Primary and secondary antibodies
Encyclopedia
Primary and secondary antibodies are two groups of antibodies based on whether they target a target of interest directly or target another (primary) antibody that, in turn, is bound to a target of interest.
, peptide
, carbohydrate
, or other small molecule
) and are typically unconjugated (unlabelled). Primary antibodies that recognize and bind with high affinity and specificity to unique epitopes across a broad spectrum of biomolecules are available as high specificity monoclonal antibodies and/or as polyclonal antibodies. These antibodies are useful not only to detect specific biomolecules but also to measure changes in their level and specificity of modification by processes such as phosphorylation, methylation, or glycosylation. A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of ADME and multi-drug resistance (MDR) of therapeutic agents.
that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification
or cell sorting
applications.
Secondary antibodies may be polyclonal or monoclonal
, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc
or Fab regions
.
Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. Identifying the optimal secondary antibody is normally done through trial and error.
Primary
Primary antibodies are antibodies raised against an antigenic target of interest (a proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
, peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
, carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
, or other small molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
) and are typically unconjugated (unlabelled). Primary antibodies that recognize and bind with high affinity and specificity to unique epitopes across a broad spectrum of biomolecules are available as high specificity monoclonal antibodies and/or as polyclonal antibodies. These antibodies are useful not only to detect specific biomolecules but also to measure changes in their level and specificity of modification by processes such as phosphorylation, methylation, or glycosylation. A primary antibody can be very useful for the detection of biomarkers for diseases such as cancer, diabetes, Parkinson’s and Alzheimer’s disease and they are used for the study of ADME and multi-drug resistance (MDR) of therapeutic agents.
Secondary
A secondary antibody is an antibodyAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification
Purification
Purification is the process of rendering something pure, i.e. clean of foreign elements and/or pollution, and may refer to:* List of purification methods in chemistry* Water purification** Organisms used in water purification...
or cell sorting
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
applications.
Secondary antibodies may be polyclonal or monoclonal
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc
Fragment crystallizable region
The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system...
or Fab regions
Fragment antigen binding
The fragment antigen-binding is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope — the antigen-binding site — at the amino terminal end of the monomer...
.
Specific secondary antibodies are usually chosen to work in specific laboratory applications. Frequently, any one of several secondary antibodies perform adequately in a particular application. They are selected according to the source of the primary antibody, the class of the primary antibody (e.g., IgG or IgM), and the kind of label which is preferred. Identifying the optimal secondary antibody is normally done through trial and error.
Applications
Secondary antibodies are used in many biochemical assays including:- ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
, including many HIV testHIV testHIV tests are used to detect the presence of the human immunodeficiency virus , the virus that causes acquired immunodeficiency syndrome , in serum, saliva, or urine. Such tests may detect antibodies, antigens, or RNA.- Terminology :...
s - Western blotWestern blotThe western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
- ImmunostainingImmunostainingImmunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...
- ImmunohistochemistryImmunohistochemistryImmunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...
- ImmunocytochemistryImmunocytochemistryImmunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...