Dimethyl suberimidate as an effective crosslinker for antibody-enzyme conjugation.
Dimethyl suberimidate (DMS), a bifunctional reagent was used for the first time to crosslink the alpha-feto protein monoclonal antibodies (AFPMAb) to horse radish peroxidase (HRP). Three batches of conjugates were prepared, purified by Sephadex gel chromatography and evaluated for their immunological reactivity. The Rz values obtained for AFPMAb-HRP conjugate were 0.39 to 1.36. Under optimised conditions the ELISA results showed the optical density of 1.9. The iso-electric focusing for the conjugate revealed different degrees of crosslinking between antibodies and HRP. It was evident that isoperoxidase-C was involved in the crosslinking process. From the dot ELISA, as low as 25 pg of AFP in the test samples could be detected with AFPMab-HRP conjugate. The conjugate prepared by DMS was stable at 0 degrees C for more than 10 months.
Sekhar
M C
MC
Centre for Biochemicals, Delhi Univ., India.
Sharma
G L
GL
Gangal
S
S
Joshi
A P
AP
Sarma
P U
PU
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004120:Dimethyl Suberimidate / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007525:Isoelectric Focusing; D000509:alpha-Fetoproteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000509:alpha-Fetoproteins; D004120:Dimethyl Suberimidate; D006735:Horseradish Peroxidase