Antibody production
A serum containing the whole antibody response of one immunised animal: many clones against many epitopes. Strong, tolerant of denatured or partly destroyed antigen, quick and cheap to raise, and a finite, non-reproducible resource that varies between bleeds and between animals.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Polyclonal
A serum containing the whole antibody response of one immunised animal: many clones against many epitopes. Strong, tolerant of denatured or partly destroyed antigen, quick and cheap to raise, and a finite, non-reproducible resource that varies between bleeds and between animals.
How it differs from the standard method
- Rabbits are the usual host for research sera; goats and sheep give larger volumes, chickens give IgY in egg yolk without bleeding.
- Typically 8–12 weeks from priming to a terminal bleed, giving tens of millilitres of serum per rabbit.
- Immunise at least two animals: individual responses vary enormously and a poor responder costs the whole experiment.
- Crude serum is roughly 3% specific IgG; protein A gives total IgG; only an antigen affinity column enriches the antibodies you wanted.
- Record and reserve the lot. When the serum runs out the reagent is gone, and sequencing it recombinantly afterwards is the only way to keep it.
1What you are actually choosing
Making an antibody looks like one technique and is really a decision about what kind of reagent you need. A polyclonal antiserum is a population of antibodies from one animal, recognising many epitopes on the antigen at once.
Polyclonal
Strong signal, tolerant of denaturation and fixation because if one epitope is destroyed others survive. But it is a finite volume from one animal, it varies between bleeds and between animals, and it cannot be remade.
Monoclonal
Renewable, consistent between batches, and defined. If your sample preparation destroys that single epitope it fails completely: there is nothing to fall back on.
Recombinant
The variable-region sequence is the reagent. Independent of any animal or cell line, shareable as a plasmid, engineerable into fragments, bispecifics and fusions, and truly reproducible.
2Immunogen design is most of the work
An antibody only ever recognises what you showed the animal. A short peptide is a hapten: it has no T-cell epitope of its own, so B cells that bind it never get the help they need to class-switch and mature, and the response is feeble.
3Adjuvant, dose and schedule
An adjuvant holds the antigen at the injection site and signals to the innate immune system that something is wrong; without one, a soluble protein is cleared before it is presented, and very high doses can induce tolerance rather than immunity. Boosting is what turns a primary response into a useful reagent: each boost, roughly three weeks apart, drives another round of germinal-centre selection, which raises the titre and takes about an order of magnitude off the dissociation constant.
4Hybridomas: whatever you coat is what you select
A fusion immortalises the B cells of one immunised mouse by joining them to a myeloma line that cannot make its own purines; only cells that got a working HGPRT gene from the B cell survive in HAT medium, so unfused myeloma dies and unfused B cells cannot grow. Hundreds of wells grow, a fraction of them secrete antibody, and you have a few weeks to find out which.
5Validation is the part people skip
A high ELISA titre against the immunogen says only that the animal responded. It does not say the antibody will find the protein in a lysate, in a fixed cell, or in a tissue section, and it certainly does not say the band at the right size is your protein.
Key terms
Practise these as flashcards →A molecule too small to raise an antibody response on its own; it must be conjugated to a carrier to recruit T-cell help.
A large immunogenic protein (KLH, BSA, ovalbumin) coupled to a hapten; it takes most of the antibody response.
A formulation that depots the antigen and activates innate immunity, without which soluble protein is poorly immunogenic.
The highest dilution of a serum or supernatant that still gives a defined signal above background in an immunoassay.
Hypoxanthine–aminopterin–thymidine medium, in which only hybridomas survive because the myeloma partner lacks HGPRT.
Plating cells sparsely enough that a growing well came from one cell; two rounds are the standard proof of monoclonality.
Somatic hypermutation and selection in germinal centres, which raises affinity over successive boosts.
The combined strength of multivalent binding; a polyclonal's apparent affinity is largely avidity, not intrinsic affinity.
Independent evidence that the antibody binds the intended target, ideally by removing the antigen genetically.
When to use it
- No commercial antibody exists against your target, or none that has been validated in the application you need.
- You need an antibody against a specific region, modification or conformation: a phosphosite, a cleaved neo-epitope, one splice form.
- You need a renewable, batch-consistent reagent for a long project or a diagnostic, which means a monoclonal or a recombinant antibody rather than a serum.
- You need a strong, robust detection reagent for western blotting or immunoprecipitation and can accept a finite supply: a polyclonal is cheap, quick and sensitive.
- You need an antibody whose sequence can be shared, re-expressed and engineered, which only a recombinant antibody gives.
Compared with alternatives
The assay used to follow the titre, screen hybridoma supernatants and measure specificity against each coat.
How the antiserum is purified: protein A for total IgG, an antigen column for the antibodies you actually wanted.
The usual first application, and where a badly validated antibody does the most damage.
Selects binders in vitro from a library, with no animal and no immunisation, and gives the sequence directly.
How a cloned antibody sequence is turned back into protein, usually in HEK293 or CHO cells.
The application where epitope destruction by fixation most often decides between a polyclonal and a monoclonal.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A serum containing the whole antibody response of one immunised animal: many clones against many epitopes. Strong, tolerant of denatured or partly destroyed antigen, quick and cheap to raise, and a finite, non-reproducible resource that varies between bleeds and between animals.
- Rabbits are the usual host for research sera; goats and sheep give larger volumes, chickens give IgY in egg yolk without bleeding.
- Typically 8–12 weeks from priming to a terminal bleed, giving tens of millilitres of serum per rabbit.
- Immunise at least two animals: individual responses vary enormously and a poor responder costs the whole experiment.
- Crude serum is roughly 3% specific IgG; protein A gives total IgG; only an antigen affinity column enriches the antibodies you wanted.
- Record and reserve the lot. When the serum runs out the reagent is gone, and sequencing it recombinantly afterwards is the only way to keep it.