Antibody purification – what you need to know to use antibodies effectively

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Quality –Consistency –Expertise

Welcome to our 15th webinar
ANTIBODY PURIFICATION: What you need to know to use antibodies effectively
Dr Andy Lane

Dr Andy Lane
•Sources of antibodies
•Different methods of purifying antibodies
•Why “purified antibodies” are not all the same
•How to purify antibodies easily without specialist knowledge or equipment
•Considerations for conjugating antibodies to enzymes, dyes and nanoparticles

Sources of antibodies
•Animal serum –polyclonal antibodies
•Ascites –monoclonal antibodies
•Cell culture supernatant –monoclonal antibodies
•Eggs –avian antibodies
•Bacterial expression systems –recombinant antibodies
Each of these sources has contains the specific antibody of interest amongst other proteins, lipids and other components.

Different methods of purifying antibodies
•Fractionation by globulin precipitation
•Ion exchange chromatography
•Size exclusion chromatography
•Protein A affinity chromatography
•Protein G affinity chromatography
•Protein L affinity chromatography
•Antigen affinity chromatography

Globulin precipitation
•First published in 1899 by James Atkinson in J. Exp. Med. –separating albumin from the anti-toxic components of a horse antiserum using magnesium sulphate.
•Ammonium sulphate and sodium sulphate are more commonly used, taking advantage of the principle of “salting-out”. At the appropriate concentration these precipitate globulins, whilst many other proteins, including albumin, remain in solution.
•Whilst this is a simple and gentle procedure, it only provides a partially pure preparation if starting from a complex mixture such as serum.
•This step is often combined with further purification using one of the other methods discussed in this webinar.

Ion exchange chromatography
•Ion exchange chromatography (IEC) uses positively or negatively charged resins to bind proteins based on their net charges in a given buffer system (pH).
•Typically, a complex mixture is added to the column in a certain set of buffer conditions (e.g. low salt), and the buffer conditions are then changed either step-wise or on a gradient basis. Different proteins are released from the column in differing conditions.
•IEC is perhaps more often used to purify polyclonal antibodies than for monoclonals–note that each monoclonal is unique in terms of charge, and will therefore be released under a specific set of conditions –something that is useful for repeated purifications of the same antibody, but also means that optimisation is required for each antibody.
•There are various IEC resins available, with perhaps the most widely used being DEAE-Sepharose.

Starting buffer counter-ions
Substances to be separated
Gradient ions
Ion exchange chromatography

Size exclusion chromatography
•Separation of complex mixtures on the basis of size or molecular shape can be achieved by size exclusion (SEC) or gel filtrationchromatography.
•The molecular sieving process takes place as a solute passes through a packed bed stationary phase. Separation depends on the different abilities of the various molecules to enter the pores of the bead-based stationary phase.
•Large molecules, which cannot enter the pores, are excluded and pass through the column quickly. Smaller molecules that can enter the pores are retarded and move through the column more slowly. Very small molecules, such as salt, are able to fully permeate and elute last.

•SEC tends not to be used for primary purification of antibodies other than IgMs. However, it may be a valuable “polishing step” following purification by other methods.
•Ranges of chromatography resins are available that separate proteins within different ranges of size (e.g. AcA22, AcA34, AcA44 from Ultrogel®, and S200, S300, S400 in the Sephadexrange)
DE-SALTING COLUMNS
•So called de-salting columns are used to undertake a buffer exchange, or to remove small size contaminants. This is simply a special case of SEC, with the most commonly used gel being SephadexG-25, often in pre-prepared PD10 columns.

Mixture of different sized proteins
Different sized proteins
Gel filtration column

Protein A/G/L chromatography
•In their native form these proteins are expressed by bacteria as part of their defensemechanisms against the mammalian immune response.
•All bind to mammalian immunoglobulins via constant regions –Protein A and G to the Fc region, and Protein L to the kappa light chain.
•Recombinant versions are used immobilised to various matrices to purify IgG by a form of affinity chromatography.

Heavy chain
Light chain
Protein G and A
Protein L

•Samples are added to the Protein A/G/L matrix in a suitable binding buffer –pH and ionic strength should be considered
•Column is washed, which effectively leaves only IgG bound
•Low pH elution is carried out, which may be with different buffer formulations and may differ depending upon the isotypeof the antibody being purified. It may be important to know the basic formulation of the elution buffer for downstream processing
•Low pH elution requires neutralisation to maintain antibody integrity. Often carried out with Tris buffer, but is this sufficient for your downstream needs?

Recombinant ProteinA
Recombinant ProteinG
Recombinant ProteinA/G
Recombinant ProteinL
RecombinantProtein A/G/L
Native Source
Staphylococcusaureus
Streptococcus
N/A
Peptostrepto- coccusmagnus
N/A
Binding Sites for Ig
5
2
6
4
13
Optimal Binding pH
8-9
5
5 to 8.2
7.5
7.5
Typical elution pH
3.0 –7.0
(isotypedependent)
2.5 –3.0
2.5 –3.0
2.0 –3.0
2.5–3.0
Ig Binding Target
Fc
Fc
Fc
VL-kappa
Fc + VL-kappa

Species
Immunoglobulin
Binding to Protein A
Binding to Protein G
Binding to Protein L
Human
IgG1
Strong
Strong
Strong
IgG2
Strong
Strong
Strong
IgG3
Negligible
Strong
Strong
IgG4
Strong
Strong
Strong
Mouse
lgG1
Weak
Strong
Strong
lgG2a
Strong
Strong
Strong
lgG2b
Medium
Medium
Strong
lgG3
Weak/medium
Medium
Strong
Rat
lgG1
Negligible
Weak
Strong
lgG2a
Negligible
Strong
Strong
lgG2b
Negligible
Medium
Strong
lgG2c
Negligible
Medium
Strong
Goat
lgG
Weak
Medium
Negligible
Rabbit
lgG
Strong
Medium
Weak
Sheep
lgG
Weak
Medium
Negligible

Antigen affinity chromatography
•In this case a specific chromatography resin is prepared to which the actual antigen that the antibodies bind to is immobilised.
•The sample containing the specific antibodies is then passed over the column, allowing only specific antibodies to bind, with all other contaminants, including other antibodies, being washed away.
•Buffer conditions are then changed to elute the specific antibodies from the column.
•Typical examples of the uses of affinity chromatography are in preparation of species specific secondary antibodies, in purifying antibodies to peptide antigens and for purifying proteins with epitope tags (e.g. Myc, HIS etc).

Matrix
Ligand
Immobilized Ligand
Sample
Complex
Impurities
Antigen affinity chromatography
Preparation of gel matrix
Application of sample
Elution of purified antibody

Why “purified antibodies” are not all the same
•Concentration
•Preservatives / stabilisers –have any been added back in to the preparation after purification?
•Buffer components –including contaminants from elution etc.
•Level of purity -e.g. Ig fraction vs. total IgG vs. specific IgG
•Unexpected contaminants –e.g. bovine IgG
•Endotoxin etc.
Many antibodies are sold by companies as “purified” –but what does that mean?

Your final buffer……..
•The final buffer that your antibody is in may have a significant effect on what you can use your antibody for. For instance, the presence of glycine in a buffer can inhibit conjugation reactions, or sodium azide may kill live cells. Some buffers may be suitable for conjugating to proteins, but not to nanoparticles.
•If you undertake a dialysis step post-purification to transfer an antibody into a particular buffer, you need to be sure that the dialysis has been effective.
•e.g. To remove (i.e. to 1nM level) 100mM glycine from 10ml of antibody you need to dialyse against 1 million litres of PBS. Alternatively, changing the dialysis 4 times will achieve the same effect with only 4 litres of PBS.
•However, a single dialysis into 1 litre leaves glycine present at 1mM.

Total IgG vs. specific IgG
•The serum produced when any polyclonal antibody is raised will contain only approximately 10% of antibodies that are specific to the antigen of interest
•Therefore if you purify all of the IgG present, only 10% of that IgG will be specific for the antigen.
•Only antigen affinity chromatography effectively provides IgG that is 100% specific for antigen –Protein A/G/L chromatography provides total IgG, albeit apparently 100% pure when analysed by SDS-PAGE.
•These differences lead to significant differences in activity –a working dilution of an antigen affinity purified antibody is likely to be 10-fold higher than that of a Protein A/G purified antibody.

Unexpected contaminants –e.g. bovine IgG
•By definition, your purified monoclonal antibody must be 100% pure. Is that right?
•Be aware that if your cell culture media contains foetal/newbornbovine serum there may be significant levels of bovine IgG present –which may co-purify with your monoclonal antibody when using media such as Protein A or G.
•Avoid these either by using a mouse IgG specific purification system or converting all of your hybridomaculture to serum free media.
•If you use ascites you avoid bovine IgG, but you will have normal mouse IgG present which also co-purifies.

http://en.wikipedia.org/wiki/Fast_protein_liquid_chromatography
Purification doesn’t have to be
this complicated…..

BSA removal kit (820-0010):
remove BSA and concentrate your antibody in one easy to use kit!
The AbSelectpurification range

AbSelectRat purification kit

BSA removal kit

What is Lightning-Link®technology?
The worlds fastest, easiest to use and most efficient conjugation technology!
•Only 30 seconds hands-on time!
•Over 50 labels available including:
Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin
Lightning-Link®
Antibodies –Proteins –Peptides
Fast –Easy-to-use –Reliable

Conjugation considerations
You need to know some things about your reagent.
Lightning-Link®, Thunder-Link® and InnovaCoat® conjugations are really simple
but you need protein in the right format to work effectively.
Concentration –1mg/ml or higher is preferred
Purity –ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation –most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiolssuch as DTT or mercaptoethanol. Trisis OK up to 20mM
Preferred buffer formulations for the antibodies to be labelled with the
various kits differ (especially for nanoparticles), and purification methods
are important to understand

AbSelect:
AbPure:

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

Innova Biosciences Ltd. Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova BiosciencesDyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

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