Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride.
In this report, we describe a two-step protocol for labeling of an affinity-purified antibody to biotin with horseradish peroxidase (HRP) using cyanuric chloride (CC) as a bridge. The enzyme was first modified with CC, and following chromatography on a PD-10 column, the activated HRP was incubated with the antibody to effect coupling of the two proteins. Assessment of the conjugate product was carried out using ELISA and SDS-PAGE electrophoresis where evidence for high antibody activity, high specific activity of the conjugate preparation, coupling of nearly all the antibody and over 90% of the enzyme was shown. The titer of the conjugate exceeded 1/100,000. High molecular weight complexes were observed in the SDS-PAGE results, indicating an efficient conjugation procedure. The presence of high molecular weight complexes indicated an efficient conjugation procedure. The protocol is simple, and the conjugation steps can be completed in 27 h once the preparatory phase has been carried out; the method is entirely generic and may be applied to labeling of any antibody.
Abuknesha
Ramadan A
RA
Department of Pharmacy, King's College London, Analytical Sciences Research Group, Pharmaceutical Science Research Division, School of Biomedical and Health Sciences, Stamford Street, London SE1 9NH, UK. ram.abuknesha@kcl.ac.uk
Jeganathan
Fiona
F
Wu
Jocelyn
J
Baalawy
Zakeya
Z
D000906:Antibodies / Q000737:chemistry*; D001710:Biotin / Q000276:immunology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D014227:Triazines / Q000737:chemistry*
D000906:Antibodies; D014227:Triazines; C019309:cyanuric chloride; D001710:Biotin; D006735:Horseradish Peroxidase