Mild and Efficient Conjugation of Rabbit Fab' and Horseradish Peroxidase Using a Maleimide Compound and Its Use for Enzyme Immunoassay

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Abstract
A mild and efficient procedure for conjugating rabbit Fab' and horseradish peroxidase using a maleimide compound was developed. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl) maleimide to introduce maleimide groups. Then, the maleimide-enzyme was allowed to react with thiol groups of Fab', and the conjugate formed was separated from unreacted components by gel filtration with Ultrogel AcA 44. In the peak fraction of the separated conjugate, 98% of peroxidase was associated with Fab' and 90% of antibodies was associated with peroxidase. The recoveries in the conjugate of peroxidase and Fab' incubated for conjugation were 65–74%. The conjugate formed appeared to be largely monomeric. Both the enzyme activity and antigen-binding activity of Fab' were fairly well preserved in the conjugate. The cross-link formed was stable at 4°C at least 6 months. Use of the conjugates obtained by this method gave greater sensitivity in sandwich enzyme immunoassay for human ferritin and human thyroid-stimulating hormone than conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided a similar monomeric preparation but was less efficient.