Choose the right secondary antibody, no longer tangled!

When you are choosing how to choose a secondary antibody, or if you don't know how to choose the most suitable secondary antibody, your eyes tell me that you are entangled, but this is not your fault. And after reading the following, you will not be entangled again, otherwise, it will be my fault.

1. Find out what the host species of the primary antibody is.

The secondary antibody points to the species of the primary antibody. If a primary antibody derived from rabbit is used, an anti-rabbit secondary antibody derived from a species other than rabbit is required. For example, a goat polyclonal anti-rabbit IgG secondary antibody can detect a rabbit polyclonal anti-Ki67 primary antibody. In other words, the host species of the secondary antibody is required to be different from the host species of the primary antibody, and the genetic relationship is as far as possible.

  2. Understand the type of primary antibody

The secondary antibody must point to the same type of primary antibody.
Polyclonal primary antibodies are usually derived from rabbits, goats, sheep or baboons and are of the same type as IgG. Secondary antibodies are typically antibodies against IgG heavy and light chains. Monoclonal primary antibodies are usually derived from mice, rabbits and rats. For example, if the monoclonal primary antibody is mouse IgG1, anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG is required.
Human immunoglobulins, subclasses, types and subtypes:

· Class or isotype: IgG (γ heavy chains), IgM (μ), IgA (α), IgE (ε), IgD (δ)

· Subclass: IgG1 (γ1 heavy chains), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4); IgA1 (α1), IgA2 (α2)

· Type: κ light chain, λ light chain

· Subtype: λ1, λ2, λ3, λ4
Other types of reactions:

· Multivalent antibodies react with all immunoglobulins

· Anti-Fc or heavy chain (α, δ, ε, γ, and μ) antibodies react only with heavy chains

· Anti-F(ab) or intact molecular antibodies react with heavy and light chains, but not with immunoglobulins

· Because all immunoglobulins use the same kappa and lambda light chains, anti-light chain (κ and λ) antibodies react with all immunoglobulins

  3. Do you need enzymatic or fluorescent detection?

The type of coupling is application dependent.
For enzymology and biotin detection, for example, in WB or ELISA, it is recommended to couple the secondary antibody to HRP, AP or biotin. Regardless of the antibody host species, avidin and streptavidin bind strongly to biotin and both amplify the signal. If a laser is used, for example, in flow cytometry, ICC/IF or IHC, it is recommended to couple the secondary antibody to a fluorescent dye for fluorescence detection.

4. Is it necessary to use F(ab) or F(ab') ₂ fragment antibodies?

The F(ab)) and F(ab') ₂ fragment antibodies abolish the non-speciality between the Fc portion of the antibody and the Fc receptor on cells such as macrophages, dendritic cells, neutrophils, NK cells, and B cells. Heterosexual bonding, and because of their small size, can penetrate tissue more efficiently. Since fragmented antibodies do not have an Fc portion, they do not interfere with anti-Fc-mediated antibody detection.

5. Do you need affinity purified antibodies or IgG fragments?

The advantages of using affinity purified antibodies or IgG fragments will depend on the type of binding desired. Affinity-purified antibodies have the lowest amount of non-specific binding, while IgG fragments contain high affinity antibodies. In fact, in affinity purification, high affinity antibodies remain immobilized on the substrate and cannot be eluted.

6. Do you need a pre-adsorbed secondary antibody?

For immunoglobulin-rich tissues and cells, secondary antibodies using pre-adsorbed serum are generally recommended for Western blotting applications. Pre-adsorbed secondary antibodies are less likely to interact with endogenous immunoglobulins, thus reducing non-specific background. When the secondary antibody is pre-adsorbed, the pre-adsorbed sample should be derived from the same species as the test sample. For example, when tested in human tissue, a human pre-adsorbed antibody will be required.

7. Do you need anti- IgG heavy and light chains, anti-light chain or anti- F(ab') ₂ secondary antibodies?
Antibodies against IgG heavy and light chains are simultaneously reactive with the heavy and light chain of the IgG subclass. Anti-light chain antibodies react with the same anti-F(ab') ₂ secondary antibody in all immunoglobulin classes. Reacts with the F(ab') ₂ part of the primary antibody