Glutaraldehyde enzyme labeling

The enzymes commonly used as labels in ELISA and other immunochemical reactions include horse radish peroxidase (HRP) and alkaline phosphatase (AP). The enzyme can be covalently coupled to the antibody using glutaraldehyde conjugation to reactive amino groups on the enzyme (lysines) in a phosphate buffered aqueous solution at neutral pH, as shown in Fig. 19 (103). Alternatively, carbohydrates present in the immunoglobulin structure can be cleaved by periodate treatment (see Fig. 20) and bound to free amino groups on the enzyme through a Schiff base reaction (103). 

Many types of mono-, bi-, and multifunctional coupling reagents are available for labeling antibodies or antigens with an enzyme. Glutaraldehyde, carbodiimide, N-succinimidyl-3-(2-pyridyldithio)propionate, and periodate oxidation of carbohydrate moieties to form active dialdehydes are several commonly used approaches in the preparation of enzyme conjugates (104-106). 

IgG antibody may be labeled with HRP using glutaraldehyde in a two-step procedure (2) this produces small conjugates, but their activity is low. Heterobifunctional reagents such as succinimidyl 4-(7V-maleidomethyl)-cyclohexane-1 -carboxylate may also be used to link the thiol group of Fab fragments to an amino group in the enzyme (3). 

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. 

Monoamine oxidase amperometric biosensor based on SPE were also modified with MWCNT by using the drop casting technique for the determination of antidepressants in model solutions and dosage forms. The authors used BSA protein which provided a matrix for the immobilization of the enzyme and protection of the enzyme activity when glutaraldehyde is used as a linker. Serafin et aZ. developed a label free dual immunosensor for the determination of human growth and prolactin hormones. The electrochemical immunosensor was based on CNT modify carbon SPE platform with the presence of poly(ethylene-dioxythiophene) (PEDOT) and gold nanoparticles. Again, the hybrid nano-material composite facilitated a proper immobilization of the antibody on the electrode matrix. 

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