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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
