15:30-15:45 online
Enter the zoom meeting, show the student ID.
They show the two topics: serum sickness and production systems
Halin:
What do you understand under serum sickness?
-I started with the historical part, how they transferred serum between animals and then from horses to humans (diphtheria), it is classified as a type III hypersensitivity reaction (mediated by IgG antibodies).
What else in the serum (other than horse antibodies) could we make antibodies against?
-proteins present in the blood, the most abundant is albumin
Time course of the immune reaction?
-BCR binds the antibody, internalizes it, presents a peptide on MHC II, activates T cell (CD4), T cell activates B cells, polyclonal response (different B cells make antibodies with different specificities against the same antibody), it takes about a week to develop antibodies; the own antibodies bind the horse antibodies and create a big immune complex
How is this immune complex cleared?
-complement activation; transfer to spleen and phagocytosis (she wanted a quite precise answer here and helped me get to it)
What are the symptoms of serum sickness? (where do the immune complexes go/accumulate?)
-she helped me with the comparison to SLE: rash, painful and inflamed joints, kidney damage (checked by urine test - what do you check for? - proteins and blood)
Compare/contrast with contact hypersensitivity?
-priming stage: antigen is present at low doses, taken up into APCs, goes to lymph nodes, presentation to T cells -> by the time the T cells get to the contact site, the antigen may not be present anymore (short exposure, unlike with IgGs which stay in blood for a long time); response to the antigen only happens at a later exposure
Neri:
Name the most important production systems:
-E.coli, yeast, mammalian cells
Production in E. coli: what do you need for it?
-non-pathogenic strain, plasmid - gene of interest, downstream of a promoter e.g. lac, IPTG can be used to switch on production, antibiotic resistance gene
What products can you produce in E.coli and which can you not?
-in general: good for simple folding/structure, non-glycosylated, e.g. insulin; not good for mAb (complex folding, glycosylation)
We saw that insulin has the A and B chain - how do you produce it in E.coli?
-either you produce the 2 chains individually and then the disulfide bonds are formed (in the periplasmic space!) or you produce a precursor and cleave parts of it
Stable vs. transient expression?
-stable: integrated into chromosomal DNA, not lost after proliferation, large yields, low flexibility, used in industry ... ; transient: gene stays extrachromosomally, lost after cell divisions (he stopped me before I listed more)
When you stably integrate a gene, can you just use it to produce your product or what do you need to do before?
-you have to show that the cells can produce the protein, use chemically defined media (was not sure what exactly he meant, so I just started listing what I remembered, he interrupted me for the next question)
Are the cells really monoclonal?
-mammalian cells are not, they can have different karyotypes (he said yes, and also can produce different yields - but the product can be the same)
I think this is everything.
They are very nice, Halin gave hints to help me to get to the answers she wanted to hear, and Neri interrupted me when he has heard what he wanted, when he wanted to ask a follow-up question or move on to the next one.
Good luck!