Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-12-23
1999-09-28
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 435 794, 435 25, 435 26, 435 27, 436517, G01N 33535, G01N 33557
Patent
active
059587156
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of quantitative measurement of an enzyme linked immunosorbent assay.
The enzyme linked immunosorbent assay technique (ELISA) is an important analytical tool that is used in a wide variety of applications.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a pictorial representation of the basic principle of an ELISA.
FIG. 2 is a pictorial illustration of the basic principle of a chemical clock.
FIG. 3 is a graph showing the effect of hydrogen peroxide concentration in the reaction mixture on the time required for generation of color.
FIG. 3a is a graph showing the rate, expressed as the time of color development, at which 0.50 absorbance units at 610 nm were achieved after the addition of various amounts of hydrogen peroxide.
FIG. 4 is a graph showing the effect of various catalase levels on the reaction time required by the chemical clock to generate the blue color.
FIG. 5 is a graph showing a comparison of a titration of antigen based on the timed catalase/iodine clock system with a standard ELISA using a spectrophotmetric determination.
FIG. 6 is a graph showing the effect of the addition of hydrochloric acid to a glycine buffer containing Neutral red indicator.
FIG. 7 is a graph showing the effect of alcohol dehydrogenase on the pH clock at a pH of 7.68 and 8.73.
FIG. 8 is a graph which is equivalent to FIG. 3. FIG. 8 shows the effect of the level of potassium superoxide on the indicator tetrazolium.
FIG. 9 is a graph which is equivalent to FIG. 4. FIG. 9 shows the effect of the level of xanthine oxidase on the time of color development.
In one commonly used protocol the analyte or antigen to be quantified is attached to a solid support, for example in the well of a microtitre plate. The analyte may be immobilised by chemical reaction, by binding to an antibody (a so-called sandwich reaction) or by direct binding to the plastics surface by virtue of the innate "stickiness" of the analyte molecule.
Unoccupied binding sites on the surface are then blocked by flooding the plate with a non-specific protein. The analyte is then treated with an antibody that binds specifically avidly and tightly to the analyte. Any residual unbound antibody is washed off the plate. If no analyte was present to bind the antibody, all the antibody would be washed away. If a significant amount of analyte is present, a corresponding significant amount of antibody will bind to the plate. The quantity of antibody binding to the plate is determined by an enzymatic colour reaction.
In most cases and particularly with commercially available kits, the enzyme is chemically coupled to the antibody. A typical example would employ an antibody coupled to horse radish peroxidase. This enzyme oxidises a colorless substrate eg the dye ABTS to produce a coloured oxidation product. The colour forming reaction is permitted to proceed for some 30 minutes before being terminated by treatment with dilute acid. The quantity of color generated by the reaction may then be determined using a spectrophotometer. The amount of color generated corresponds directly to the amount of enzyme present and thus to the amount of bound antibody and to the amount of analyte of interest. This system allows quantitative analysis of an analyte of interest.
Use of the ELISA technique allows detection and quantification of a wide variety of molecular species and it is typically used to detect and quantify substances such as pesticides, mycotoxins, microbial cells or viruses. Because of the suitability of the ELISA technique for detecting analytes or antigens that would be difficult to assay by other methods dedicated ELISA systems are frequently sold as complete test kits and are often purchased on a "one-off" basis. However, all quantitative ELISA systems suffer from the same drawback that at the end of the analysis they require the use of spectrophotometric equipment for the final assessment and quantification step. Spectrophotometers are expensive pieces of equipment and may not always be available. This is especially likely given
REFERENCES:
patent: 4496658 (1985-01-01), Kondo et al.
patent: 4590157 (1986-05-01), Chandler et al.
patent: 4722893 (1988-02-01), Shigeta et al.
Billingham et al, 1991, Philosophical Transaction of the Royal Society of London, 340:569-91.
BRF International
Saunders David
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