Ig purification Flashcards
how are pAbs made?
Inject Ag into animal of choice –> Ag activated B cell response –> plasma cell clones that secrete pAbs –> collect animal serum containing the pAbs
What determines which animal you will use to produce your pAbs?
based on the amount of Abs you want, so you select an animal based on their size
What are nano-Abs, what animals produce these, why are they useful for research?
nano-Abs = heavy chain only and signle chain Vh fragments
animals = camelids and sharks
use = access regions too small for conventional Ab.s
Why would we use IgY form birds?
- when a bird is immunized with a mammalian Ag the Ag is more “foreign” –> higher avidity
- high specificity
- low background (won’t bind to mammalian FcRs)
- high yield
- highly stable
- non-invasive (produced and purified in egg yolk)
Describe the differences between IgY and IgY(deltaFc)
IgY:
- 2H + 2L
- H = 2V + 4C regions
IgY(deltaFc):
- lacks two terminal domains
- major serum Ab
Describe how you’d make a pAb to a phosphopeptide epitope?
synthesis the peptide of interest (phospho and non-phospho) –> conjugate the phosphopeptide to a carrier –> immunize the animal with the carrier –> collect the antiserum (contains phospho-Abs. unwanted Abs, and unspecific Ab.s) –> affinity purify the Abs with the phosphopeptide (gets rid of unspecific Abs) –> deplete the unwanted peptide Abs. using the non-phosphopeptide –> specific phospho-Abs
Describe mAb production
Immunize a mouse with your Ag –> isolate plasma cells from the mouse –> combine the plasma cells and myeloma cells and culture them on HAT medium which inhibits de novo synthesis of NTs (forces cells to make guanine using the salvage pathway which requires HGPRT from B cells) –> creates hybridomas –> assay for desired Ab in culture supernantant –> reclone Ab+ hybridomas –> expand postive clones –> mAbs
Describe ammonium sulphate (AS) precipitation method to purify Abs. How does it work, which isotypes does it isolate (why?), what are a couple of advantages?
how does it work: salt fractionation
- ammonium salts precipitate out proteins at different concentrations depending on physical properties of the protein
- in aqueous environments, salt ions interact strongly with their complementary charge on a water molecule –> if the [salt] is great enough, ions will effectively complete for the water molecules that are bound to the proteins –> without water molecules to associate with, proteins will seek complementary charges on other proteins –> aggregating together –> precipitating out of solution
isolates IgG because it is quite hydrophobic (fewer charges) so it precipitates out before many other proteins
advantages = doesn’t damage the protein too much and concentrates your protein
Describe how caprylic acid (CA; octanic acid) purifies Abs. Which protein doesn’t it precipitate out of? What are a couple of advantages?
weak acid buffer + addition of short fatty acids (CA) –> precipitate most proteins, except for IgG
advantages = cheap and fast
Describe how ion exchange chromatography works.
- DEAE has a net (+) charge and is part of the anion exchange matrix –> DEAE will bing to (-) charged proteins
- the net charge on a protein will change in response to pH and salt concentration
- each protein has a distinct isoelectric point and thus has a distinct pH or salt concentration which it will no longer bind an ion exchange column
- create fractions to find your protein of interest
What is an isoelectric point?
when the pH or salt concentration has reached a point where all the charges on a protein are balanced –> neutral charge
How can you use protein A, G, or L to purify Abs?
Create a protein A-, G-, or L-sepherose column –> run proteins through the column (eluent = other proteins) –> elute the proteins stuck in the column by changing the pH or salt concentration (eluent = Abs)
Describe how immunoaffinity purfies Abs
covalently bind Ag to support matrix –> pass anti-serum through the column (eluent = everything but the Abs that bind to the Ag) –> elute the desired Abs by changing the pH or salt concentration of the buffer
Describe how dialysis works.
dialysis tubing has a MW cut-off that indicates the size of the pores in the membrane –> all proteins and contaminants that are smaller than the hole will leak out –> water will also leak in to dilute the salt
