Primary and secondary antibodies are largely utilized for the purpose of research. Counting on their binding ability and classification, antibodies are divided into two groups – primary antibody and secondary antibody. Whereas a primary antibody gets bonded with an antigen (proteins); a secondary antibody gets bonded with a primary antibody. When each epitope (a small part of an antigen) gets connected with the light chain area (Fab Zone) of a primary antibody, a secondary antibody gets connected with the heavy chain area (Fc Zone) of a primary antibody.
Production of Primary & Secondary Antibody
A particular host species is required to produce primary antibodies. Generally, rabbits, mouse, goats, and/or chickens are used to produce a primary antibody. The host gets immunized to produce primary antibodies. But, the production of secondary antibodies is completely different. Here, an antibody injection is injected into a host species (This host is totally different from the host that is used to produce the primary antibody). For an instance, if the primary antibody got produced from a rabbit, the secondary antibody would be produced from a goat, a mouse, or a chicken.
Note: A secondary antibody that is produced against a specific primary antibody (IgG) can easily recognize all fragments of the same type of IgG. Secondary antibodies can recognize both the light (Fab) and heavy (Fc) chains. Secondary antibodies are readily cheaper compared to primary antibodies and widely useful for different immunoassays.
Differences in Immunoassays for Primary & Secondary Antibodies
The binding of primary antibodies to antigens can be visualized by direct and/or indirect immunoassays.
· In a direct immunoassay, the primary antibody gets bonded to a signal epitope. A successful binding process activated the signal molecule and helps in easy detection.
· During indirect immunoassays, the secondary antibodies help detect primary antibodies in activating the signal molecules in a complex way.
Now it’s time to know how secondary antibodies produce visual signals.
The visual signal process of secondary antibodies is always an indirect immunoassay. Let us have a look at methods by which secondary antibodies produce signal molecules.
· Radioisotopes: Via this method, indirect detection of antigens is processed with radioisotopes. X-ray films are used to detect the rays.
· Enzyme: During the Western blotting process, a secondary antibody gets conjugated with an enzyme. Here, horseradish peroxide is used as an enzyme. After the completion of bonding between the secondary antibody and the enzyme, the enzyme splits the pale substrate to produce a completely visible colorful signal on a blot.
· Fluorochromes: FITC (fluorescein isothiocyanate), rhodamine, texas red, or R-phycoerythrin are used to label secondary antibodies during an immunoassay. At a definite wavelength, the bond of secondary antibody with antigen excites a fluor. A light gets emitted at a different wavelength.
Conclusion,
It is always advised to choose a secondary antibody that can easily match the class/subclass of the primary antibody. It helps to complete the immunoassay level successfully. A secondary antibody that is high in specificity and less in cross-reactivity is best to process the immunoassay. Primary and secondary antibodies should be produced from different hosts to have successful signal detection in an immunoassay.
