Part 3 First 41

Question 1

What are the two categories of immune response and what do they mean

Answer 1

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

Question 2

What are the two catergories of adaptive immunity

Answer 2

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

Question 3

Where do B and T cells come from

Explain pathway

Answer 3

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

Question 4

How many classes of Ig are there and what are they

Answer 4

5

IgA D E G M

They all show up at diff times in response to a foreign substance and have diff biological functions

Question 5

What are IgM and igG

Answer 5

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

Question 6

Explain the IgG curve

Answer 6

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

Question 7

What are antibodies

Structure

Answer 7

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

Question 8

Explain the MW of antibodies

Answer 8

For igG:

Heavy chains are 55 kDa

Light are 25

Since two it’s

110 + 50

So total 160 kDa

Question 9

Back then Explain the problem they encounter when purifying antibodies and how the problem was solved

Answer 9

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

Question 10

Why would the blood of a person with type of lymphoid cancer called multiple myeloma have large amount of a dominant AB molecule

Answer 10

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

Question 11

Who does a B cell make a antibody

Answer 11

The single B cell becomes committed and makes a single specific igG molecule

This single igG molecule recognizes a single epitope

Question 12

What is an epitope

What is a mono vs poly clonal antibody

Answer 12

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

Question 13

What is edman sequencing

Answer 13

Sequencing individual amino acids of the peptide on mass spec

Question 14

What did they find when sequencing the AB from lymphoid cancer patients and comparing their amino acid sequence

Early day of igG research

Answer 14

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)

Question 15

Explain how the variable and constant regions of an antibody come together

Answer 15

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

Question 16

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

Answer 16

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

Question 17

Why are AB a powerful tool in Biochemistry

Answer 17

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

Question 18

Give an example to show show AB can be used as a tool in biochemistry

Answer 18

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

Question 19

What is the process of making polyclonal antibodies

Answer 19

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

Question 20

What is a done during a test bleed

Answer 20

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

Question 21

One B cell make ___ antibody and

Answer 21

1 antibody and many copies of this antibody that get secreted out of the cell

Question 22

What is polyvalent AB heterogeneity

Answer 22

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

Question 23

What is the issue with making monoclonal antibodies

Answer 23

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

Question 24

Different in cell froth and proliferation

Answer 24

Growth is the cell is growing

Proliferation is its dividing

Question 25

What are hybridoma cells How do they help

Answer 25

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

Question 26

Explain process of how to make monoclonal antibodies

Answer 26

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

Question 27

What is the salvage pathway De novo pathway

Answer 27

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

Question 28

What are the uses of antibodies in research Uses of Mab

Answer 28

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.

Question 29

What is an example of a mab

Answer 29

Keytruda Stands for fighting woman/ strength Treats lung caner

Question 30

What is now in most drugs

Answer 30

Mab

Question 31

What are the two case studies to examine the use of antibodies

Answer 31

Antibodies to define the role of PP1 binding protein of K9 Making antipeptide antibodies

Question 32

How did they use affinity chromatograph to purify the PP1 from the hela cell nuclei

Answer 32

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

Question 33

What did the SDS page gel of the RARA, RVRW and SCN peptide elution show

Answer 33

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)

Question 34

After finding k9 on the SDS page gel from the affinity chromatography what happened What was the control and why

Answer 34

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

Question 35

After collecting the k9 antibody serum (from the boost) what did they do

Answer 35

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)

Question 36

What would the SDs page of an antibody look like

Answer 36

SDS page of the antibody would have two bands at masses 55 and 25 (heavy and light chain)

Question 37

How did they test the quality of the k9 antibody they purified

Answer 37

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

Question 38

Why would they run the western blot of the k9 antibody with diff concentrations of the k9 protien and the crude extract

Answer 38

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

Question 39

What did the western blot of the preimmune serum antibodies show

Answer 39

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

Question 40

What did the western blot of affinity k9 pure antibodies show What would a ponceau stain of the crude extract look like

Answer 40

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

Question 41

How much of protien should a really good antibody recognize

Answer 41

1 ng

Question 42

What is the short linear motif the pp1 binds to

Answer 42

RVRW

Question 43

In purifying pp1 using MC What is the RARA meant to be

Answer 43

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

Question 44

How can we tell that we’re actually purifying the correct thing from the SDS page gel band for example k9

Answer 44

Do mass spec and see if the peptides align with the K9 Then use can spend the rest of your life studying it

Question 45

Explain the other way to do an immunoprecipitation experiment

Answer 45

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?

Question 46

What is a crude extract

Answer 46

Extract the has a huge number of diff protiens in a dynamic range that are expressed in diff levels in the cell

Question 47

What’s a way in immunoprecpitiation to just get the antigen and no antibody

Answer 47

Covelently cross link/couple the antibody to protien A then elute All that’s left is the antigen

Question 48

Explain how and why they did IP with K9 to find PP1

Answer 48

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

Question 49

What are the two possibilities of PP1 and K9 binding together

Answer 49

K9 binds directly to PP1 K9 binds to pp1 through a binding partner

Question 50

What is the large subcellular structure inside the nucelous

Answer 50

Nucleolus

Question 51

Where is K9 located and how did the find this

Answer 51

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

Question 52

Explain the process of cells needing to divide

Answer 52

The nuclear membrane on the nucleus has to disappear for divisions Then has to reform the membrane Nucleoli also has to do this

Question 53

What is co immunpreocotpiation of k9 with aPis as control show in the coomasie SDS

Answer 53

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

Question 54

What did they find out about k9 in terms of its role

Answer 54

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

Question 55

When making pp1 specific antibodies using the invidual peptides what are we doing and why

Answer 55

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

Question 56

What is keyhole lymphet hemocyanin

Answer 56

The KLH protien Carried o2 in the hemolymph Has zero relationship to hemoglobin but has still evolved as an oxygen carrier

Question 57

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

Answer 57

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

Question 58

When doing peptide affinity chromatography what part of your peptide needs to be free to bind to the functional group on the bead

Answer 58

The N-term, lys, arg (any free NH2)

Question 59

When doing peptide affinity chromatography with an antigen how is it done

Answer 59

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

Question 60

What’s the minimum number of amino acids that can be in an epitope

Answer 60

6

Question 61

What did the dot blots with pp1 alpha beta gamma show and what was the control

Answer 61

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

Question 62

What did the western blot with pp1 alpha beta gamma and crude hela show What did they change

Answer 62

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 63

Question at the end What does alpha handing to alpha and beta mean

Answer 63

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

Question 64

Explain how do make the anti aplha antibody just alpha specific and not bind to beta

Answer 64

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