[Construction of Fab antibody against Rh antigen].
OBJECTIVE
To Construct Fab antibody against Rh antigen.


METHODS
The variable regions of light and heavy chains were amplified by RT-PCR from the PBMCs of volunteers with high titer (1:256-512 by inditect agglutation) antibody to Rh antigen. Meanwhile, the genes of constant regions of light and heavy chains were isolated from pComb3xTT and pComb3xlambda phagmid carrying the templates respectively. Vkappa light chain and Fd heavy chain were linked by the first splicing overlapping extension PCR (SOE), and a full-length Fab gene was created by the second SOE. The Fab gene was ligated to phagmid pComb3HxSS and transformed to E.coli XL1-Blue by electroporation. The obtained human Fab phage antibody library was panned using Rh(-)/Rh(+) RBC four times. the phage antibodies against Rh antigen were highly enriched. Indirect agglutation test, western blot analysis and sequencing analysis were performed to detect the specificity of Fab against Rh.


RESULTS
The repertoire of human phage display Fab library was 7.4 x 10(6);. After panning, A Fab clone which could bind to Rh antigen specifically was obtained.


CONCLUSIONS
A Fab antibody that specifically aggulated Rh(+) RBC is obtained, this makes it possible to produce Rh antibody with high quantity and effection in our country.
Chen
Qi
Q
Physiological Department of Medical College, Yangzhou University, Yangzhou 225001, China. yzchenqi@126.com
Zhang
Jia-min
JM
Zhang
Hai-yan
HY
Zhu
Zi-yan
ZY
Li
Hou-da
HD
D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D005818:Genetic Engineering; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D012204:Rh-Hr Blood-Group System / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D012204:Rh-Hr Blood-Group System