Efficient Preparation of Rabbit Fab'-Horseardish Peroxidase Conjugates Using Maleimide Compounds and Its Use for Enzme Immunoassay

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Abstract
A mild and efficient two-spet procedure for conjugating rabbit Fab' and horseradish peroxidase using maleimide compounds was developed, and the conjugate obtained was evaluated as an analytical tool in enzyme immunoassay. The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxyeyclogexylmethyl) maleimide to introduce maleimide groups. Then, the maleimideenzyme was allowed to react with thiol groups of Fab'. MOre than two thirds of Fab'and the enzyme used was converted to conjugates with a minfaml polymerization, and both activities of the enzyme and antibodies were well preserved in the conjugate. The conjugate Obtained by this method was more suitable for enzyme immunossay thatn polymerized conjugates prepared by the periodate method. The conjugation using N-hydroxysuccinimide ester of m-maleimidobenzoic acid provided similarly less polymerized preparations but was less efficient.