What Is A Secondary Antibody?

The experimental usage of secondary antibodies (2° Ab) is to bind primary antibodies (1° Ab). With enzymatic, cross-linking or light-emitting labels on them, the binding effect of primary antibodies can be amplified and converted into many sorts of signals, then being processed by instruments for various purposes. In most cases, primary antibodies are supplied as unlabeled monoclonal and sometimes as biotinylated to work with avidin/streptavidin which play the role of secondary antibodies. On contrary, secondary antibodies are usually polyclonal and chemically conjugated with functional labels. Therefore, applications of antibody reagents are diversified and deepened by a suitable pairing of primary and secondary ones.

Host species is the animal species in whose bodies we inject primary antibodies generate secondary antibodies. The host species chosen to generate a batch of secondary antibody should be distinct from that is used for raising the primary antibody. For example, if the primary antibody is raised in mice, to appropriately recognize murine antibodies, rabbits or goats are the recommended host species.

Citation:Janeway's Immunobiology, 9th Ed Chapter 5: The Generation of Lymphocyte Antigen Receptors

An Immunoglobulin (Ig, a scientific term of antibody) is a heterotetrameric globulin molecule which consists of two identical heterodimers with a heavy chain (HC) and a light chain (LC) connected by disulfide bonds. The HC or LC of an antibody can be divided into variable (V) and constant (C) regions, which are generated by different molecular mechanisms. The antibody V region, collectively the heavy-chain variable region (VH) domain and the light-chain variable domain (VL), is responsible for antigen binding, whereas the antibody C region, assembled by the CH and CL, may mediate immunological effector functions as well as provide the V region essential support in structure.

The reactivity description of a secondary antibody product is usually toward which host species and class, more specifically the subclass, of the primary antibody. Properly matching these two property ends is the key to choose a useful secondary antibody. In common mammals, there are five HC classes (aka. isotypes): IgM, IgD, IgG, IgE, and IgA, and two LCs classes: κ and λ. In addition, subclass is another issue to be addressed. For example, human has four IgG subclasses: IgG1, IgG2, IgG3 and IgG4, while those of the BALB/c mouse are IgG1, IgG2a, IgG2b and IgG3. These classes and subclasses originate from a cluster of gene suites on separate chromosome loci.

Immunoglobulins are highly conserved among different species. Therefore, it is possible that one anti-IgG antibody might cross-react to IgG of multiple species. In order to enhance the species specificity, secondary antibody can be pre-absorbed by other species-derived immunoglobulin- or protein-immobilized resin to reduce cross-reactivity. Pre-absorption of secondary antibody is an essential procedure for multi-color staining experiments or tissue immunostaining.

Secondary antibody can be labeled with various conjugates, such as enzymes (HRP or AP), fluorescein (FITC, PE, FAM, TAMRA, TRITC) or biotin. The label of secondary antibody is based on its application. For ELISA, dot blot or western blotting, enzyme-labeled secondary antibody is the most common-used secondary antibody. For flow cytometry and indirect Immunofluorescence Assay (IFA), it’s common to use fluorescein-labeled secondary antibody. According to laser and filter of equipment, the best-fitted fluorescein for experiments should be chosen. Leadgene provides fluorescein dye with excitation wavelength ranging from 405, 450, 488, 594 to 680 nm.

Secondary antibodies are contained in sera collected from immunized animals and then purified by affinity chromatography columns that are packed with of Protein A/G resin beads. In cases of customers’ requests, preliminarily purified antibodies will be further refined by absorbing non-specific and cross-reactive binders, enriched by the immunizing antigen targets, or quality-enhancing by other specified procedures.