However, the Fab format substantially reduced the affinity by
two orders of magnitude. On account of the chosen orientation of the assay, the affinity constant determination can be
affected by avidity effects. See Note 11.
4 Notes
1. Replacing used culture medium for fresh F17 (without antibiotic), ensuring a sufficient amount of nutrients to keep the cells
growing until the addition of fresh culture medium at day 2.
2. Using polystyrene tubes instead of polypropylene tubes is
important to ensure that the PEI-DNA complexes do not
stick to the tube what would lead to a lower transfection rate
due to a lower total amount of available complex.
3. Directly mix the DNA with PEI to form stable PEI-DNA
complexes. Be sure that the DNA is not precipitating. Turbidity or visible aggregates indicate that the quality of the DNA
preparation is not sufficient. Make sure the DNA sample is not
contaminated with chromosomal E. coli DNA.
4. The optimal combination in our facility comprises the OpIE2
promoter, the IE1 terminator, and a FlashBac compatible
backbone [20].
Table 2
Summary of antibody yields and apparent affinity to KLK7
Antibody
Volume yield (mg/L)
K D (nM)
k on (1/M·s)
k off (1/s)
scFv-Fc-LUP37-A10
7.36
0.304
1.45 Â 10
5
4.39 Â 10
À5
scFv-Fc-LUP37-B10
7.42
5.53
9.84 Â 10
4
5.44 Â 10
À4
scFv-Fc-LUP37-C11
8.10
0.231
1.32 Â 10
5
3.05 Â 10
À5
scFv-Fc-LUP37-D11
6.96
0.179
1.34 Â 10
5
2.39 Â 10
À5
IgG-LUP37-A10
0.40
0.247
1.08 Â 10
5
2.67 Â 10
À5
IgG-LUP37-B10
0.47
–
–
–
IgG-LUP37-C11
1.05
0.012
3.68 Â 10
5
4.43 Â 10
À6
IgG-LUP37-D11
2.02
0.419
6.74 Â 10
4
2.82 Â 10
À5
Fab-LUP37-A10
1.33
18.8
7 .95 Â 10
4
1.50 Â 10
À3
Fab-LUP37-B10
1.75
–
–
–
Fab-LUP37-C11
4.55
27.8
1 .05 Â 10
5
2.91 Â 10
À3
Fab-LUP37-D11
4.47
25.3
1 .56 Â 10
5
3.96 Â 10
À3
74
Jens Ko ¨ nig et al.
two orders of magnitude. On account of the chosen orientation of the assay, the affinity constant determination can be
affected by avidity effects. See Note 11.
4 Notes
1. Replacing used culture medium for fresh F17 (without antibiotic), ensuring a sufficient amount of nutrients to keep the cells
growing until the addition of fresh culture medium at day 2.
2. Using polystyrene tubes instead of polypropylene tubes is
important to ensure that the PEI-DNA complexes do not
stick to the tube what would lead to a lower transfection rate
due to a lower total amount of available complex.
3. Directly mix the DNA with PEI to form stable PEI-DNA
complexes. Be sure that the DNA is not precipitating. Turbidity or visible aggregates indicate that the quality of the DNA
preparation is not sufficient. Make sure the DNA sample is not
contaminated with chromosomal E. coli DNA.
4. The optimal combination in our facility comprises the OpIE2
promoter, the IE1 terminator, and a FlashBac compatible
backbone [20].
Table 2
Summary of antibody yields and apparent affinity to KLK7
Antibody
Volume yield (mg/L)
K D (nM)
k on (1/M·s)
k off (1/s)
scFv-Fc-LUP37-A10
7.36
0.304
1.45 Â 10
5
4.39 Â 10
À5
scFv-Fc-LUP37-B10
7.42
5.53
9.84 Â 10
4
5.44 Â 10
À4
scFv-Fc-LUP37-C11
8.10
0.231
1.32 Â 10
5
3.05 Â 10
À5
scFv-Fc-LUP37-D11
6.96
0.179
1.34 Â 10
5
2.39 Â 10
À5
IgG-LUP37-A10
0.40
0.247
1.08 Â 10
5
2.67 Â 10
À5
IgG-LUP37-B10
0.47
–
–
–
IgG-LUP37-C11
1.05
0.012
3.68 Â 10
5
4.43 Â 10
À6
IgG-LUP37-D11
2.02
0.419
6.74 Â 10
4
2.82 Â 10
À5
Fab-LUP37-A10
1.33
18.8
7 .95 Â 10
4
1.50 Â 10
À3
Fab-LUP37-B10
1.75
–
–
–
Fab-LUP37-C11
4.55
27.8
1 .05 Â 10
5
2.91 Â 10
À3
Fab-LUP37-D11
4.47
25.3
1 .56 Â 10
5
3.96 Â 10
À3
74
Jens Ko ¨ nig et al.