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Antibody Service FAQ

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  1. What is single domain antibody and its special features?
  2. How can I get human origin antibody?
  3. How can I get my antibody modified for specific purpose?



1. What is single domain antibody and its special features?[Top]

Single domain antibody (sdAb), also named domain antibody, is antibody fragment that only contains single variable domain of the whole antibody. The first sdAb is derived from the antibodies of camelids, which is a dimer of two heavy chains, devoid of light chains. This type of antibody was also found in cartilaginous fishes, such as sharks. Up to date, the majority of generated sdAbs are engineered from camelids or cartilaginous fishes, called VHH. Besides, sdAbs from the light chains of common IgG are also proven to be active in antigen binding.

Although sdAb is much smaller (usually 12-15KD) than other forms of antibodies, such as Fab and scFv, it has all the required elements for antigen binding. For this reason, sdAb has many advantages including 1) high thermostability and resistance to denaturant (urea), protease and the low pH environment in digestive tract; 2) high tissue penetration, can cross brain blood barrier; 3) more soluble in water; 4) recognition of small epitope deep in molecular that cannot be bound by other forms of antibodies; 5) easily tracking the target in living cells/tissues; 6) high production yield. These merits of sdAb render it with great potential in biochemistry research and development of novel diagnostic and therapeutic approaches.   

2. How can I get human origin antibody?[Top]

In clinical application, antibodies must be human origin to avoid the trigger of immune response. Obviously, it is not practical to immunize human with interested antigens due to ethical restrictions. To address this problem, four alternative approaches have been developed, including

(1) Human antibody gene (fragment) pool is acquired from B cells in the blood of healthy donors by PCR amplification with universal primers on conservative sequences. Subsequently, phage display or yeast display is performed to sort out the antibody with the highest affinity to the antigen, followed by antibody gene sequencing. It is noteworthy that this antibody gene is from the undifferentiated B cells, so the sorted antibodies needs to undergo further artificial affinity maturation procedure in vitro by introducing random mutations to the ‘naïve’ antibody. Alternatively, the antibody gene pool can be obtained by in vitro gene synthesis of the randomly combined VDJ sequences in human genome.

(2) The second approach is named CDR (complementarity determining region) grafting. The basic concept of this method is dependent on the fact that the specificity and affinity of antibodies to antigens are largely determined by the CDR sequences. Accordingly, CDRs of a fully developed non-human origin antibody are grafted into human antibody backbone without CDRs. In order to further minimize the immunogenicity of these chimeric antibodies, some relative conservative sequences within CDRs can be replaced by the corresponding human sequences. Following, this chimeric antibody will be displayed on the mammalian cells as the whole IgG, and more mutations are introduced into framework regions after computer aided protein structure modeling. Finally, the antibody with desirable properties is selected by FACS.

(3) Due to the high sequence homology of non-human primate (NHP), such as monkey, to human beings, the antibody developed from NHP is very convenient to be germline humanized with several mutations predicted by computer modeling within the frameworks domain.

(4) The transgenic mice with human antibody gene loci substituting for the original mice loci are able to generate the repertoire of human antibodies. By immunizing this kind of transgenic mice, fully human origin antibody can be harvested.

3. How can I get my antibody modified for specific purpose? [Top]

The antibodies already in your hands can undergo further modifications to acquire new features for different purposes.

(1) Antibodies can undergo affinity maturation to further increase the specificity and affinity or obtain the appropriate binding properties. The maturation is achieved by introducing randomly or computer aided selected mutations into variable domain of antibody and the mutants are selected with yeast display or mammalian cells display technology.

(2) Based on the understanding of proteins structure and the prediction of 3D structure of antibodies, mutations can be introduced to existed antibodies to enhance their stability to high temperature or low pH environment. Besides, the structure of antibodies with increased stability from other species, such as cartilaginous fish, also provides valuable clues in antibody improvement.

(3) Antibodies can be altered to other animals’ origin antibodies without changing the affinity and specificity to antigens. For instances, humanization of non-human origin antibodies for clinical applications, murinization of human-origin antibodies to test their features in mice for the instructions of clinical therapeutics, and caninization of non-dog origin antibodies for treatment of diseases of dogs.

(4) Antibodies can be labeled with extra chemical groups to acquire new features. For example, antibodies labeled/conjugated with small molecular therapeutic medicines can target specific cell type without affect normal cells. Antibodies labeled with biotin can be easily detected or recruited by avidin or streptavidin. Fab, the fragment of antibodies without Fc, can be PEGylation to increase its Bioavailability and stability in blood. Antibodies can be attached to gold particles for the further application in electron microscopy, nanotechnology, and materials science. 

(5) Some artificial antibodies that do not exist in nature can be widely used in research and clinics due to their unique properties, for instances, bi-specific antibody, single domain antibody (sdAb) and single chain variable fragment (scFv). (please refer to question 4 in Antibody&Antigen FAQ for more information)


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