With the advent of radioimmunoassay (RIA) (Yalow and Berson, (1960), J. Clin. Invest., 39:1157), the immunoassay became recognized as an exquisitely sensitive tool in the measurement of clinically important substances found at low concentrations in various body fluids.
U.S. Pat. No. 3,654,090 which issued to Schuurs on Apr. 4, 1972, teaches the use of an enzyme-substrate system to replace the radioactive label used in the RIA.
Both of these assays make use of a competitive reaction between a limited number of antibody molecules and both a labeled antigen and unlabeled antigen (either known amounts used to construct a standard curve or unknown amounts contained in test samples). The more unlabeled antigen in the reaction mixture, the less labeled antigen will be bound to the limited number of antibody molecules. One must be equipped with a method to detect the ratio of bound to free labeled antigen.
The earliest methods used a physical separation of antibody-bound labeled antigen from free labeled antigen. This type of assay in which a separation step is employed is referred to as a heterogeneous assay.
The assays referred to above all make use of antigen or hapten (a low molecular weight substance which is not immunogenic but is capable of being bound by specific anti-hapten antibodies) which is identical or immunochemically analogous to the analyte (the unknown being tested for). The antigen or hapten is labeled with reagent means for determining the extent to which the labeled antigen or hapten is bound to the antibody. The use of such substances presents problems in the various assay systems already developed in the art.
For example, various drawbacks are associated with the use of radioisotopes, such as high cost, limited shelf-life, radiolysis of the sample, and licensing and disposal restrictions. The drawbacks associated with the use of enzymes as labels include: sensitivity to temperature and buffer variation, limited shelf-life in solution, and susceptibility to degradation by lytic enzymes that may be present in the sample to be analyzed.
FTIR spectrometers have been found to be very useful in the analysis of weak signals from opaque samples. Surface analysis can also be carried out routinely by reflectance techniques. Detection limits in the nanogram to picogram range have been cited in the literature for FTIR spectrometers.
It would be highly desirable if there could be a solid-phase immunoassay system using FTIR techniques. That is, an interferometrically coded signal from a mid-inpared source transmitted through, or reflected from, a solid support surface where the resulting attenuation of the signal by a label attached to an antibody or antigen would provide a measurement of an antigen-antibody complex on the solid support surface. Such a system would provide a heterogeneous immunoassay method that would offer several advantages with respect to existing heterogeneous immunoassay methods. Such a system would be unaffected by background fluorescence or phosphorescence, unlike existing heterogeneous immunoassay systems that employ fluorescent tags.