture inefficiency, this work examines replacing the streptavidin-biotin-IgG capture matrix with an HSA-Protein G-IgG capture matrix.
Nucleic Acid
Capture efficiency is a problem for many biosensor assays, either too few cells are captured or those that are captured are under reported. Methods to improve both capture of targets and reporting of targets captured are under development by ABL researchers. Nucleic acid based labeling methods are currently being investigated as a means of increasing reporter signal for captured targets. Nucleic acid probes labeled with fluorescent dyes compatible with biosensor assays are being used to boost reporter signals for targeted analytes.
Phage
Bacteriophage are being tested as part of a continuing effort to find alternative capture 
elements that might increase the sensitivity, specificity and/or reliability of biosensor assays compared to standard antibody-based assays. Bacteriophage are viruses that specifically infect bacteria. Infections are initiated by attachment of the virus to the surface of a specific bacterium. Strict host specificity is one advantage of using phage as a capture element. Preliminary experiments reveal that phage can be used to capture target bacteria in ELISA-based assay formats.