Antibody is a very often used diagnostic agent in our experiment ,if there are tested antibodies in our lab and can be used out of hand ,it’s a very lucky thing.However,if we want to make new test,faced with various antibodies,how can you single out your Ab.Right?Following,let me humble editor share with you some tricks.
Antibody,namely Immunoglobulin,Ig,is structured with two about 50kDa heavy chains and two 25kDa light chains being linked by hydrophobic bond and disulfide bond, identify epitope through N-terminal of these two kinds of chains. Epitope could be linear epitope formed by continuous amino acids and also could be conformational epitope that although has discontinuous amino acid sequence ,is with a similar space.
According to the disparities of the heavy chains ,antibody can be divided into different varieties and subvarieties.For example ,human antibody can be categorized as IgG,IgA,IgM,IgD,IgE five types.,and IgG further can be identified as IgG1-IgG4 four subvarieties The most often used antibodies are category IgG as well as a small part of IgM and others. Aside from identifying proteins, antibody can also distinguish polysaccharose , antibiotic and DNA
The simple method of choose a a antibody is by looking up documents to see if there’s same usage,another way is referring to the test data of antibody companies.In addition ,there are some other factors should be took into consideration .
1.The source of primary antibody: general experiments have no specific requirements of the species of primary antibody ,however, when in immunohistochemical experiments, if choose a primary antibody that’s from the same specie with the sample tissue,it will affect the outcome of the experiments because the secondary antibody identifies the immunoglobulin of the tissue, so for this kind of experiments you should choose a primary antibody from a different specie.Also,in Co-IP experiment ,because there’s anti protein in the sequent western blot, if the size of targeted protein is around 50kDa and of the same source of primary antibody, the heavy chain of primary antibody will disturb the outcome, so the primary antibody of the same specie should be avoided.
2.Secondery antibody:secondary antibody should be the antibody that anti immunoglobulin that’s of the same specie with primary antibody ,for example ,if the primary antibody is mouse IgG antibody,the secondary antibody ought to be anti mouse IgG antibody,if primary antibody is IgG1 subvarieties, secondary antibody can take it for target ,you can also use anti-IgG that’s effective with the four subvarieties. Based on the above ,we can confirm the antibody according to the needed marker such as enzyme and fluorophore.
3.Monoclonal antibody/polyclonal antibody/recombinant antibody
Monoclonal antibody is the antibody that identifies single epitope.At present ,the production of monoclonal antibody is by fusing B cell which secrets single antibody with myeloma cell to form hybridoma, and cultivating cell line.Compared to polyclonal antibody ,monoclonal antibody has higher specificity and less miscellaneous bands, is more stable in a period of time. But because of the singularity of epitope identified by monoclonal antibody ,if the epitope changes due to chemical process or it’s coverd by the combination of different proteins or protein and DNA,monoclonal antibody will not be able to function.
Polyclonal antibody is also called antiserum or serotype antibody, it’s produced by purifying the serum of immune animal, can identify multiple epitopes of antigens with a higher affinity than monoclonal antibody and spare you the concern of epitope coverage, further more,polyclonal antibody can exemplify the signal of low expression target protein. Although with all the advantages,polyclonal antibody sometimes can introduce relatively high background signal. When lack of reference material,antigen sequence of polyclonal antibody can be produced by Blast to confirm if there’s homologous protein that may interfere with the experiment,in addition,it’s likely to see disparity in different batch of the polyclonal antibody.
Although the monoclonal antibody produced by hybridoma cell line can maintain a stable antibody functionality in a period of time,our GLORY SCIENCE CO.,LTD scientists also take various measures to guarantee antibody quality, for example ,we would do restrict quality examination of each batch of antibody ,guarantee the preserved quantity of the low subcultured hybridoma in the liquid nitrogen,prevent the high subcultured ones from producing antibody, shorten the time of in vitro and animal in vivo culture.
Recombinant antibody:this is expressively produced through obtaining gene of monoclonal antibody of high affinity and specificity and establishing a special antibody expressive carrier.