Efficient labelling of antibodies with horseradish peroxidase using cyanuric chloride

An efficient and mild method for labelling of immunoglobulin G (IgG) with horseradish peroxidase (HRP) using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, CC) as a bridging molecule is described. The enzyme was treated first with cyanuric chloride to introduce dichloro triazine and after removal of excess reagent, the activated enzyme was mixed with the IgG preparation and incubated to effect linkages with amine groups in the antibody protein. Various amounts of coupling reagent were tested to optimise the conjugation method using commercially available enzyme and affinity-purified sheep IgG antibody preparations to three different test haptens. The conjugates were assessed by solid phase Enzyme Linked Immunosorbent Assays (ELISA) and commonly used peroxidase substrate preparations. The binding activity of the conjugates rose with increasing coupling reagent added during the enzyme activation step. Use of the conjugates prepared by the new method gave comparable sensitivity in direct competitive ELISAs for the three test haptens to assays carried out using indirect ELISA with commercial anti-sheep-HRP conjugates. No deterioration of enzyme activity or hapten-binding activity in the conjugates was observed after storage in 50% glycerol at − 70 °C for up to 18 months. This study presents a relatively simple and efficient conjugating method for labelling antibodies with HRP and provides an additional and probably a better alternative to the periodate, glutaraldehyde and succinimide-maleimide procedures.