Abstract
We describe methods for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge of Fab’ using reaction with maleimide groups or the thiol-disulfide exchange reaction [l]. The Fab’-horseradish peroxidase conjugates prepared by these methods were demonstrated to be superior in both sandwich enzyme immunoassay and immunohistochemical staining to conjugates prepared by methods in which the enzyme was conjugated through Fab’ amino groups [l], with detection limits in the attomole range [2]. Hinge conjugation is, however, not suitable for small amounts of affinity-purified Fab’, due to loss of thiol groups during gel filtration and concentration. We describe a micro-scale method for affinity purification after Fab’-peroxidase conjugation and its application to a sandwich enzyme immunoassay of insulin in human serum. The non-specific binding of the affinity-purified conjugate was lowered by gel filtration, and the sensitivity for insulin was 1 nU/tube and 100 nU/ml of serum.
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