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Antibody, Y shaped protein complex belonging to immunoglobulin (Ig) superfamily, exists in many kinds of organisms, especially higher animals. Specific antibody can bind specifically to particular exogenous pathogen like virus or bacteria to either directly neutralize the pathogens by blocking its active sites or facilitate the elimination of pathogens by other immune cells, such as macrophages. Antibody is produced by B lymphocytes (plasma cells). B cells initially express antibodies on the cell membrane as B cell receptors (BCR). When BCRs are coupled with desired pathogens, B cells are differentiated into plasma cells, producing a myriad of antibodies. Part of plasma cells transform into memory cells which respond relatively fast to the same substance upon later infection.
Most antibodies are Y shaped and comprises four subunits, two heavy chains and two light chains. Each light chain and a moiety of each heavy chain pairs together via one disulfide bond to form the upper branch of ‘Y’, also named as Fab (antigen-binding fragment). The rest part of two heavy chain form the root of ‘Y’ via two disulfide bonds, also named Fc. Collectively, one antibody has two Fabs and one Fc. The top part (N termini) of Fab are the variable domains of light chain (VL) and heavy chain (VH) with the antigen-binding site on the tip, and the rest part, involving some part of Fab and the whole part of Fc, is the more constant domain (CL and CH). Accordingly, Fab decides the specificity and affinity of antibody to antigen while Fc determines the locations of antibody.
2. How many kinds of native antibodies exist in vivo? [Top]
In placental mammals, five different types of heavy chain (α, δ, ε, γ, and μ) divide antibody into five isotypes, including IgA, IgD, IgE, IgG and IgM.
|monomer tetramer||Most abundant Ig, distributing in mucosal areas, also found in saliva, tears, and milk||
Preventing pathogens invading into tissues from epiderm
|IgD||N/A||monomer||On naïve B cell membrane||Activating basophils and mast cells to produce antimicrobial factors|
|IgE||N/A||monomer||N/A||Interacting with allergens, facilitating histamine releasing from mast cells and basophils, preventing worms infection|
|IgG||IgG1,IgG2a, IgG2b IgG3, IgG4||monomer||Serum and the only antibody found in fetus||The major antibody fighting against pathogens|
|Monomer On B cell membrane and pentamer secreted in serum||Provide early elimination of pathogens before the generation of sufficient IgG|
It is surprising that nearly all the pathogens or microbial can trigger the immuno-response in human/mammals, indicating the ability of organisms to generate a pool with a myriad of antibodies. Indeed, it was estimated that human body is able to produce 10 billions different kinds of antibodies. Complex mechanisms are employed by human/mammals to achieve the diversification of antibodies with limited genomic size.
The gene loci responsible for coding the heavy chain or light chain includes the gene region for variable domains and constant domains. The variable domain gene region is constituted by variable (V), diversity (D) and joining (J) segments for heavy chain, and only V and J segments for light chain gene. V, D or J segments comprises multiple copies of tandemly arranged V, D or J genes flanked by conserved Recombination Signal Sequences (RSS) which can be recognized by V(D)J recombinase and subsequently processed by other recombinational factors to randomly delete spare V, D and J genes. By this manner, randomly selected one V, one D and one J (only V and J for light chain) genes are linked together to form the variable gene region for transcription. Given that human has 44 V gene copies, 27 D gene copies and 6 J gene copies, antibodies pool with imaginable diversity can be produced. This process is called V(D)J recombination.
After V(D)J recombination, when B cells expressing naïve antibodies on the cell membrane encounter the antigen, the variable domain genes undergo a process called somatic hypermutation (SHM), which also require helper T cells. The SHM is executed by the AID enzyme and the machinery of DNA mismatch repair (MMR) which recruits error-prone DNA polymerase to re-synthesis certain part of variable genes. It was estimated that one nucleotide mutation will be introduced into one variable gene upon one round of cell division. By this manner, the naïve antibodies acquire new features to be slightly different in antigen binding affinity. The B cells with ‘novel’ antibodies that acquire higher affinity to specific antigen then possess survival advantage over the B cells with naïve antibody and antibody acquiring lower affinity. This process is also called affinity maturation, through which the B cells expressing specific antibody with high affinity to certain pathogen are maintained and amplified.
When the B cells are activated and matured by encountering antigens, the constant gene region of antibody is rearranged to obtain isotypes other than the original IgM and IgD on the naïve B cells to orient antibodies to perform multiple functions in immuno response. This rearrangement process is accomplished by class switching recombination (CSR). Randomly selected two nucleotide motifs (switch regions) locating between constant regions genes are recognized and processed by several enzymes to generate two DNA double strand breaks at switch regions. The gene region between these two switch regions is then removed and the rest part is re-linked by a process called Nonhomologous end joining. By this manner, different isotypes are produced.4. Is there any artificial antibody and what are their applications? [Top]
With the development of biomedical technologies, apart from the in vivo naturally occurred antibodies, a series of artificial antibodies has been introduced and applied in research and clinics.
|Antibodies||Structure||Features and application|
|Single domain antibody (sdAb)||(single VH or VL domain)||
Small, highly accessible to hard-reaching epitopes; efficient tissue penetrating and brain blood barrier crossing; high resistance to temperature and chemical environment changes; high selectivity than other small molecular medicines.
Good candidates for brain medicines and discovery for oral delivery medicines.
|Bispecific antibody (BsAb)||(trifunctional antibodies, two different Fabs linking, Bispecific T cell engager)||
Containing two or three paratopes;
delivering cytotoxic T cells or macrophages to target cells; applied for cancer, bacteria or virus infection therapy
|Single-chain variable fragment (scFv)||(linked VH and VL)||Easily produced and expressed on cell membrane, so good candidates for surface display systems; also used in immunohistochemistry and chimeric antigen T cells (CART) for cancer, bacteria or virus infection therapy|
|Fab-scFv Fusion (Tribody)||(linked VH and VL)||Two different scFv are co-expressed with each chain of Fab domain respectively; middle size between IgG and bispecific antibodies; balanced tissue penetration efficiency and retention time, so high tissue accumulation.|
|Single chain antigen-binding fragment without cysteins (scFabΔC)||Linked Fab without disulfide bond||
High stability and affinity compared with scFv;
The soluble form can be easily produced by E.coli; allowed for phage display and detection by common antisera reagents.
Due to their binding specificity to pathogens/antigens, antibodies are widely used in the biochemical research.
|Western blotting||Detecting the existence of specific protein/peptide in tissue lysates.|
|Immunocytochemistry||Detecting the existence of specific protein/peptide in cells, on the cell membrane or in extracellular matrix, detecting protein-protein interactions by Proximity ligation assay or co-localization assays|
|Immunohistochemistry||Detecting the existence of specific protein/peptide in the tissue sections.|
|Immunoprecipitation||Examining protein-protein or protein-chromatin interactions in cells/tissues, including chromatin immunoprecipitation and RNA immunoprecipitation.|
|ELISA||ELISA (enzyme-linked immunosorbent assay) to quantify the concentration of certain antigen in solutions, or to qualify the antibody affinity to given antigen|
|Flow cytometry||Calculating or sorting out the cell population with specific proteins/DNA|
|EMSA||EMSA (electrophoretic mobility shift assay) to detect the interactions between DNA and protein.|
At present, with the development of precise/personalized medicine, the demands of antibody in disease diagnostics and treatment keep increasing. In the field of disease diagnostics, antibody is helping clinicians to determine the existence of specific biomarkers in tissues or blood through in vivo or in vitro examinations. In the field of disease treatment, especially cancer and autoimmune disease (e.g. Celiac disease), antibody is taking advantage of its ability to block the bio-activity of targets or label tumor cells. For example, some tumor cells are good at avoiding the detection by immune system, and specific antibody can label these cells for immune system. Moreover, antibodies are able to affect the survival and growth of cancer cells by blocking the growth factor receptors that for cancer self-growth or blood vessels’ growth (angiogenesis). Furthermore, artificial bispecific antibody and chimeric antigen receptor T (CAR-T) cells can guide cytotoxic T cells to specific cancer cells. Besides, antibodies conjugated with radioactive substances or chemicals are also proven a useful tool for precise cancer treatment. In the tissue transplantation, antibodies are successfully used to alleviate the rejection reaction.
Compared to the conventional chemotherapy, antibody based treatment possesses apparent advantages in minimizing side effect and enhancing the efficacious. Nevertheless, antibody therapy inevitably has some limitations. Two major shortcomings are 1) cancer cells may evolve to harbor resistance to the antibody through mutating the antibody-target receptors or exploring alternative pathway for blood vessels formation or uncontrolled growth; 2) It is, sometimes, challenging in practical to generate the suitable antibody for specific target.7. What is the difference between the applications of polyclonal antibody and monoclonal antibody?[Top]
Briefly, the polyclonal antibody (PAb) indicates that antibodies are heterogeneity in a collection, thus recognizing different epitopes of the antigen. In contrast, the monoclonal antibody (MAb) means all the antibodies are identical to each other in a collection, thus binding to one specific epitope of the antigen.
|Production||Manipulation||Affinity to Target||Modification||Cross Reactivity|
|PAb||quick & cheap||Easy to store, tolerant to PH and ion changes in buffer||Overall high affinity to antigen, resistant to conformational changes of target protein||Easy to be labeled without changes in binding features||high|
|MAb||slow & expensive||Sensitive to storage conditions||Relative low affinity and sensitive to conformational changes of targets||Can be label but may affect the binding capacity to targets||low|
Apart from the differences listed above, MAb is easier to reproduce with the already obtained hybridoma from the intial production process, while immunization of animals is required every time producing PAb. Because MAb only recognize one epitope of antigen, so it is always utilized to quantify the antigen concentration. Of note, due to its low cross activity, MAb is much more popular in clinical applications than PAb.8. What is antigen?[Top]
Basically, antigen is a group of substances that can be recognized by host immune system (B cells, T cells or antibodies). Of note, not all the antigens can trigger host immune response, which means antigens are not necessary to be immunogens. One important criteria for antigens to be immunogenicity is its molecular weight should be more than 10,000 Da. Typically, antigens can be categorized into exogenous antigens, endogenous antigens, autoantigens and tumor antigens based on its sources, and into proteins, polysaccharides and lipids based on its chemical properties.9. What kind of antigen is suitable for immunization to generate antibody?[Top]
Basically, all the antigens that can trigger immune response can be used to generate antibodies. For generation of antibodies for clinical or research applications, antigens should meet two primary requirements 1) the molecular size should be more than 10,000 Da; 2) the purity is suggested to be more than 90%. Different types of substances has been used in antibody generation, including hatpens (12-25 amino acids small peptides) linked to bigger helper peptides, recombinant proteins, native proteins, bacteria, virus and even naked plasmids which can directly express antigen proteins in vivo.
|Native proteins||Recombinant protein||haptens|
|Antibody generation success rate||80%-95%||60%-80%||30%-60%|
|Suitable to be ligand||Best||Good||Fair|
A brief comparison of Native proteins, recombinant proteins and haptens as antigens10. How does antibody bind to its target antigen?[Top]
The antibody binds to antigen through the interaction between the antigen-binding site on the antibody and the epitope on the antigen. The antigen binding site, also called paratope, is a small region (typically 15 to 22 amino acids) in the variable domain of the light chain or heavy chain. The antigen binding site involves a set of complementarity determining regions (CDRs), also called hypervariable region. Every light chain and heavy chain has three CDRs (CDR1, CDR2 and CDR3, of which CDR3 is the most variable) flanked and sterically supported by four framework regions. The affinity of antibody to specific antigen is determined by the level of complementation (like lock and key) and the force strength of non-covalent bonds between paratope and epitope. So the interaction between antibody and antigen is relative weak compared to the covalent force between atoms in one molecule. Thus, this interaction is reversible in most cases.11. How to determine the affinity of certain antibody to antigen?[Top]
The interaction between antigens and antibodies is largely reversible, so the reaction can be simplified as:
[Ab], [Ag] and [Ab-Ag] describe the concentration of the antibody, antigen and the antibody-antigen complex in a given reaction, respectively. When the reaction reaches equilibrium or steady state, the antibody antigen association speed equals to antibody-antigen complex dissociation speed. So at this stage, the ratio between [Ab-Ag] and [Ab][Ag] (Ka) can indirectly reflect the antibody affinity to the specific antigen. Usually, high affinity antibodies have the Ka>107/M.
Several methods have been employed for calculating the Ka. First one is called equilibrium dialysis. Briefly, antibody solution filling in a dialysis bag is dialyzed against the solution of radioactive labeled antigen. The principle is that antibodies in the dialysis bag cannot travel out but the antigen can travel between both sides of the bag. When the dialysis reaction reaches balance, the radioactive level in the bag reflects the antibody-binding antigens and the free antigens while the level outside the bag reflects the free antigens. Given that the concentrations of free antigens inside and outside the bag should be same, the concentration of antibody-antigen complex can be calculated. This method is commonly used when the antigen is haptens or small molecules.
Alternative approach to measure the affinity of antibody is fluorescence quenching. Briefly, proteins/antibodies may contain tyrosine and tryptophan emitting fluorescence excited by ultraviolet. When non-fluorescent quenchers/antigens bind to fluorescent antibody, they may form a complex with less fluorescence. In this manner, through measuring the fluorescence signal change, the affinity of antibody for antigen can be measured.
Nevertheless, both of the two methods above have limitations in the property of antigens. In equilibrium dialysis, antigen should be small for diffusing through the bag, and in fluorescence quenching, antigens must have unique fluorescent features. To address these limitations, alternative technology called surface plasmon resonance (SPR) has been developed. Briefly, antibodies/antigens are immortalized on a layer of dextran and antigens/antibodies is allowed to flow on the layer. The SPR detector is so sensitive that any change in the amount of substances on the layer can be recorded, so the antibody-antigen binding are measured in real time. In this manner, antibody-antigen binding kinetics can be calculated.
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