Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles

Abstract

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

“…The framework gene and anti-S-layer scFvs differed in CDR1 and CDR3 of both heavy and light chains and CDR2 of the heavy chain (Table 2), which corresponds to the phage library design rules (19). Only one clone, PolyF5, originated from the ScFvP repertoire based on the presence of a tryptophan (W) as the last amino acid in the CDR-L3 loop (26). Accordingly, the remaining selected binders originated from the ScFvM repertoire.…”

“…Panning is the process of the selection and isolation of specific antibodies by their binding activity (27). S-layercoated microtiter wells were panned with a 1:1 mixture of pEB32x ScFvP and ScFvM libraries (26 ) not coated with S-layer protein for 1 h at 25°C with slow shaking (Delfia Plateshake, low mode; Perkin-Elmer), after which the unbound fraction was transferred to S-layer-coated wells. After 2 h (for the first panning) and 1 h (for the subsequent pannings) of incubation, the wells were washed 3 times (for the first panning) and 4 times (subsequent pannings) with TBT-0.1 and then once with TBS-0.1% Tween 20.…”

Molecular tools for selective recovery and detection of lignin-derived molecules

“…The binders for ferulate, coumarate and caffeate were isolated from a synthetic antibody phage library (ScFvM) described by Huovinen et al 35 E. coli XL1-Blue (Stratagene, USA) cells were used for phage infection and production, cloning, and protein expression. The VCS-M13 helper phage (Stratagene) was used to rescue the phagemid carrying phage in biopanning.…”

“…Antigenbinding site architectures of the antibodies in these libraries can be designed to favor the recognition of certain types of antigens differing for instance in terms of size. 35 With a suitable antibody library, specific binders can be obtained even for very small haptens, such as skatole (MW = 131.2 Da), 36 which typically are very difficult targets for antibody development. Thus, scFv binders for small molecules that are relevant for the lignin industry, such as hydroxycinnamates, might also be found from synthetic antibody libraries, even though they differ only in the degree of methoxylation and the number of hydroxyl groups.…”

Non-competitive ELISA with broad specificity for microcystins and nodularins

“…coli cells carrying the clone SA51D1 construct in pLK06H (Huovinen et al, 2013) vector were grown in 50 mL shaking flask in SB medium supplemented with 100 µg mL -1 ampicillin, 10 µg mL -1 tetracycline and 0.05% glucose. The cells were induced with IPTG (isopropyl-β-D-1-thiogalactopyranoside induction) to the final concentration of 100 µM and incubated overnight at 26°C, with shaking at 300 rpm.…”