Linked assay of enzyme reactions

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

Enzyme-linked immunosorbent assay is a heterogenous immunoassay. Reactions involve a solid phase to which components are sequentially presented and successively bound. This method is very effective in the determination of the total alkaloid content. The positive characteristics of this method are the use of non-toxic reagents and basic equipment with low costs, a small sample volume and the ability to measure alkaloids in crude sample extracts. According to the literature, compared with results obtained from GLC, the precision of ELISA for quinolizidine alkaloids is not as high as that of the gas chromatography procedure, but is adequate for plant breeding purposes. The use of enzymes in developing the methods of quinolizidine alkaloids analysis looks likely to increase in the future. 

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5-28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 

GO often is used in solution phase chemical reactions as well as being immobilized on dip-sticks and electrodes. Although its overall clinical usage is widespread, its use as conjugated to antibodies in enzyme-linked assay systems is minor compared to the popularity of other enzymes like horseradish peroxidase and alkaline phosphatase. 

Fig. 1. Comparison of enzyme-linked immuno sorbent assay (ELISA, left) and immuno-polymerase chain reaction (IPCR, right). During ELISA, an antibody-enzyme conjugate is bound to the target antigen. The enzyme converts a substrate in solution to a detectable product. In IPCR, the antibody-enzyme conjugate is replaced by an antibody-DNA conjugate. The subsequent addition of a DNA polymerase enzyme (e.g., Taq), nucleotides and a specific primer pair uses the antibody-linked DNA marker sequence as a template for amplification of the DNA. The PCR product is finally detected as an indicator of the initial amount of antigen.

If neither the substrates nor products of an enzyme-catalyzed reaction absorb light at an appropriate wavelength, the enzyme can be assayed by linking it to another enzyme-catalyzed reaction that does involve a change in absorbance. The second enzyme must be in excess, so that the rate-limiting step in the linked assay is the action of the first enzyme. 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

A merging of chemistry and biology is essential to effectively probe the immune system for catalytic antibodies (Fig. 3). Haptens that are successful in eliciting catalytic antibodies are variations of the central theme that transition state stabilization in the antibody combining site will yield functional catalysts for a desired chemical reaction. The evolution of hapten design will be discussed further in subsequent sections. Once the hapten is selected and synthesized, it is attached to an immunogenic carrier protein, usually via an amide bond, for hyperimmunization. A preliminary screen for antibodies that bind the hapten using an enzyme-linked immunosorbent assay (ELISA) is followed by another screen for catalysis of the reaction for which the hapten... 

Enzyme-Linked Immunosorbent Assay. LlSAis a heterogeneous EIA technique that is widely used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates separation of bound- and free-labeled reactants. In the most common approach to using the ELISA technique, an aliquot of sample or calibrator containing the antigen to be quantitated is added to and allowed to bind with a solid phase antibody. After washing, enzyme-labeled antibody is... 

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