**Author details**

at a lower speed (about 180 rpm) can be performed although oxygen supply is reduced as well. Alternatively, PP deep well plates can be used for screening. To prevent evaporation, plates can be tape-fixed in an additional plastic bag or use PBS-filled plates at the bottom and top when using microplate holders. The highest yield of soluble scFvs can be obtained with special temperature-controlled shakers with a small orbital diameter (0.12 inch) and shaking

**Note 21:** Usually, antigen-specific binding is defined as signals at least five times higher than

**Note 22:** The 2% glucose guarantees repression of antibody expression within the first 3 h of growing. To induce antibody expression, medium must be replaced by induction medium without glucose and optimized concentration of IPTG. Many scFvs show very low yield in the supernatant after expression in *E*. *coli* and periplasmic extraction is highly recommend prior to ELISA, e.g., by resuspending bacteria pellets in 180 μl/well periplasmic preparation buffer (see Protocol G) for 30 min on ice. However, as shown by Hust et al. [32], buffered 2xYT-SAI and growing cultures overnight at 30°C can improve production in *E*. *coli* for some scFvs even

**Note 23.** Adjusting PCR conditions to obtain single scFvs bands on agarose gel is important for successful sequencing of PCR fragments, e.g., by increasing annealing temperature. Alternatively, scFv bands can be recovered by QIAquick Gel Extraction Kit or sequencing can

**Note 24:** Plasmid DNA can be used for electroporation into self-made electrocompetent HB2151 if scFv-phage screening with TG1 bacteria was performed beforehand. If already in HB2151 after soluble screening, the glycerol stock can be used to obtain single colonies on 2xYT-GA plates for small-scale production of soluble scFvs (see Protocol G). Alternatively, plasmid DNA can be used for subcloning into bacterial expression vectors without pIII gene. **Note 25.** For time reasons, we recommend soluble expression of scFvs in nonamber suppressor strain such as HB2151 when using phagemid with the amber stop codon between the scFv and the pIII protein. Otherwise, scFvs can also be subcloned into expression vector without the pIII gene. If clones are still in TG1 after phage screening, phagemid DNA can be transformed into self-made competent HB2151. Use standard protocol for generation of chemically or electrocompetent HB2151. Alternatively use commercially available competent nonamber

by NEB).

suppressor strains for soluble production (e.g., SS320 by Lucigen, or Express *Iq*

**Note 26.** Reducing the temperature for soluble scFv expression is important for proper fold-

**Note 27:** As a cheaper option for dialysis in small scale, we recommend using 2 ml PP tubes without lids, filled with scFv preparations, and sealed with square cut dialysis membrane fixed with rubber band and parafilm. If using other tags for purification, dialyze in recom-

**Note 28:** Determine best number/growing conditions for your cell line. Gently tilt plate about five times horizontally after seeding to guarantee that uniform monolayers of cells are

the background. For a typical result, see [18], Figure 4B and C.

without performing periplasmic extraction.

be done using plasmid DNA (see F10).

ing and stability of produced scFvs.

mended buffer prior purification.

at high speed (1000 rpm).

98 Antibody Engineering

Philipp Diebolder1 and Adalbert Krawczyk<sup>2</sup> \*

\*Address all correspondence to: adalbert.krawczyk@uni-due.de

1 Department of Radiation Oncology, Washington University School of Medicine, St. Louis, USA

2 Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
