Enzyme conjugate preparation

Despite the obvious disadvantages of glutaraldehyde-mediated conjugation, the crosslinker continues to be used to form enzyme-antibody complexes and in other applications. Many diagnostic tests still utilize antibody-enzyme conjugates prepared through glutaraldehyde... 

In this article the preparation of one class of carbohydrate-enzyme conjugates, prepared by attachment of dextran to enzymes, is described in some detail and the properties of enzymes modified in this way are discussed. The molecular basis of enzyme stabilization by coupling with dextran is also considered. 

Immunoassay. All samples were analyzed for trlazlne herbicides using RES-I-MUNE Immunoassay kits (ImmunoSystems Inc., Scarborough, Maine). The kits use polyclonal antibodies coated on the walls of polystyrene test tubes and an atrazine-enzyme conjugate prepared by covalently binding atrazine to horseradish peroxidase by a modified carbodilmide technique (12-13. Other reagents in the immunoassay kits include three standards (negative control, 0.1 ug/L atrazine solution and 1,0 ug/L atrazine solution), substrate, chromogen, and a "stop" solution of 2.5 N (normal) sulfuric acid. 

The following protocol represents a generalized method for protein thiolation using SATA. For comparison purposes, contrast the variation of this SATA modification method as outlined in Chapter 20, Section 1.1 for use in the preparation of antibody-enzyme conjugates. 

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... 

The following protocol illustrates the use of SIAB in preparing antibody-enzyme conjugates using P-galactosidase. 

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. 

A variation of the above method can be used, wherein the enzyme is first activated with SMCC and conjugated to a thiolated (strept)avidin molecule. This approach probably is the most common way of preparing (strept)avidin-enzyme conjugates, and since the preactivated enzymes are readily available (Thermo Fisher), it also may be the easiest. 

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

The following sections describe some of the more common procedures of preparing DNA-enzyme conjugates. 

---