**4. Results**

measured by indirect ELISA with the PcrV antigen coated at 1 μg/mL. In the second step, *K*d, the dissociation constant, was measured using binding equilibrium studies (competition ELISA) to determine the concentration that gives 50% inhibition of maximum binding.

**44.5- 32.4- 18.4-**

**(kD)**

**1- n iot carf el b lu oS**

**1- et lu E**

**Figure 3.** Expression and purification of scFv166. *E. coli* lysates were loaded onto a 4–15% gradient Tris-HCl gel and, after electrophoresis, the gel was stained with Coomassie Blue. For the immunoblot analysis, after polyacrylamide gel electrophoresis, the protein was blotted onto a nitrocellulose membrane and immunostained with a horseradish peroxidase-conjugated anti-c-Myc IgG antibody, and the blot was developed with a chemiluminescent substrate. The secreted scFv166 (298 amino acids) was detected as a 29.7 kD-band in the elute-1 and elute-2, designated by arrows. Soluble fraction: the osmotically shocked lysate; elute: the eluted solution from an Ni-NTA agarose column; soluble fraction after elution: the solution passed through an Ni-NTA agarose column (two sets of the lysate and the column

> **50- 25-**

**(kD)**

**50-**

**(kD)**

**25- 37-**

**Figure 4.** Immunoblot of *P. aeruginosa* proteins reacted with scFv166. Precipitated *P. aeruginosa* PA103 proteins were resuspended in 100 μL of PBS. After adding 100 μL of SDS-PAGE sample buffer and boiling for 5 min, the proteins in the sample were separated by SDS-PAGE with *E. coli*-derived recombinant PcrV (rPcrV) as a reference. After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and immunostained with scFv166 and a horseradish peroxidase-conjugated secondary anti-c-Myc IgG antibody and the blot was developed with a chemiluminescent substrate. The bindings of scFv166 to both native PA103 PcrV (294 amino aicds, 32.4 kD) and recombinant PcrV (rPcrV, 306 amino

**1- n iot carf el b lu oS**

**n iot lu er etf a**

**2- n iot carf el b lu oS**

**scFvm166-HLL 29.8kD**

**2- et lu E**

**rPcrV PA103**

**2- n iot carf el b lu oS**

**n iot lu er etf a** **) %1. 0( decud in l ort noc**

**) %1. 0( il oc. E decud In**

**44.5- 32.4- 18.4-**

**SDS-PAGE**

**Immunoblot with scFv166**

**(kD)**

**Immunoblot with anti-Myc IgG**

elute were analyzed and labeled "−1" and "−2", respectively).

acids, 33.8 kD) were detected as shown in arrows.

**SDS-PAGE** 

126 Antibody Engineering
