Antibody-enzyme conjugation purification

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

Once transfectomas have been generated, they must be screened for the production of the desired fusion. The binding site provided by the expression of the VNP heavy chain in J558L cells and association of the heavy chain with the resident light chain means that the protein fusion can be captured on a solid phase by NP or NIP, and detected using commercially available antibody to mouse X chain and an appropriate enzyme conjugate, in a simple ELISA screening procedure. Similarly, purification can be achieved by affinity adsorption of the fusion onto an NP matrix. An immunoblot method is described here for characterization of selected transfectomas, which allows the mol-wt of the fusion product to be estimated. 

The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 ]Xg/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions. 

Figure 9 shows the curve for tobacco residues purified by TLC before analysis by the assay. It is clear that the interference seen previously was practically eliminated. However, such extensive sample preparation makes use of this assay in its present form cumbersome, at best. We are presently investigating an alternative form of immunoassay, the enzyme immunoassay (EIA) ( , 21) In this assay methoprene is conjugated directly to an enzyme and the anti-methoprene antibody is bound to the solid support. Free methoprene and methoprene-enzyme conjugate are in solution and compete for immobilized antibody binding sites. Unbound methoprene is washed from the assay prior to addition of substrate. Preliminary results under these conditions indicate that tobacco extracts of acetonitrile/water (9 1) do not require further purification steps prior to application to the EIA. 

Sorensen (1993) Method for isolation and purification of enzyme-antibody conjugates. US Patent No. 5,266,686. 

Dissolve the enzyme to a concentration of 1 - 2 mg/ml in Soln. A (if it is delivered in another buffer, dialyze against Soln. A). Mix 1 mg -galactosidase in Soln. A with 0.25 mg SMCC activated antibody (concentration about 1 mg/ml for activation see Protocol 4.1.3). Shake at RT for 2 h, dialyze or desalt on a Sephadex G-25 column against Soln. B and concentrate to about 2 mg/ml. Mix 9 vol. of the concentrated conjugate with 1 vol. Soln. C and store without further purification at 4 °C. 

Collect 0 5-1 mL fractions The first protein peak to elute contains the conjugate, however, the first or second fraction may contain some aggregates Analyze each fraction absorbing at 280 nm for biotin binding and assay it for the antibody or enzyme activity HPLC may also be performed for further purification, if necessary... 

Free enzyme in the conjugate will interfere mainly in the methods where the activity of the free fraction is measured. In most cases, however, the activity is measured in the bound fraction, and the presence of free enzyme will cause only minor interference in the form of increased background. If the enzyme is smaller than the labeled compound, free enzyme can be eliminated by gel filtration as above. Immunoadsorbent purification could be useful in separating enzyme-labeled (and free) antigen (antibody) from free enzyme, provided that an elution buffer can be found that does not inactivate the enzyme label. 

Antibody specificity and affinity are modified by manipulating the immunization schedule. After the immunization procedure is finished, antibodies need to be characterized in depth to ensure specificity and affinity, which will help select the best-performing antibodies before they are used in assay development. Serum containing antibodies can be used as is, without further treatment, but antibodies can also be purified. Several methods are available to purify antibodies from serum, but the most common procedures use the IgG-binding protein A (or protein G). Purification of antibodies is required when they have to be conjugated to other molecules, such as enzymes, latex and gold particles, radioactive isotopes, or fluorescent labels. Undesired cross-reacting antibodies can be eliminated by im-munoaffinity purification. 

Reagent for enzyme-linked immunoassay Immunoadsorption of anti-(human immunoglobulin G) antibody-jS-D-galactosidase conjugate prepared for enzyme-Unked immunoassay Purification by immunoadsorption of Cl r, an activated component of the first component of the complement system... 

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