Panning and identification of humanized antinuclear antibody Fab fragment / 第二军医大学学报
Academic Journal of Second Military Medical University
; (12): 87-91, 2010.
Article
in Zh
| WPRIM
| ID: wpr-840971
Responsible library:
WPRO
ABSTRACT
Objective:
To prepare humanized antinuclear antibody Fab fragment.Methods:
The reconstructed humanized antinuclear antibody (ANA) Fab phage display library was enriched by 4 rounds of panning and was identified by indirect ELISA method. Phasmid DNA isolated from positive clones was deprived of g III gene. After self-ligation the recombinant plasmid was used to transform E. coli. XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab fragment. Finally, soluble human antinuclear antibody Fab in the supernatant was identified by indirect ELISA method and immunofluorescence.Results:
The eluted phages were enriched by more than 200 folds after 4 rounds of panning. Two positive clones were isolated from the ANA Fab library. Electrophoresis after Xho I digestion proved that the self-ligation was successful after deletion of g III gene. The results of indirect ELISA indicated that the 2 positive clones of Fab had specific anti-dsDNA activity. Indirect immunofluorescence showed homogeneous fluorescence within nuclei of Hep2 and monkey hepatic cells and in the Crithidia kinetoplast.Conclusion:
We have successfully prepared soluble, specific human antinuclear antibody Fab fragment, which pares a way for preparation of high affinity antinuclear antibody Fab fragment.
Full text:
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Index:
WPRIM
Language:
Zh
Journal:
Academic Journal of Second Military Medical University
Year:
2010
Type:
Article