ISSN:
1434-0879
Keywords:
Single-chain Fv antibody
;
Expression
;
Polymerase chain reaction
;
Hybridoma
;
Bladder carcinoma
;
ELISA
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the V H and V K genes were 366 and 324 bp, respectively. Compared with other published sequences, the V H gene was a member of mouse heavy-chain V H subgroup II and originated from the rearrangement of V H, Dsp2.2 and J H4. The V K gene was V K subgroup IV and from V K and J K4. The V H and V K genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00304776
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