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Antibody frequently asked questions

General antibody questions

What is the difference between target species reactivity and antibody host species?

While antibody host species indicates the species that produced the antibody, the species reactivity refers to the ability of an antibody to react with a target protein from defined species.

An antibody raised against a certain human antigen will bind to the human homolog and not homologs of the same protein from other species.

Some antibodies can instead detect a homologous epitope from multiple species. This is referred to as species cross-reactivity and often occurs when immunoglobulins from different species share conserved sequences and similar quaternary structure. It is crucial to select an antibody with the appropriate species reactivity for the experimental model involved.

For example, if using a mouse cell line, you will need an antibody with mouse species reactivity, while if using a mouse cell line overexpressing a human target protein, an antibody with human reactivity which does not react to the mouse homolog is recommended.

Are there any recommended dilutions for usage?

These depend on the intended application and must be determined individually.

Can I use the antibody in an application it is not validated in?

Using an antibody in an application that was not validated might not generate the desired results due to sample preparation potentially affecting epitope structure or availability, or due to binding conditions potentially affecting the strength of the antibody-antigen interaction.

The experimental design and protocol used for the antibody’s validation experiments are crucial, including the cell type used, treatments (if applicable), positive and negative controls, and sample processing. Nevertheless, validation in your experimental conditions remains the most important factor when checking validity of the antibody.

Can monoclonal antibodies be supplied in other formulations such as, “bulk” size or without sodium azide?

If you do have individual requirements, we are more than happy to discuss them so please do not hesitate to contact us.

Are bovine serum supplements used?

Yes, from USFDA approved serum suppliers.

What are the storage conditions for antibodies upon receipt?

Most antibodies are quite robust and should retain functional activity if kept refrigerated at 2—8°C for up to 12 months. For longer term storage, it is recommended to store the material frozen in smaller aliquots. Avoid repeated freezing and thawing cycles, which will adversely affect the material.

Due to the large inventory size, individual stability studies have not been conducted on every antibody.

How are the antibodies stored and dispatched?

They are stored at -20°C and dispatched at ambient temperature in padded jiffy bags containing cool packs.

How is the antibody concentration determined?

By UV absorption at 280 nm, in a 1 cm path length cuvette using an extinction coefficient of 1.4.

Monoclonal antibodies

How are the monoclonal antibodies produced?

By in vitro production methods; we do not produce in ascites fluid.

How are the monoclonal antibodies purified?

IgG by Protein A (murine) or Protein G (rat) affinity chromatography, which typically give a > 95% degree of purity.  IgM is purified using a thiophilic chromatographic method.

How are monoclonal antibodies supplied?

Monoclonal antibodies are supplied as 1.1 ml aliquots (to include overage) at a concentration of either 1.0mg/ml or 2.0mg/mL ±0.1mg/mL, in Phosphate Buffered Saline (PBS) containing 0.02% sodium azide as preservative.

CAUTION:  Sodium azide is toxic if ingested and due care should be exercised.

Can monoclonal antibodies be supplied in other formulations such as, “bulk” size or without sodium azide?

If you do have individual requirements, we are more than happy to discuss them so please do not hesitate to contact us.

Polyclonal antibodies

What are polyclonal antibodies?

Polyclonal antibodies are a heterogeneous mixture of antibodies directed against various epitopes on the same antigen, with different specificities and affinities.

This constitute an advantage in certain applications, such as detection with secondary antibodies. Indeed, conjugated polyclonal antibodies are usually preferred for secondary detection, as the signal amplification can increase the sensitivity of the assay.

Polyclonal antibodies are relatively quick, inexpensive and easy to produce in comparison to monoclonal antibodies.

How is a polyclonal antibody produced?

An animal is immunised with the target such as a protein. Blood is taken from the animal once it has had time to seroconvert, meaning the animal has produced an immune response specific to the target and antibodies against the target can be detected in its blood.

The harvested blood is allowed to clot at room temperature for 15 – 30 minutes, and then centrifuged, for example at 2,000 x g for 10 minutes.  The serum contains a mixture of antibodies specific to the target, non-specific antibodies and other non-immunoglobulin proteins such as albumin. This is known as ‘whole’, ‘crude’ or ‘unpurified’ serum and is one form of a polyclonal antibody preparation.

Crude serum can be used for some applications, however, enrichment of the antibody component is often required for better performance in applications.  This enrichment can be done using a number of antibody purification methods based on selecting antibodies by their size or affinity purification.

Affinity purification is done by selecting antibodies based on their immunoglobulin class, for example IgG, using proteins that specifically bind to this class of antibody, this is known as ‘class-specific affinity’ purification.  Alternatively, ‘antigen-affinity’ purification is done by incubating the serum with immobilised antigen, then washing, at this point only antigen specific antibodies are retained and these can be eluted off to produce a highly antigen specific polyclonal preparation.

Recombinant antibodies

What are recombinant antibodies?

recombinant antibody is a type of monoclonal antibody where the sequence has been identified and then produced synthetically. It is produced with a phage display method, which guarantees batch to batch consistency.

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