pFUSE-Lucia-CHIg-mG2a

Heavy chain constant region expression plasmid - Mouse IgG2a - Lucia tag

pFUSE-Lucia-CHIg-mG2a is a cloning plasmid that expresses the   constant region of the murine IgG2a heavy chain. It contains a multiple cloning site upstream of the constant region to enable cloning of the heavy chain variable region.

This plasmid is designed for antibody generation when co-transfected into mammalian cells with the light chain variable region cloned into pFUSE-CLIg.

pFUSE-Lucia-CHIg-mG2a contain the secreted luciferase (Lucia) gene upstream of the MCS and the CH region to serve as a tag to facilitate the detection and quantification  of recombinant antibodies.

Specifications

• Constant region of the mouse IgG2a heavy chain
• Lucia secreted luciferase tag

Lucia luciferase is a secreted protein, therefore, the VH sequence should not include a signal sequence.

Plasmids is selectable with Zeocin™ in E.coli and mammalian cells.

These products are covered by a Limited Use License (See Terms and Conditions).

pFUSE Contents

• 20 µg of lyophilized DNA
• 1 ml of Zeocin® (100 mg/ml)

Product is shipped at room temperature

Description

Antibody generation using pFUSE-Lucia-CHIg & pFUSE-CLIg

1- Obtaining VH and VL sequences

To obtain the cDNA sequence of the VH and VL regions from an antibody producing hybridoma, total RNA or mRNA is extracted and reverse transcribed to cDNA. PCR is performed with 5’ degenerate primers to anneal to the unknown VH and VL regions and the 3’ primers designed to anneal to the ‘known’ CH and CL regions. Alternatively 5’ RACE can be used. The resulting amplicons are sequenced.

2- Cloning into pFUSE-CHIg and pFUSE2-CLIg

Once the VH and VL sequence are known, inserts for cloning into the plasmids can be generated.

3- Antibody production

Cotransfect mammalian cells, such as 293 and CHO cells, with the recombinant plasmids pFUSE2-CLIg encoding the light chain and pFUSE-CHIg encoding the heavy chain. Antibody production depends greatly on the ratio of heavy chain and light chain expression. Typically, pFUSE-CHIg to pFUSE2-CLIg ratio of 2:3 is used to cotransfect mammalian cells. Use blasticidin and Zeocin™ to select pFUSE2-CLIg and pFUSE-CHIg respectively.
Antibody production can be analyzed by different techniques including SDS-PAGE, flow cytometry, ELISA, or a bioactivity assay: the luciferase levels in the supernatant could be determined using QUANTI-Luc™.

4- Antibody purification

Many antibody purification methods are available, among them mouse IgG2a isotype-specific affinity chromatography using Protein G or Protein L.

Details

1- Schematic representation of a Lucia luciferase-tagged antibody

2- Luciferase activity of Lucia-tagged anti-hTNF-α antibodies.

CHO cells were stably co-transfected with a heavy chain expressing plasmid, pFUSE-Lucia-TNF-CHIg-hA2m1 (TNF-CHIg-hA2m1) or pFUSE-Lucia-TNF-CHIg-hG1 (TNF-CHIg-hG1) and a light chain expressing plasmid, pFUSE2-TNF-CLIg-hk (TNF-CLIg-hk) to generate Lucia-tagged anti-hTNF-α antibodies, Lucia-anti-hTNF-α-hIgA2m1and Lucia-anti-hTNF-α-hIgG1, of human IgAm2 and human IgG1 isotypes, respectively. pFUSE2-CLIg-hk (CLIg-hk), which expresses no VL, and pSELECT-blasti-mcs (mcs) were used as negative controls. Supernatants
were collected and the luciferase levels determined using QUANTI-Luc™. Only the cells co-producing a Lucia-anti-hTNF-α heavy-chain and an anti-hTNF-α light chain displayed luciferase activity.

3- Neutralizing activity of anti-hTNF-a antibodies

The activity of anti-hTNF-α-hIgA2m1 and Lucia-anti-hTNF-α-hIgA2m1 antibodies, purified from the supernatants of CHO transfected cells, was determined by performing an TNF-α neutralizing assay. Both antibodies display similar neutralizing activities, thus fusion of the Lucia luciferase tag at the N-terminus of the heavy chain does not alter the functionality of the antibody.