Primary Antibodies

An antibody is a "Y-shaped" glycoprotein, produced by the body's immune system in response to invasion by foreign molecules, that is capable of binding to specific antigens. Each antibody is composed of four polypeptide chains, two identical heavy chains and two identical light chains, which vary in sequence and length. An antibody can be broken down into two F(ab) regions, the top sections of the "Y" which contain the variable region which binds specifically to a particular epitope on the antigen, and an Fc region, the bottom of the "Y" which provides a binding site for endogenous Fc receptors (and secondary antibodies).

Figure 1: Graphical representation of an antibody.

Monoclonal antibodies are produced by injecting an antigen into a host animal in order to initiate a humoral immune response, and by subsequently extracting the antibody-producing spleen cells and fusing them in vitro with cultured malignant myeloma cells to create immortal hybridoma cell lines. This provides a stable, long-term supply of monoclonal antibodies. Monoclonal antibodies are identical, recognizing a single epitope, and are derived from a group of identical cloned cells. A clone ID is given to each clone of hybridoma cells which identifies the monoclonal antibody it produces.

Polyclonal antibodies are produced by injecting an antigen into a host animal in order to initiate a humoral immune response. After the initial immunisation, the host animal will receive a secondary, and potentially a tertiary, immunisation in order to produce higher concentrations of antibodies against the particular antigen. Polyclonal antibodies are a collection of multiple different antibodies, derived from the immune response of multiple B-cells, which each recognize a different epitope on the same antigen.

Monoclonal vs Polyclonal Antibodies

In mammals, antibodies are classified into five main classes or isotypes according to the heavy chain they contain. These are: IgA (alpha), IgD (delta), IgE (epsilon), IgG (gamma), and IgM (mu). Each class differs in: the sequence of constant domains, the number of constant domains, the hinge structure, and the valency of the antibody.

The light chains of an antibody are classified as either kappa or lambda based on their polypeptide sequence. Typically, only one type of light chain is present in an individual antibody and, as such, the two light chains are identical.

IgG (consisting of IgG1, IgG2a, IgG2b, IgG3, and IgG4) is the most abundant antibody in normal human serum, accounting for 70-85% of the total immunoglobulin pool. IgG is involved in placental transfer, with the mother’s IgG transferring to the foetus in order to provide immune protection during the initial stages of development.

Figure 2: Graphical representation of IgG.

Each oval represents a protein domain in the antibody. These are systematically labelled by their chain type (L = Light chain or H = Heavy chain) and whether their sequence changes to bind an antigen (V = Variable domain or C = Constant domain), with CH domains numbered from the variable region. The domains are joined by linker regions of protein and disulfide bonds, represented by lines.

The antibody's structure is also described in terms of the fragments made by protein degradation or enzymatic cleavage of linker regions, namely the antigen-binding fragments (Fab), the variable fragments (Fv), and the constant fragment (Fc).

IgA (consisting of IgA1 and IgA2) accounts for around 5-15% of the antibody pool, existing either as a monomer or a dimer. IgA is the predominant antibody in mucous secretions, such as saliva, tears, and milk.

Figure 3: Graphical representation of IgA.

IgD only accounts for approximately 1% of the total plasma immunoglobulin, however, it is found in large quantities on the membrane of B-cells. IgD has the same basic structure as IgG but has an extended hinge region, which is susceptible to proteolytic digestion. IgD is thought to serve as an antigen receptor for the activation of B cells.

Figure 4: Graphical representation of IgD.

IgE only accounts for approximately 0.002% of serum antibodies and is found on the basophils and mast-cells. Similar to IgM, IgE has two additional constant domains instead of a hinge region. IgE is thought to play a role in immunity to parasites but is more commonly associated with type I immediate hypersensitivity, responding to innocuous environmental antigens such as pollen and peanuts, which can result in extreme reactions such as anaphylaxis or allergic rhinoconjunctivitis.

Figure 5: Graphical representation of IgE.

IgM accounts for between 5-10% of the immunoglobulin population and is the predominant antibody in the body's primary immune response. It is often represented as a pentamer, with a five-chain structure held together by a J chain, however, it can also exist in a hexameric form, without the J chain, and as a monomer on the surface of B-cells. The large size of soluble IgM (~ 900kDa) mainly confines this antibody to the intravascular pool.

Figure 6: Graphical representation of IgM.

Each clone number represents a specific cell line cloned from ascites that was used to manufacture the antibody. Since monoclonal antibodies are produced by more than one host, and more than one cell line, each cloned cell line receives a unique clone number to identify it.

Secondary antibodies should be raised against the host species of the primary antibody you are using. For example, if your primary antibody is a rat monoclonal you will require an anti-rat secondary antibody. We recommend checking the datasheet of the secondary antibody to ensure that it has been validated in the application you will be using.

Isotype controls are used to confirm that the binding of the primary antibody is specific and not a result of other protein interactions or non-specific Fc receptor binding.

The isotype control antibody should match the primary antibodies host species, isotype, and conjugation. For example, if the primary antibody is a HRP-conjugated rat IgG1, then you will need a HRP-conjugated rat IgG1 isotype control.

We recommend checking the datasheet first, which will often have a suggested positive control. It is important to ensure that the tissue or cell line used is from a tested species.

If no positive controls are suggested, we recommend looking at the UniProt website. This database often has a list of tissues that the protein is expressed in. These tissues can be considered suitable positive controls.

Many of our antibodies have dilution instructions on their datasheets. In these instances, we recommend following these instructions. For antibodies that do not have recommend dilutions, we recommend using the chart below to decide a starting dilution.

Most unpurified antibodies (i.e. whole antiserum, culture supernatant, or ascites fluid) will not have a concentration stated on their datasheets, as it will not have been determined. These antibodies can vary significantly in specific antibody concentrations. As rough concentration estimates: tissue culture supernatants are 1-3 mg/ml, ascites are 5-10 mg/ml, and whole antisera are 1-10 mg/ml.

It is important to remember that the dilutions / concentrations in the chart above are recommended simply as a starting point. It may be necessary to adjust the dilution / concentration based on experimental results.

We recommend always storing the antibody as directed on the datasheet. We are unable to guarantee how the antibody will perform if it is stored under different conditions.

The size of the aliquots will depend on how much one typically uses in an experiment. The aliquots should be no smaller than 10µl. The smaller the aliquot is, the more the concentration is affected by evaporation and adsorption of the antibody onto the surface of the vial.