Esophageal Cancer
Copyright ©The Author(s) 2004.
World J Gastroenterol. Sep 15, 2004; 10(18): 2619-2623
Published online Sep 15, 2004. doi: 10.3748/wjg.v10.i18.2619
Table 1 Oligonucleotide primers used for PCR of human immunoglobulin genes
A. Primary PCRs
Human VH back primers (sense)
HuVHlaBACK5’-CAGGTGCAGCTGGTGCAGTCTGG-3’
HuVH2aBACK5’-CAGGTCAACTTAAGGGAGTCTGG-3’
HuVH3aBACK5’-GAGGTGCAGCTGGTGGAGTCTGG-3’
HuVH4aBACk5’-CAGGTGCAGCTGCAGGAGTCGGG-3’
HuVH5aBACK5’-GAGGTGCAGCTGTTGCAGTCTGC-3’
HuVH6aBACK5’-CAGGTACAGCTGCAGCAGTCAGG-3’
JH forward primers (anti-sense)
HuJHl-2FOR5’-TGAGGAGACGGTGACCAGGGTGCC-3’
HuJH3FOR5’-TGAAGAGACGGTGACCATTGTCCC-3’
HuJH4-5FOR5’-TGAGGAGACGGTGACCAGGGTTCC-3’
HuJH6FOR5’-TGAGGAGACGGTGACCGTGGTCCC-3’
Human V kappa back primers (sense)
HuV κ laBack5’-GACATCCAGATGACCCAGTCTCC-3’
HuV κ 2aBack5’-GATGTTGTGATGACTCAGTCTCC-3’
HuV κ 3aBack5’-GAAATTGTGTTGACGCAGTCTCC-3’
HuV κ 4aBack5’-GACATCGTGATGACCCAGTCTCC-3’
HuV κ 5aBack5’-GAAACGACACTCACGCAGTCTCC-3’
HuV κ 6aBack5’-GAAATTGTGCTGACTCAGTCTCC-3’
Human J kappa forward primer (anti-sense)
HuJ κ 1FOR5’-ACGTTTGATTTCCACCTTGGTCCC-3’
HuJ κ 2FOR5’-ACGTTTGATCTCCAGCTTGGTCCC-3’
HuJ κ 3FOR5’-ACGTTTGATATCCACTTTGGTCCC-3’
HuJ κ 4FOR5’-ACGTTTGATCTCCACCTTGGTCCC-3’
HuJ κ 5FOR5’-ACGTTTAATCTCCAGTCGTGTCCC-3’
Human V lambda back primers (sense)
HuV λ 1BACK5’-CAGTCTGTGTTGACGCAGCCGCC-3’
HuV λ 2BACK5’-CAGTCTGCCCTGACTCAGCCTGC-3’
HuV λ 3aBACK5’-TCCTATGTGCTGACTCAGCCACC-3’
HuV λ 3bBACK5’-TCTTCTGAGCTGACTCAGGACCC-3’
HuV λ 4BACK5’-CACGTTATACTGACTCAACCGCC-3’
HuV λ 5BACK5’-CAGGCTGTGCTCACTCAGCCGTC-3’
HuV λ 6BACK5’-AATTTTATGCTGACTCAGCCCCA-3’
Human J lambda forward primers (anti-sense)
HuJ λ 1FOR5’-ACCTAGGACGGTGACCTTGGTCCC-3’
HuJ λ 2-3FOR5’-ACCTAGGACGGTCAGCTTGGTCCC-3’
HuJ λ 4-5FOR5’-ACCTAAAACGGTGAGCTGGGTCCC-3’
B. PCR assembly
Hu JH-Linker primers
HuJH1-2Linker5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTGCC-3’
HuJH3Linker5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAAGAGACGGTGACCATTGTCCC-3’
HuJH4-5Linker5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCAGGGTTCC-3’
HuJH6Linker5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTCCC-3’
Linker-Hu V κ primers
Linker-HuV κ 2-3-6 BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAWRTTGTGHTGACKCAGTCTCC-3’
Linker-HuV κ 1-4 BACK5’-gTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCSWGATGACCCAGTCTCC-3’
Linker-HuV κ 5 BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAAACGACACTCACGCAGTCTCC-3’
Linker-Hu V λ primers
Linker-HuV λ 1-2BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGYSYTGACKCAGCCKSC-3’
Linker-HuV λ 3BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCYTMTGWGCTGACTCAGSMMCC-3’
Linker-HuV λ 4-5BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCASGYTRTRCTSACTCARCCGYC-3’
Linker-HuV λ 6BACK5’-GTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGAATTTTATGCTGACTCAGCCCCA-3’
C. Reamplification with primers containing restriction sites
Human VH back (Sfi) primers (sense)
HuVH1aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG-3’
HuVH2aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTCAACTTAAGGGAGTCTGG-3’
HuVH3aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGG-3’
HuVH4aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG-3’
HuVH5aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGC-3’
HuVH6aBACKSfi5’-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG-3’
Human J kappa forward (Not) primers (anti-sense)
HuJ κ 1FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTTGGTCCC-3’
HuJ κ 2FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3’
HuJ κ 3FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTTGGTCCC-3’
HuJ κ 4FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCACCTTGGTCCC-3’
HuJ κ 5FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC-3’
Human J lambda forward (Not) primers (anti-sense)
HuJ λ 1FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC-3’
HuJ λ 2-3FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC-3’
HuJ λ 4-5FORNot5’-GAGTCATTCTCGACTTGCGGCCGCACCTAAAACGGTGAGCTGGGTCCC-3’
Table 2 Immunore activity of soluble scFv determined by ELISA
SampleA405nm(mean ± SD)
Eca109HeLaNHEE
Soluble scFv0.78 ± 0.120.21 ± 0.09d0.23 ± 0.07b
PBS0.14 ± 0.040.13 ± 0.080.15 ± 0.02