Superfund Research Program
Immunoassays for Human and Environmental Health Monitoring
Project Leader: Bruce D. Hammock
Grant Number: P42ES004699
Funding Period: 1995-2023
Final Progress Reports
The Immunoassays for Human and Environmental Monitoring project utilizes both classical and innovative technologies to design and develop assays that can be used to detect environmental contaminants and their breakdown products in the environment or in our bodies. The assays, which use antibodies to detect chemicals, are used in both the laboratory and for on-site testing. One innovative technology takes advantage of the fact that camelids produce a unique type of antibody. Unlike classical antibodies, the single domain antibodies are about 1/10th the size and bind to their antigen via the variable heavy chain domain (VHH). The binding properties of the VHH are similar to the classical antibody, but their small size offers many advantages. Since VHHs are relatively small and encoded on a single polypeptide chain they are easily expressed by recombinant methods. In immunoassays the classical antibody that binds to the chemical of interest is detected using a second antibody that is chemically attached to an enzyme. Dr. Gee and her research team have successfully substituted the VHH for the classical antibody in this type of assay for the flame retardants tetrabromobisphenol A and polybrominated diphenyl ether congener 47 (BDE-47). Alternatively the researchers have demonstrated that the VHH can be genetically engineered to include the enzyme (in this case alkaline phosphatase), thus eliminating the second antibody making the assay much faster. Other detection methods, such as PCR, were also used since the gene for the VHH is well-defined. The smaller, more stable and robust VHH are ideal in biosensors. This was demonstrated with the VHH for BDE-47 in a new type of sensor called an impedance biosensor. These biosensors are advantageous because they do not contain as complex components as in other sensor types making them less susceptible to damage when used in the field. The BDE-47 VHH were also employed on a lab-on-a-chip device interfaced to a smartphone that can be advantageous in low resource environments. These demonstrations further prove the utility of VHH for analysis of environmental contaminants.
A second new technology is the phage-amplified immunoassay (PHAIA). Here a bacteriophage is engineered to express peptides that will bind the complex of the antibody (either classical or VHH) with the chemical. Such a test results in a positive signal when the chemical is present. Recently, the research team demonstrated this technology for malachite green and leucomalachite green, chemicals that are used to control fungi in aquaculture. Usually the bacteriophage that is bound is detected with a secondary antibody that recognizes only the bacteriophage. Another method to detect the bacteriophage bound to the complex is to conduct a PCR on the well-known bacteriophage DNA. In a novel approach the result of the PCR amplification can be detected by a color change in a detection method called loop-mediated isothermal amplification (iLAMP). Thus if a sample contains the organophosphate insecticide, the assay will turn from violet to sky blue that could be easy to interpret in a field test. Application of these new technologies to classical immunoassays will result in faster, sensitive and more robust methods to detect environmental contaminants.