Glutaraldehyde-Mediated Conjugation

Glutaraldehyde was one of the first and still is one of the most commonly used crosslinking agents available for creating antibody-enzyme conjugates. The crosslinking process using 

In a fume hood, add 10 p, of 25% glutaraldehyde (Sigma) per milliliter of antibody/enzyme solution. Mix well. 

To reduce the resultant Schiff bases and any excess aldehydes, add sodium borohydride (Aldrich) to a final concentration of 10 mg/ml. 

Note Some protocols do not call for a reduction step. As an alternative to reduction, add 50 pul of 0.2 M lysine in 0.5 M sodium carbonate, pH 9.5, to each milliliter of the conjugation reaction to block excess reactive sites. Block for 2 h at room temperature. Other amine-containing small molecules may be substituted for lysine—such as glycine, Tris buffer, or ethanolamine. 

To remove any insoluble polymers that may have formed, centrifuge the conjugate or filter it through a 0.45-p,m filter. Purify the conjugate by gel filtration or dialysis using PBS, pH 7.4. 

Purify the activated enzyme from excess glutaraldehyde hy gel filtration (using Sephadex G-25) or hy dialysis against PBS, pH 6.8. 

---

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... 

Despite the obvious disadvantages of glutaraldehyde-mediated conjugation, the... 

Phytobaemagglutinins.—Conditions have been established for the modification of concanavalin A with dextranase by a glutaraldehyde-mediated reaction. The soluble, intermolecularly cross-linked conjugate possessed both concanavalin... 

Crude preparations of gangliosides have been treated with albumin via a carbodi-imide mediator. The reaction was stopped by precipitation with ammonium sulphate. Following resuspension of the precipitate, unsubstituted albumin was mixed with the albumin-ganglioside conjugate and the suspension treated with glutaraldehyde and then coated onto cellulose filters. These ganglioside affinity filters could be used for the identification of toxigenic strains of Clostridium botulinum types C and D. 

---