Modified Antibodies

The number of sulfhydryls created on the immunoglobulin using thiolation procedures such as this one is more critical to the yield of conjugated enzyme molecules than the molar excess of maleimide-activated enzyme used in the conjugation reaction. Therefore, it is important to use a sufficient excess of Traut s reagent to obtain a high density of available sulfhydryls. 

Dissolve the antibody to be modified at a concentration of 1—10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, containing 10 mMEDTA. High levels of EDTA often are required to completely stop metal-catalyzed oxidation of sulfhydryl groups when working with serum proteins—especially polyclonal antibodies purified from antisera. Presumably, carryover of iron from partially hemolyzed blood is the contaminating culprit. 

Add 2-iminothiolane (Pierce) to this solution to give a molar excess of 20—40 x 

Antibody-Enzyme Conjugate Formation through Thioether Bond 

Purify the thiolated antibody by gel filtration using a desalting matrix such as Sephadex G-25. Perform the chromatography using 0.1 M sodium phosphate. 

---

Tc(V) gluconate and glucoheptonate are often used when the reaction should be carried out in a neutral aqueous- or mixed aqueous/organic medium, and a rapid exchange reaction and high radiochemical purity of the product is required. In radiopharmaceutical preparations, Tc(V) tartrate is also quite often used. For labelling modified antibodies Tc(V) tricine has recently been particularly recommended [33],... 

When reactions between the benzaldehyde and hydrazinium nicotinate groups are done in relatively dilute solution, for example using a modified antibody molecule at lmg/ml ( 6 tM) containing one of the two reactants, then to obtain maximal yield of the hydrazone conjugate, the second component should be added in sufficient excess to drive the reaction to completion. Kozlov et al. (2004) found that at least an 8-fold molar excess or above of the second reactant over the first reactant is necessary to obtain a yield of about 80 percent hydrazone bond formation. 

Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solution to obtain the desired molar excess of reagent over the antibody. Typically, antibody modification procedures are done with 10- to 20-fold molar excess, but for dilute antibody concentrations, this may have to be doubled, depending on how many hydrazine or aldehyde groups are desired to be introduced on the modified antibody. 

Deprotect the acetylated sulfhydryl groups on the SATA-modified antibody according to the following protocol ... 

Figure 21.9 PDTP may be used to modify antibody molecules using a carbodiimide reaction with EDC. The derivative is the same as that obtained using SPDP activation and is highly reactive toward sulfhydryls.

Figure 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

Roffler, S.R., and Tseng, T.-L. (1994) Enhanced serum half-life and tumor localization of PEG-modified antibody-enzyme conjugates for targeted prodrug activation. Antibody Engineering Conference. San Diego, California. 

The reactive NP core provides an alternate use for catalytic NPs as sensitive electrocatalytic tags for biosensors. Brozik and coworkers have developed a reagentless electrochemical immunoassay by using electrocatalytic NP modified antibodies that are sensitive to the oxygen reduction reaction.74 Gold/palladium core-shell... 

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

---