Antibodies enzyme conjugation with

ELISA - Enzyme linked immunosorbent assay - Reaction of antibody - enzyme conjugate with ligand. Free ligand or antibody affects enzyme activity (NAD NADH). 

An antibody-enzyme conjugate with specificity against the first antibody is added. The second antibody is species specific. This means that it binds to any immunoglobulin of the species from which the first antibody is derived, e.g., goat anti-mouse antibody. Conjugated to the second antibody is an enzyme such as alkaline phosphatase which can be detected by a colorimetric assay. These conjugated anti-Ig antibodies are available commercially. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

For identification the lectins must be labeled. The best labels are biotin (streptavidin-enzyme conjugate) or digoxigenin (anti-digoxigenin antibody-enzyme conjugate), but in the case of Concanavalin A, direct labeling with HRP is possible (see below). 

RT for 30 min. Remove blocking solution, rinse once with Soln. B and add 100 pl/well of a dilution series (e.g., 1 500,1 1500,1 4500, 1 13 500,1 40 500,1 121 500,1 364 500 in TBS) of antibody-enzyme conjugate to the wells. Shake at RT for 30 min, remove conjugate solution, knock out the plate on paper tissue, and rinse three times with Soln. B. 

Fig. 11. Principle of antibody directed enzyme prodrug therapy. Hatched circles represent cells that have not bound antibody-enzyme conjugate. Internalized drug is abbreviated with the letter d . (From [172] with permission)...

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

Specific detection of nitrocellulose membrane-bound proteins using a conjugated enzyme. (1) Proteins are transferred from electrophoresis gel to nitrocellulose membrane. Blocker proteins bind to unoccupied sites on the membrane. (2) The membrane is incubated with a primary antibody directed against the protein of interest. (3) A secondary antibody is directed against the primary antibody. (4) The second antibody is conjugated with an enzyme to provide a detection mechanism. Substrate solution is added to the blot. The conjugated enzyme (HRP or AP) catalyzes the conversion of substrate (S) to product (P) to form a colored precipitate at the site of the protein-antibody complex. 

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