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Membrane Protein Antibody Production by Whole Cell Antigen Presentation

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Creative Biolabs provides Membrane Protein Antibody Production by Whole Cell Antigen Presentation Services. With years of experience in the area, our scientists have developed whole cell antigen presentation technology, which is a world leading membrane protein antibody production technology with antigens presented on the surface of mammalian cells. Our technology is contributing to the growing interest in targeting membrane proteins as therapeutic targets with antibodies.

The traditional membrane protein antibody production methods involve in vitro antigen production when immunizing the animals. However, the drawbacks of in vitro antigen production hamper the production of membrane protein antibodies.To serve as a “bait” to isolate antibodies, the antigen should remain in a pure, homogenous and conformationally stable form as the native membrane protein structure. However, this is hampered by the requirement for membrane proteins being within the plasma membrane. Low cell surface expression is another limiting factor and an obstacle for obtaining sufficient quantities of membrane proteins for purification. Still, the instability of protein during purification process is another issue. Purified membrane proteins have been produced for a limited amount and display functional activity as determined by radioligand binding. The results turned out that a small percentage of functional receptor remains in a larger quantity of unfolded protein. Another critical issue is the requirement for detergents to maintain protein folding in a functionally active form following purification of membrane proteins. Long chain detergents that confer stability can mask extracellular epitopes in a certain level.

To obtain antibodies reactive to the native extracellular structure of membrane proteins, Creative Biolabs has developed whole cell antigen presentation technology. Our technology involves immunization by injection of cultured cells stably expressing the antigens, which avoids all the drawbacks of in vitro antigen production. We have successfully generated hundreds of antibodies that bind specifically to native conformation of membrane proteins.

Our technology involves two methods in isolating anti-membrane protein antibodies:

Phage Display Library Screening
The technology has been used for screening phage libraries. Basically, a large library of human phage antibodies is exposed to mammalian cells displaying the membrane proteins. Inert phage displayed antibodies are washed away, while positive antibodies are screened against cells expressing the target proteins. Positive clones are screened by testing antibodies with flow cytometry.

Mouse / Rat Hybridoma Production
The technology has also been used for hybridoma production. Commercially available cell lines (e.g., HEK293, CHOK1, NIH3T3, Sf9) are stably over expressed with membrane proteins. The cells are then used to immunize mice/rats. The immune spleen cells are fused with mouse myeloma cells to generate hybridoma cells. Hybridomas are screened by testing antibodies in the culture supernatants with flow cytometry.




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