Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
This page provides general guidelines and tips for making decisions about the kinds and types of custom antibodies that are best for the intended applications, available sources of antigen, desired scales of production, and overall budget.
The goal of custom antibody production is to acquire an antibody that performs well in a particular qualitative or quantitative immunodetection method. Thus, antibody performance in the intended application is the foremost consideration when choosing among antibody production options. Secondarily, both the available forms of antigen (protein vs. peptide) and types of production (polyclonal vs. monoclonal) must be considered with respect to the costs involved in producing the needed amount of antibody.
Polyclonal antibody production (PAb)
| Monoclonal antibody production (MAb)
| |
| Peptide antigen (P) |
|
|
| Recombinant protein antigen (R) |
|
|
Polyclonal antibodies represent the total population of immunoglobulins produced by an animal in response to an antigen. Crude serum usually contains several particular antibody clones against the injected antigen, as well as against other antigens to which the animal has been exposed in its environment. Polyclonal antibodies are nearly always the best choice for applications involving qualitative detection or purification, such as western blotting or immunoprecipitation. They are far less expensive to produce than monoclonal antibodies and they generally have higher affinity and broader utility across assay methods. A population of antibodies having greater specificity can be obtained by secondary purification (positive or negative) from the antiserum. The main disadvantages of polyclonal antibody production are that it yields only a limited amount of antibody (i.e., the duration of the immunization schedule), having variable consistency.
Several different host species can be used for polyclonal antibody production, including rabbit, guinea pig, rat, mouse, goat, and chicken. These provide the opportunity to design assays to take advantage of host-specific secondary antibodies. Larger animals yield more antiserum per bleed than smaller ones.
Becausemonoclonal antibody development yields cell lines that produce exactly one specific molecular form of antibody which can be maintained and cultured indefinitely, it is the best option for applications requiring consistent performance in quantitative detection, regardless of production batch. Screening protocols are essential to establishing antibody populations that function in the intended assay use. However, monoclonal antibodies are more expensive to develop and produce than polyclonal antibodies, and they are not as broadly applicable in different assay methods. For example, a monoclonal antibody produced for use in ELISA might not work at all for western blotting. As a general rule, choose monoclonal antibody development only when you:
For the purpose of antibody production, polypeptides are considered proteins if they are larger than nine kilodaltons (9 kDa) and do not need to be conjugated to a carrier protein to be made immunogenic. Nearly any purified protein (>90% pure) can be used as an antigen for antibody production. Gene-specific expression of recombinant proteins (or recombinant protein fragments) is often used to ensure that the purified protein antigen is precisely the one intended.
Protein antigens are generally best when the goal is to elicit production of as many different antibody clones to detect as many different possible epitopes on the target protein. The result is production by the animal host of a broad range of antibodies (a) that can be screened to select particular clones for different applications during monoclonal antibody development or (b) can be used as a polyclonal population (antiserum) to provide the broadest possible affinity and utility for multiple applications.
Given that whole proteins are more likely than peptides to present normal secondary and tertiary structure, they are more likely to elicit production of antibodies that bind certain epitopes that are present only in the native protein target, as is usually the case in ELISA and immunoprecipitation.
When they can be designed based on knowledge of the target protein structure and function (as with the Antigen Profiler System), peptide antigens offer the greatest control of antibody production for specificity and performance in particular assay applications. Peptides allow focused production of antibodies against point mutations or polymorphisms, post-translational modifications, and highly-conserved proteins (by specifically designing against the few variable regions).
Peptides for antibody production (usually 4 to 20 amino acids) are simple and affordable tosynthesize and conjugate in quantities sufficient for immunization. Peptides are too small by themselves to elicit an immune response, so they must be crosslinked to an immunogenic carrier protein (e.g., KLH or BSA) for immunization. Despite this added step, peptide synthesis and conjugation services are much less costly than services for developing, expressing, and purifying recombinant proteins from cDNA.
Because peptides represent specific epitopes based on primary sequence structure rather than whole protein secondary or tertiary structure, they elicit production of antibodies whose specificity is less likely to be dependent upon the target protein being in its native, biologically-active form. As such, anti-peptide antibodies are more likely to bind both native, fixed, and denatured targets for use in many different applications, including immunohistochemistry, western blotting, ELISA, and immunoprecipitation.
Peptides also provide the only practical way to obtainmodification-specific or monospecific antibodies, such as an antibody that binds only the phosphorylated form of the target protein. This is possible because both modified and unmodified forms of the peptide can be synthesized and then used to screen or purify the antibody with the desired specificity. Examples of monospecific conditions that require synthesis of matched peptide antigens for antibody production include: phosphorylation, acetylation, glycosylation, prenylation, myristoylation, ubiquitination, sumoylation, protein cleavage (neo-epitopes), ligand binding, drug binding, polymorphisms, mutations, splice variants, isoforms, species cross-reactivity, and highly conserved proteins.
Western blotting is the most common application for antibodies. Protein samples (usually cell lysates) are electrophoresed by denaturing SDS-PAGE (polyacrylamide gel electrophoresis), transferred onto a nitrocellulose or PVDF membrane, and then probed with antibody for detection. Because target proteins are denatured and linearized (devoid of most high-level structure), most epitopes of the primary sequence fully exposed for antibody binding.
Immunohistochemistry is the second most common application for antibodies. In this method, paraffin-embedded or frozen tissue sections are probed with antibody to detect endogenous protein. When samples (target protein epitopes) are denatured and crosslinked (fixed) with formalin, this non-native condition can be anticipated and mimicked by the conjugation method used to prepare peptide antigens.
ELISA is the assay of choice for quantitative assessment of target proteins or specific modification states of target proteins. In this method, target proteins are quantitatively captured from biological samples to microplate wells and then detected, most commonly using two different antibodies. Because ELISA samples are fresh cell extracts or biological fluids, use of antigens that retain and present the native tertiary structure of the target protein are extremely important for this application.
Immunoprecipitation has the same tertiary structural concerns as ELISA. The method involves capture and temporary immobilization to agarose beads of native proteins from fresh biological samples. However, the intent with immunoprecipitation is nearly always to capture the whole population of target protein. (Usually, assessment of specific forms or modification-states of target proteins is made in a subsequent detection step, such as western blotting). As such, monoclonal antibody production is usually not necessary.
Flow cytometry is one of the more difficult applications to optimize and test with custom antibody production. Large numbers of peptides have to be evaluated, or monoclonals must be screening using very rigid conditions. Researchers who need reagents for this application are asked to discuss the details with a technical expert before proceeding.