Application of colony lift assay in the medullary thyroid carcinoma screening of single-chain variable fragment antibody phage library

Figures & data

Figure 1. Principle of the colony lift assay. A cell-coated filter was placed on an IPTG-containing plate (AI plate). Bacteria infected with the selected phage library were plated on the AG plate, and a replica of the bacterial filter (master filter) was made and placed on top of the cell-coated filter. Upon overnight incubation, the master filter was removed, placed on a fresh AG plate and stored for further use. The cell-coated filter was stained with scFv-tag specific antibody coupled to HRP and developed using H₂O₂ activated DAB. Subsequently, colonies corresponding to obtained spots were identified on the master filter.

Figure 2. Simple principle of the colony lift assay: (A) bacterial clones from MTC scFv library; (B) master filter; (C) MTC-coated filter and (D) double strength YT + Amp + IPTG + agar.

Figure 3. Colony assay and lift on MTC-coated filter and master filter: (A) MTC-coated filter, positive spots from scFv-stained MTC-coated filter, scFv produced by corresponding positive clones on master filter; (B) master filter, clones growing on master filter used for colony lift.

Table 1. Frequency of positive clones screened from MTC scFv library by random pick and colony lift assay.

	Random picking			Colony lift assay
Selection round (SR)	Tested clones	Positive clones	Positive rate	Tested clones	Positive clones	Positive rate
1	96	0	0 (0∕96)	318	2	0.63 (2∕318) *
2	96	3	3.13 (3∕96)	198	12	6.06 (12∕198) *
3	96	10	10.42 (10∕96)	162	20	12.35 (20∕162) *
4	96	56	58.33 (56∕96)	148	94	63.51 (94∕148) *

Notes:*P > 0.05; n = 4; chi-square test, colony lift assay vs. random pick (SR1, χ² = 0.607, P = 0.436; SR2, χ² = 1.151, P = 0.283; SR3, χ² = 0.218, P = 0.640; SR4, χ² = 0.660, P = 0.417).