Primary antibodies are developed to detect a specific target antigen such as a protein, peptide, carbohydrate, nucleic acid or small molecule. They can also be designed to identify post-translational modifications which include phosphorylation, acetylation, glycosylation or methylation, making them ideal reagents to study complex processes such as protein activation or silencing.
Typically created by immunising a host species such as a rabbit, mouse or rat, primary antibodies are categorised as either monoclonal or polyclonal. They may also be produced recombinantly using bacterial systems or mammalian cell lines such as Chinese Hamster Ovary (CHO) cells. Recombinant technology provides the capacity to engineer in desirable antibody features such as improved sensitivity or enhanced thermostability.
Primary antibodies can be selected for research studies according to properties such as antigenic target, host species, clonality, isotype, subtype, species reactivity, tested applications, conjugate availability, and more. All these factors should be considered carefully to ensure that meaningful, high-quality data is achieved.
While primary antibodies are designed to recognise a specific target antigen, secondary antibodies are developed to identify a primary antibody according to the host species in which it was raised. For this reason, secondary antibodies are usually referred to as being anti-mouse, anti-rabbit, or anti-whichever species they are intended to detect.
To demonstrate successful binding of the primary antibody to its target, secondary antibodies are generally conjugated to a detection moiety such as an enzyme, fluorescent dye, biotin or streptavidin. Commonly-used enzymes include horseradish peroxidase (HRP), alkaline phosphatase (AP) and glucose oxidase, which are used to catalyse the conversion of a substrate to generate a measurable readout.
Secondary antibodies which are conjugated to fluorescent dyes afford the opportunity for multiplexing, since they can be used to detect primary antibodies from different host species within the same experiment, provided spectral overlap is avoided. The biotin-streptavidin interaction can be exploited in many ways, for example using a layered approach to boost signal.
In vivo antibodies
In vivo-grade antibodies are typically intended for use within animal models, where they may be employed to study the effects of neutralization, blocking and activation/proliferation, or to image specific cells or tissues. To avoid adverse effects, they should be of high purity; free of endotoxin, pathogens, preservative, stabilizer, and carrier protein; and should not aggregate.
For downstream antibody activity to be realised, it is also important that in vivo-grade antibodies exhibit the desired functionality following introduction to the model species. Recombinant antibodies can be advantageous for in vivo work, for example allowing species or subtype to be switched, extending the serum half-life, or facilitating deeper penetration into tissues for in vivo imaging. Recombinant bispecific antibodies can be used to recruit effector cells or cytotoxic agents to a specific in vivo target.
Monoclonal and polyclonal antibodies
Monoclonal antibody preparations are composed of identical immunoglobulins, all of which bind the same epitope on a target antigen. They are traditionally produced by fusing the spleen cells of an immunised host, usually a mouse, with immortal myeloma cells. By culturing the resultant cell pool in defined media and screening clones for antigen-specificity, antibody-producing hybridomas can be isolated and expanded. These may be cultured in vitro indefinitely, affording a consistent antibody supply.
Since monoclonal antibodies have restricted epitope recognition, they mostly demonstrate minimal cross-reactivity with other proteins. They also deliver reproducible performance between experiments and between antibody batches, provided experimental conditions remain constant.
Polyclonal antibodies consist of a heterogeneous pool of immunoglobulins, which are directed against multiple antigenic epitopes. They are secreted by different B-cell lineages and are extracted directly from the immunised host’s serum by methods such as Protein A/G purification or antigen-specific affinity purification. Since animal hosts have only a finite lifetime, an indefinite supply of polyclonal antibody cannot be guaranteed.
Due to their heterogeneous nature, polyclonal antibodies may demonstrate greater cross-reactivity than monoclonals. This increases their tolerance to antigenic changes and makes them more likely to detect across a range of species, however it can also result in increased background staining. Polyclonal performance often varies between batches, making lot-to-lot optimisation necessary.
Secondary antibodies are generally conjugated, since the presence of a detection moiety is required to confirm primary antibody binding to an antigenic target. Many primary antibodies are also conjugated, allowing for shortened experimental timelines through removal of the need for a secondary antibody incubation step. Directly-conjugated primary antibodies are ideal to support multiplexing, since they permit several antibodies from a shared host species to be used simultaneously.
Widely-used conjugates include enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP) and glucose oxidase; fluorescent dyes and proteins; and biotin or streptavidin. The choice of conjugate will be dictated by the nature of the immunoassay, with enzyme conjugates often used for techniques such as Western blot and ELISA, and fluorescent conjugates typically employed for flow cytometry and fluorescence microscopy.
In situations where a conjugated antibody is not available, it is now possible to purchase kits to perform antibody conjugation in-house. Provided the antibody is at a suitable starting concentration and supplied in an appropriate buffer, conjugation to an enzyme, fluorescent dye, biotin or streptavidin can easily be achieved with minimal hands-on time.