Characterisation of Antibodies with BRAND® PCR Plates

By: AG Arndt/ Krauss
Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg, Im Neuenheimer Feld 460, 69120 Heidelberg

Introduction

The generation of fully human antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. However, it is possible to isolate these antibodies from human B-cellls in contact with specific antigens.

In this study, variable antibody genes of igG were cloned from B-cell repertoires in lymph nodes of head and neck cancer patients. The sequence analysis of the antibodies of lymph nodes revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germ line sequences.

To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, these antibodies were tested for their affinity to the target antigen of HSV-1 (Herpes simplex viruses) infected cells.

The majority of the scFv (single chain variable fragment)-antibody fragments bound with nanomolar affinity to the target antigen of the HSV strains.

Material and Methods1

For antibody isolation IgG donor repertoires from lymph nodes of head and neck cancer patients were used. Thereby independent gene pools of variable domains of light and heavy chains (VH/ VL-kappa and VH/ VL-lambda) could be generated. Bacterial cells transformed with these genes were grown on agar plates and cultivated overnight. These transformed and cultivated E.coli cells were in the next step stored in glycerol aliquots at -80 °C.

Exemplary transformed samples of aliquots were selected and cultivated in shaking flasks, before a tenfold dilution of these E.coli cells were transferred on agar plates. Eight clones of each plate were picked and further analysed with a colony PCR to clarify whether the genes of both the heavy chain and the light chain were integrated (fragment size ~ 1 kb).

Table 1. PCR Program characteristics

PCR-Program Time
Initial Denaturation 95 °C 5 min
Denaturation 95 °C 15 sec --------I
Annealing 51 °C 15 sec I à 35 cycles
Elongation 72 °C 15 sec --------I
Final Elongation 72 °C 10 min
Cooling 4 °C unlimited

Table 2. PCR mixture

  Capacity
KAPA 2 G Fast Ready Mix (buffer, polymerase, dNtPs, gel loading dye) 12.5 μL
Primer A 0.5 μL
Primer B 0.5 μL
Template picked single colony
H2O 11.5 μL
Total capacity 25.0 μL

To visualize expression of scFVs in the selected colonies, the colony PCR was subjected to agarose gel electrophoresis.

 

Result

All eight clones (in triplicates) showed a band at a fragment size of ~ 1 kb. For this reason, they include the genes of the heavy chain as well as the genes of the light chain.

DNA marker (100bp DNA Ladder (N3231) of NEB

Figure 1. [right]: DNA marker (100bp DNA Ladder (N3231) of NEB at position 1;14 and 27. The 1,5% agarose gel ran at 140 V for 40 minutes) with marker description; [left]: agarose gel (triplicates each)

Subsequently, the isolated B-cell fragments were incubated with HSV-1 infected cells. The equilibrium constant of binding (KD) of the scFvs with the target antigen of HSV-1 infected cells was determined on the basis of a flow cytometry affinity measurement. The KD value was in the nanomolar range indicating a high binding affinity.

Discussion

BRAND® PCR plates (Cat. No. 7813 75) are perfectly suitable for a colony PCR. The smooth well surfaces minimize attaching of reactants to the well surface, so that the PCR reaction can process completely. Further the high-quality polypropylene of this PCR plate is free of PCR inhibitors that could reduce the yield. In addition, these PCR plates were sealed with the optimal fitting sealing mat (Cat. No. 7814 05) to reduce evaporation losses to a minimum.

In the light of the above the antibody fragments of all transformed clones could be reproduced with a high yield and verified with an agarose gel.

1 Philipp Diebolder, Armin Keller, Stephanie Haase, Anne Schlegelmilch, Jonathan D Kiefer, Tamana Karimi, Tobias Weber, Gerhard Moldenhauer, Roland Kehm, Anna M Eis-Hübinger, Dirk Jäger, Phillippe A Ferderspil, Christel Herold-Mende, Gerhard Dyckhoff, Roland E Kontermann, Michaela AE Arndt, and Jürgen Krauss. Generation of "LYmph Node Derived Antibody Libraries“ (LYNDAL) for selecting fully human antibody fragments with therapeutic potential. mAbs 6:1, 130-142; January/ February 2014; ©2014 Landes Bioscience

Materials