This invention relates to the detection of components of the antigen-antibody reaction by solid phase immunoassay techniques, more particularly to enzyme immunoassay.
Many immunoassay procedures for detecting antigens, antibodies and haptens in body fluids are known in the immunoassay art. Radio-immunoassay techniques are numerous and have been shown to be highly sensitive. Numerous enzyme-immunoassay techniques are also known, including competitive, double antibody solid phase ("DASP") and sandwich procedures. Both classes of solid phase immunoassays have been performed using various solid supports, including finely divided cellulose, solid beads or discs, polystyrene tubes and microtiter plates.
Enzyme immunoassays have included color formation as an indicator of a result. Degree of color formation has been used for quantitative determinations. Colorimeters have been used for automatic quantitative determinations based on color gradations of analytical solutions.
Dot enzyme-linked immunosorbent assays using nitrocellulose filters as the solid phase have recently been described by Hawkes et al., "A Dot-Immunobinding Assay for Mono-clonal and Other Antibodies," Anal. Biochem. 119, 142-147 (1982). Hawkes et al. described putting antigen spots on nitrocellulose filters, cutting out areas containing spots, and either placing the cut-out portions in the wells of microtiter plates for enzyme immunoassasy or, where a range of different antigens are to be screened, using nitrocellulose strips. Similarly, Pappas et al., "Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA): a Micro Technique for the Rapid Diagnosis of Visceral Leishmaniasis," J. Imm. Meth. 64, 205-214 (1983), have disclosed a dot assay for parasites. Both Hawkes et al. and Pappas et al. asserted advantages of nitrocellulose dot (or spot) assays, including not only a reduced need for bound reagent, but also the almost-whiteness of nitrocellulose as a background for color reading. Pappas et al. also stressed the improved binding of bound reagent to nitrocellulose as compared to microtiter plate wells. Hawkes et al. disclosed a quantitative procedure in which reflectance of spots was determined by a thin layer scanner.
Also known in the art are pregnancy tests which utilize dipsticks coated or partially coated with bound antibody to HCG, such as the PREGNASTICK pregnancy test kit sold by Monoclonal Antibodies, Inc. Enzyme immunoassay procedures are used to change the color of the coated portion of the dipstick.
The myriad assays in the prior art, while providing in some instances very high sensitivity, suffer from a variety of drawbacks. Radio-immunoassay procedures require the handling of radioactive materials, as well as their disposal, and expensive equipment. Many enzyme-immunoassay procedures produce soluble color solutions, are used with colorimeters, and require incubation periods longer than desired. Enzyme immunoassay pregnancy tests utilize single-use kits, which are thrown away after a single use. The nitrocellulose-based dot tests utilize a solid support which may absorb reactants, which can lead to background color and reduced sensitivity. Further, nitrocellulose is fragile, making it difficult to handle.
It is a principal object of this invention to provide an improved immunoassay kit and protocol which eliminates drawbacks, discussed above, of the prior art and is capable of achieving an immunoassay which is sensitive, fast, inexpensive, which does not require expensive and delicate instruments, which is usable for multiple assays, and which can be made for a qualitative or a quantitative test. This and other objects of this invention will be better understood by reference to the description which follows.