Antibodies site-directed conjugation

The conjugation of deacetylvinblastine to a monoclonal antibody that recognizes a tumor-associated antigen results in an agent with substantial antitumor activity in mice with relatively little toxicity (la). Conjugates of this type are of obvious interest for future clinical trials as site-directed cancer chemotherapeutic agents. 

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. 

Heterobifunctional cross-linking reagents also may be used to site-direct a conjugation reaction toward particular parts of target molecules. Amines may be coupled on one molecule while sulfhydryls or carbohydrates are targeted on another molecule. Directed coupling often is important in preserving critical epitopes or active sites within macromolecules. For instance, antibodies may be coupled to other proteins... 

One of the earliest published attempts to create antibodies with catalytic activity had as its goal the generation of a transaminase. Raso and Stollar prepared V-(5-phosphopyridoxyl)-3 -amino-L-tyrosine 154 as a mimic of the Schiff s base intermediate that is formed during the pyridoxal-dependent transamination of tyrosine and showed that it was a site-directed inhibitor of the enzymes tyrosine transaminase and tyrosine decarboxylase.132 Partially purified polyclonal antibodies, elicited against y-globulin conjugates of the hapten, recognized both the... 

Fig. 2.4. Competitive, homogeneous EIA. The symbol E represents enzyme, A antigen or hapten, S and CO substrate and enzyme cofactor, and Av and B avidin and biotin. Enzymes indicated with have modulated (increased or decreased) activities. In (a) hapten is conjugated to the enzyme and reaction with the antibody (shaded structure) modulates the enzyme activity. This interaction is, however, prevented if antigen is present in the sample. Similar principles apply if substrate (b) or cofactor (c) are conjugated with the antigen. In (d) antibody is directly linked to the enzyme, and the reaction of this antibody, particularly with high-molecular weight antigens, will block the active site of the enzyme. In (e) antibody, directed to haptens labeled to avidin, prevents avidin from inhibiting the biotin-containing enzyme, unless antibody is neutralized by the same hapten present in the sample.

---