Anticorps CUSTOM CAMELIDS VHH FRAGMENT

 

Bulle About VHH

Antibody Format

Camelids produce conventional antibodies made of 2 heavy chains and 2 light chains bound together with disulphide bonds in a Y shape. However, they also produce a unique subclasse of immunoglobulin G, also known as heavy chain IgG (hcIgG).

These antibodies are made of only two heavy chains  without the CH1 region, and an antigen binding domain at their N-terminus called VHH.

The unique feature of hcIgG is the capacity of their monomeric antigen binding regions (VHH) to bind antigens with specificity, affinity and especially diversity that are comparable to conventional antibodies without the need of pairing with another region. Using molecular engeenering VHH fragaments can be generated.


Bulle Why use VHH fragments ?

Bind the hidden epitope

Their steric hindrance is reduced, allowing them to reach hardly accessible antigens that conventional antibodies can not, such as active sites of enzymes (useful to inhibit enzyme activity) as well as smaller enzymes, such as lysozymes or ribonucleases.

Better permeability

Their capacity to penetrate tissues is increased compared to conventional antibodies, allowing a more precise staining of tissue sections when performing immunohistochemistry.

More stable

The high refolding capacity of the VHH region allow them to be thermodynamically more stable and resist to high temperatures (up to 70°C / 158°F)  and extreme pH, such as gastric acid.


Bulle VHH development

Llama immunization

1- CAMELID IMMUNIZATION

Immunization

According to our 88-day exclusive protocol, the immunization step consists in 4 injections and 2 test bleeds.

Monitoring

The immunoreactivity is monitored by the detection method developed for the project.



2- PHAGE LIBRARY CONSTRUCTION

PBMCs isolation and RNA extraction

Peripheral blood mononuclear cells (PBMCs) are isolated from the final bleed and total RNA is extracted.

cDNA synthesis and VHH amplification

RT-PCR and VHH antibody fragment amplification

Specific Primers are design to perform retro-transcription within the region of the coding for VHH and second nested PCR allows cDNA amplification.

Phage vector cloning

VHH coding genes are integrated into our phage vector which is then used to transform competent bacteria for phage library production.

Phage Display

VHH Panning

3- PANNING

Target interaction

The antigen is immobilized in the bottom of microtiter plate wells, phages preparations are loaded into the wells to allow them to bind the antigen through the VHH expressed at their surface.

Amplification

After washing off unbound phages, remaining phages are transduced into competent bacteria for enrichment and amplification.

Selection

At the end of each panning stage, cDNAs coding for VHH specific to the antigen are isolated



4-HIGH SCALE PRODUCTION

Subcloning in high yield expression vector

Inserts are integrated into our high yield expression vector, which is then used to transform competent bacteria.

Culture, lysis and affinity chromatography purification

Specific VHH are purified from culture supernatants by affinity chromatography.

All cDNAs coding for specific VHH are kept frozen in our laboratory.

VHH Expression


Bulle Brochures

For more informations :

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VHH Technology