Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates

Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation procedures. The conjugates were tested by enzyme immunoassay and their efficiency was characterized by the antibody and enzyme concentrations needed to obtain an arbitrary OD value. The suitability of antibody for conjugation through NH2 groups was tested by fluorodinitrobenzene (FDNB). HRP conjugates were produced by two variants of the sodium periodate procedure and two variants of the glutaraldehyde method, as well as by the heterobifunctional linker N-succinimidyl 3-(2-pyridyldithio)pro-pionate (SPDP). Two of the four antibodies were coupled by a third variant of the periodate method, through their carbohydrate moieties. The periodate-mediated conjugations, using sugar moieties on the enzyme, provided the most efficient HRP conjugates, regardless of the antibody subclass or isoelectric point. The glutaraldehyde procedures consistently gave the worst results. AP conjugates were prepared using the same methods. The most efficient and reproducible AP conjugates with all four monoclonal antibodies were obtained using the SPDP procedure. The efficiency of the other methods differed from one antibody to another.