A novel method for CRM9 and IgG linkage

    HU Jie1, ZHANG Runª²Ling1, DU Huiª²Fen2, YUAN Ming2, LI Keª²Sheng2

    1Department of Clinical Laboratory, Second Hospital, Lanzhou Medical College, Lanzhou 730030, China, 2Biotechnique Center, Medical Academy of Gansu, Lanzhou 730050, China

    ¡¾Abstract¡¿ AIM: To search the method of conjuncting rabbit antiª²Hb IgG and diphtheria toxin mutation (CRM9) by chemical crosslinkers MBS (mª²Maleimidobenzoylª²Nª²hydoxysuccinimide ester, NHSª²Estersª²Maleimide crosslinkers) and SPDP [Nª²succinidimidylª²3ª²(2ª²pyridyldithio) proprionate)] and to study the results achieved by the two methods. METHODS: Rbª²antiª²Hb Igª²G was linked with diphtheria toxin mutation (CRM9) by MBS and SPDP respectively. The outcomes were eluted through Sephacryl Sª²300 to purify the linkageª²protein. The degree of IgG and CRM9 linking was observed. The conjuncting conditions were testified by 8% SDSª²PAGE and agarose electrophoresis. The antibodyª²part of the linkageª²protein, which was mixed in different ratio, was tested by ELISA. RESULTS: In image of SDSª²PAGE, the linkageª²protein, IgGª²MBSª²CRM9, conjuncted by MBS lagged behind IgG. In ELISA, the A450 of the five groups had obvious deviation. The activity of linkageª²protein¡¯s antibody had little influence on group A and group B, the ratio of IgG and CRM9 being 1¡Ã1 and 1¡Ã3. The antibody¡¯s activities of group C (1¡Ã5) and group D (1¡Ã8) decreased greatly. CONCLUSION: Rabbit antiª²Hb IgG can conjunct CRM9 by MBS. The best ratio of IgG to CRM9 is 1¡Ã1-1¡Ã3. Under this condition conjunction is better and has little influence on the activity of antibody. This provides a valid method for the new conjunction of immunity toxin. ......