| The ability to conjugate a protein to another carrier or molecule has been widely used in life science research and medical science. In immunoassay, the conjugation of antibody and enzyme will directly effect sensitivity and reliability of the method.Present methods, such as glutaraldehyde-modified,EDC-modified and sodium periodate-oxidized, have inevitably formed much polyer, reduced the efficiency and decreased activity of the conjugation. Heterobifunctional conjugation reagents like SPDP, MBS can overcome this shortage, but enzyme will crosslink somewhat randomly to nearly all parts of the antibody molecule,this in turn leads to an influence of antibody and enzyme.So we must establish a site-directed conjugation schemes for application.This research conjugating condition of IgG and HRP was studied and optimized making use of SMCC-modified.In this research,monoclonal antibody was produced by immuning asctic fluid,the activity and titer of antibody was determined by ELISA,and the competition inhibition curve was established. The DTT reduction reaction of IgG has been studied from feed ratio,reaction temperature,reaction time,and investigate its influence on the structure,activity of immunoglobulin. HRP has been conjugated with antibodies using SMCC as a crosslinking reagent,which would be used in ELISA.The results showed that reaction temperature and reaction time have little effect on the structure and activity of immunoglobulin,but there is notable effect on them if we change the feed ratio.In the condition of blocking free sulphydryl, the activity of antibody begin to decline when the feed ratio is 600, and the activity is almost completely loss of when the feed ratio is 6000; but the free sulphydryl would re-aggregation if we do not block it, then the difference of structure and activity of antibody are not obvious when the feed ratio is between 600～12000. Finally, we determined that the feed ratio is 8000, reaction time is between 20～30min, reaction temperature is 25℃. |
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