Part 3 First 41 Flashcards
What are the two categories of immune response and what do they mean
Innate:
quick response to foreign invaders without having to encounter them beforehand ,
first line of response,
non specific (since react to all invaders same way)
Adaptive:
this needs a lag period where it prepares to attack the invader,
highly specific (can discriminate between two similar molecules) ,
has memory where if invasion happens again there is a fast response
What are the two catergories of adaptive immunity
Cell mediated:
Done and Mediated by t-cells (T-lymphocytes) that when active, recognize and kill infected cells
Humoral:
Done by antibodies from the ig superfamily
Mediated by b-cells (b lymphocytes) that when active, differentiate into plasma cells that secrete a single antibody that recognizes a single epitope
Where do B and T cells come from
Explain pathway
Hematopoietic stem cells
The hematopoietic stem cell comes from the bone marrow
Turns into a lymphoid progenitor cell
Than that differentiates to T-cells and B-cells
B -cells can differentiate into plasma cells which are an antibody factory
How many classes of Ig are there and what are they
5
IgA D E G M
They all show up at diff times in response to a foreign substance and have diff biological functions
What are IgM and igG
IgM:
are the first antibodies made by B-cells after antigen stimulation
show up in the blood after a lag of a few days,
short half life of 5 days
IgG: the predominant antibody found in the blood during the secondary response to antigens
Explain the IgG curve
Upon initial stimulus with and antigen (like a vaccine)
Log scale so a lot
In the primary response
The IgM appears first then comes down to basal level
The igG responds slower then comes back to basal level
Since immune system has memory, has a bigger secondary response after a varying period of time
IgG more and IgM less now
We’d take a bleed at the secondary response if trying to make antibodies
What are antibodies
Structure
Made by B cells and their differentatiated plasma cells
Called immunoglobulins
Made of 2 identical heavy chains (gamma Y) and two identical light chains (lambda or kappa K) connected by disulfide bonds
To make a Y shape
With one side having are light and heavy and the other with one light on heavy
Explain the MW of antibodies
For igG:
Heavy chains are 55 kDa
Light are 25
Since two it’s
110 + 50
So total 160 kDa
Back then Explain the problem they encounter when purifying antibodies and how the problem was solved
They tried to find the ab specificity by purifiying and getting its sequence
But there are 1000s of antibodies in the blood that are too similar to be separated, so leave a big jumble in SDS page
Found that the blood of a person with type of lymphoid cancer called multiple myeloma had large amount of a dominant AB molecule
So they started purifying the antibodies in the blood to the cancer patients because each patient over expresses one type of antibody
Why would the blood of a person with type of lymphoid cancer called multiple myeloma have large amount of a dominant AB molecule
Since cancer is a monoclonal disease where the cells of a tumor come from the overproliferation of a single mutated cell
The specific lymphoid cell for the lymphoid cancer that’s being over expressed would also overexpress the single antibody that it makes (since lymphoid cells make b and T cells and B cells make antibodies )
Making a single type of antibody in large abundance
Who does a B cell make a antibody
The single B cell becomes committed and makes a single specific igG molecule
This single igG molecule recognizes a single epitope
What is an epitope
What is a mono vs poly clonal antibody
The a minimum 6aa long sequence on the antigen that is recognized by the antibody
The antibody only recognizes this single epitope on the antigen but the antigen can have more than one epitope
Mono is where one antibody recognizes a single epitope on the antigen
Poly is where the antigen protien has multiple epitopes on it that mutiple diff antibodies bind to their specific one. This collection of antibodies on the protien is a polyclonal antibody
What is edman sequencing
Sequencing individual amino acids of the peptide on mass spec
What did they find when sequencing the AB from lymphoid cancer patients and comparing their amino acid sequence
Early day of igG research
Half of each kappa light chain (the n term part with 110 amino acids) had a constant amino acid sequence (CL)
but the other half of the chain (c term) varied from each patient (VL)
Same for the lambda light chains
The heavy chains were the same but had 1/4 variable (VH) and 3/4 constant (CH)
Explain how the variable and constant regions of an antibody come together
The variable parts of one heavy and one light chain on the antibody come together to make the antigen binding site (working en of the molecule)
The constant region of the antibody is bound by protien A with high affinity (protien made by S.aureus)
Protien a always bound since always constant
What is the Fab fragment and the Fc fragment
What is the interaction between the antibody and the antigen
What about between the heavy and light chain
fab fragment: fragment antigen binding , the variable region of the AB that has a close fit to the ag
Fc: constant region with the Ch chains
Non covalent forces
Disulfide bonds
Why are AB a powerful tool in Biochemistry
Since they have very specific and very tight binding to the side chains of the antigen they can select a few protiens out of the hundreds in the cell to bind to
by distinguishing between two polypeptides that differ by even just one amino acid or covenlent modification
The heavy and light chain come together to bind the antigen
Give an example to show show AB can be used as a tool in biochemistry
You made an AB that’s made to recognize a phosphorylated RRVpSFAD (a phosphorylated serine)
When doing western blot with the phosphorylated peptide vs the unphosphorylated, only the phosphorylated was recognized
What is the process of making polyclonal antibodies
Inject an animal (rabbit) with an Ag (a pure protien) this is the primary injection
Boost with the Ag 1 month later
(1 month so that the IgG levels in the blood after the primary response go back to basal levels and none left in the blood, if some left they’d just bind at low levels to the Ag instead of having the secondary response)
Take a test bleed two week after secondary response (after the boost) since now the igG Has the highest response to the Ag and lots of it in the blood
Then boost with Ag again two weeks after (so after igG back down to basal levels again to get proper boost and high levels)
Keep purifying the antibodies from the blood
Basically trying to get the most amount and most specific antibody
What is a done during a test bleed
After taking the blood, they clot
There is a plug at the bottom of the tube
When centrifuging, the serum goes to the top and the blood cell go past the plug
Then we test the serum goes
One B cell make ___ antibody and
1 antibody and many copies of this antibody that get secreted out of the cell
What is polyvalent AB heterogeneity
When multiple B cells (not just one) are activated and commit to making multiple antibodies that each recognize single different epitopes on the single antigen
This is the polyclonal antibody
What is the issue with making monoclonal antibodies
More difficult because need to isolate a single b-cell/plasma cell making one type of antibody and put in in culture to divide
But antibody producing cells don’t divide in culture since they are normal cells
Different in cell froth and proliferation
Growth is the cell is growing
Proliferation is its dividing
What are hybridoma cells
How do they help
In order to make monoclonal antifboides
You fuse the b-lymphocytes (the one that commit to make a single type of ab) with a myloma cancer cell (one that grows and proliferates rapidly in culture and makes ab)
This fusion is the hybridoma cell
now the cancer cell can rapidly divide in culture and secrete antibodies using the information from the fused b-lymphocyte to make the antibody that we select for
Explain process of how to make monoclonal antibodies
Don’t need a purified ag protien because the screening process will be done later on
Inject the ag into a mouse to make specific ab producing cells
Remove the spleen of the mouse after a few weeks, the spleen has the B cells and the salvage pathway
They also made a culture of myeloma cells which are HGPRT-mutants, they cannot do the salvage pathway since not HGPRT enzyme
Then fuse the myeloma cells with the spleen cells using PEG
Add the fused cells to a HAT medium
HAT medium (has hypoxanthine, aminopterin, thymidine) the aminopterin inhibits the de novo pathway (no nucleotide sysnthesis, no way to grow)
The only cells that grow now are fused hybridoma cells since used the salvage pathway from the spleen b-cells to survive
They separate the surviving hybridoma cells and screen then for production of the antibody against the antigen
Then clone the hybrid cells that make the specific antibody, store
What is the salvage pathway
De novo pathway
To divide they need to duplicate their genome meaning they need nucleotides, the salvage pathway salvages these nucleotides from RNA
Regularly replication and transcription using nucleotides
What are the uses of antibodies in research
Uses of Mab
Western blots
immunoprecpitication (on agarose bead antibody collects the target)
Co-immunoprecipitation: on agarose bead antibody collects the target and the target brings its partners with it
Immunolocalization in cells: seeing where certain proteins are inside the cell
Monoclonal antibodies are useful as therapeutic agents to treat diseases like cancers, psoriasis, crohns.
What is an example of a mab
Keytruda
Stands for fighting woman/ strength
Treats lung caner
What is now in most drugs
Mab
What are the two case studies to examine the use of antibodies
Antibodies to define the role of PP1 binding protein of K9
Making antipeptide antibodies
How did they use affinity chromatograph to purify the PP1 from the hela cell nuclei
Want to see the regulatory protiens that control the PP1
Have a small molecule toxin called MC (microcystin) that is on a bead, binds to PP1
Elite with a chaotropic agent called NaSCN which elites out pp1 and all of its binding partners
Or since pp1 docks to RVRW sequence, all regulatory subunits/binding partners of PP1 can be eluted with the RVRW peptide that binds to this same docking site to displace the binding partners from PP1
What did the SDS page gel of the RARA, RVRW and SCN peptide elution show
RARA control shows nothing because the binding to PP1 needs hydrophobic residues, partners stay on PP1
RVRW elutes out two binding partners of PP1 called Kif18a (Kynesin motor protien) and K9
SCN chaotropic agent takes everything else off the matrix (pp1 and other PP binding partners)
After finding k9 on the SDS page gel from the affinity chromatography what happened
What was the control and why
Further purification of k9 with cation exchange, make sure it’s the correct mass using mass spec
Now pure k9 is used to make an antibody
Control was preimmune serum from the rabbit , since we want to make sure that there are no pre existing antibodies in the serum that could bind to the k9
To make sure that the antibody we made is actually the correct one and not just from pre-existing non k9 antibodies in the pre immune serum that can bind k9
If there were bands that means we also purified non k9 antibodies since they’d purify with the real k9 antibodies
After collecting the k9 antibody serum (from the boost) what did they do
Purified the k9 antibody using affinity chromatography with the k9 antigen
Also purify the pre immune serum antibodies with protien A affinity chromatography (so basically all antibodies would be collected, since the rabbit has all kinds of antibodies in serum before introduction of the k9 antigen)
Elute using a pH shift (immunoprecipitation)
What would the SDs page of an antibody look like
SDS page of the antibody would have two bands at masses 55 and 25 (heavy and light chain)
How did they test the quality of the k9 antibody they purified
Has the pre immune serum antibodies western blot and the K9 purified antibodies western
Made the antibody solutions at a concentration of 2 ug/mL in each blot
Tested both antigens against the pure k9 protien antigen and the crude hela cells in each blot
Why would they run the western blot of the k9 antibody with diff concentrations of the k9 protien and the crude extract
The pure k9 is at ng concentration because already in abundance
but the hela cells are in micrograms because the k9 in there would be lower abundance since there are other protiens in the crude
What did the western blot of the preimmune serum antibodies show
Control
No bands in k9 lane because there should be no antibodies in the pre immune serum recognizing k9
Odd but there are also no bands in the hela extract , should be some since the many antibodies in the pre immune serum could recognize other protiens
What did the western blot of affinity k9 pure antibodies show
What would a ponceau stain of the crude extract look like
The antibodies recognized the pure k9 in high abundance
Also recognized the k9 in the crude hela extract
This is good because it shows that the antibody specifically recognized the single k9 protien antigen even in a crude extract with many other protiens in it
Would have all kinds of red and blue bands since all diff type of protiens in the crude extract I’m dynamic range
How much of protien should a really good antibody recognize
1 ng
What is the short linear motif the pp1 binds to
RVRW
In purifying pp1 using MC What is the RARA meant to be
A control for non specific binding to the beads
Shows that the RWRW peptide was effective in displacing the K9 protien from pp1
To see if the eluting protiens (K9 and KIF18a are non specific binding
How can we tell that we’re actually purifying the correct thing from the SDS page gel band for example k9
Do mass spec and see if the peptides align with the K9
Then use can spend the rest of your life studying it
Explain the other way to do an immunoprecipitation experiment
The antibody is mixed with the cellular extract at 4h at 4C to keep it cool to inhibit activity of proteases
Then that is sent over a protien A sepharose bead
After the non working end of the antibody and antigen are bound to the covalently coupled protien A bead, the beads are washed with buffer (to remove non specific binders)
Then elute the antibody with its antigen from the bead by boiling with SDS cocktail
Because the SDS had reducing agents , degraded igG and it’s antigen is relessed because held by disulphide bonds
Answer the the second why?
What is a crude extract
Extract the has a huge number of diff protiens in a dynamic range that are expressed in diff levels in the cell
What’s a way in immunoprecpitiation to just get the antigen and no antibody
Covelently cross link/couple the antibody to protien A then elute
All that’s left is the antigen
Explain how and why they did IP with K9 to find PP1
Had the crude hela cell extract
Split it exactly in half and do preimmune serum IP AND K9 IP to each sample
The PIS IP is a control
Found that the anti K9 IP showed K9 and PP1 on western blot
Means that k9 binds pp1
What are the two possibilities of PP1 and K9 binding together
K9 binds directly to PP1
K9 binds to pp1 through a binding partner
What is the large subcellular structure inside the nucelous
Nucleolus
Where is K9 located and how did the find this
In the nucleolus of the nucleus
Probed the nucleoli and cytosol extract with k9 antibody on western page and found a band only with nucleoli
Also use immunolocalization of hela cells to see the k9 was in the nucleolus
Explain the process of cells needing to divide
The nuclear membrane on the nucleus has to disappear for divisions
Then has to reform the membrane
Nucleoli also has to do this
What is co immunpreocotpiation of k9 with aPis as control show in the coomasie SDS
Anti k9 showed bands of k9 but also many other bands
Meaning k9 had binding partners
Also showed the igG heavy chain and light chain bands
What did they find out about k9 in terms of its role
It’s in the nucleolus
Found that its part of the pre 60s ribosomal subunit processing complex (helps make ribosomes)
This make sense since the ribosomes are made in the nucleolus and k9 is in the nucelous
So k9 takes pp1 to the nucelous complex and help do a dephosphorylation role
When making pp1 specific antibodies using the invidual peptides what are we doing and why
So we have alpha beta gamma pp1 which are just diff pp1 peptides in humans that are conserved
Because the n and c term are usually exposed and freely available, if trying to do IP need to make it so the epitope for the antibody is free to bind the antibody.
This is why use n and c term and not the middle sequence because could be folded in the middle and not free to bind during ip
Also for this case the only diff in sequence between each peptide was the n and c term so need to use those sequence to make specific diff antibodies
What is keyhole lymphet hemocyanin
The KLH protien
Carried o2 in the hemolymph
Has zero relationship to hemoglobin but has still evolved as an oxygen carrier
When trying to make our antibody using a peptide (so we can do peptide affinity chromatography)
Why do we couple the antigen peptide with carrier KLH
If the peptide where injected alone there would be barely and immune response and barely any antibodies made
KLH is potently immunogenic meaning it give a massive immune response when covelently coupled to the peptide of interest
This makes it so that many antibodies to that peptide are made in the blood upon injection
When doing peptide affinity chromatography what part of your peptide needs to be free to bind to the functional group on the bead
The N-term, lys, arg (any free NH2)
When doing peptide affinity chromatography with an antigen how is it done
Add the diluted crude serum to the column
Mostly high level or your antibody binds to the antigen peptide on the column
But could also be other antibodies present in the blood, remove them as non specific binders
Elute with pH 2 to disrupt the interaction between antibody and the peptide antigen
Collect the antibodies and immeadiately put them back in ph 7 solution because don’t want them left too long in low ph and denature
What’s the minimum number of amino acids that can be in an epitope
6
What did the dot blots with pp1 alpha beta gamma show and what was the control
Had three dot blots with each diff concentration of pp1 a b gamma
Did anti alpha them beta then game on the three blots
Also Had an unrelated peptide on each blot as a negative control (to see if antibody also reconizes that and is just recognizing everything )
Found the the anti aplha only recognized alpha and same for all other antibodies recognizing their specific epitopes
What did the western blot with pp1 alpha beta gamma and crude hela
show
What did they change
For the alpha antibody , Found that the alpha and beta pp1 was detected, the crude hela extract also showed bands
These bands in crude could have a similar epitope to the PP1 which the antibody binds
In beta antibody , beta is recognized in the beta pure protien and in the hela crude
Same for gamma
Added more concentration of alpha beta gamma and did experiment again, saw the anti alpha binds highly to alpha and beta but not gamma
Shows that the gamma epitope sequence is very unrelated to the gamma and alpha
Even if a faint signal is seen at high concentration that could just be nonspecific binder, or the secondary antibody binding to something in the membrane
But the anti aplha does recognize beta
Question at the end
What does alpha handing to alpha and beta mean
Okay
Means there must be a region in the alpha peptide that the antibody binds to that has its epitope sequence similar to a sequence in beta (so it’ll then bind both alpha amd beta)
But the beta antibody must only be recognizing an epitope more unique to beta
Explain how do make the anti aplha antibody just alpha specific and not bind to beta
Some antibodies recognize alpha , alpha and beta, and beta
If we add the beta peptide to the solution of antibodies, the antibodies that bind beta all bind beta and the only ones left are one the only bind alpha
Now anti alpha won’t bind to beta and only bind alpha
