----start immuno 1.13.97----
Dr. Phil Scott - office rm 216 Rosenthal?
email pscott@vet.upenn.edu
phone 8-1602


material today: directly from the purple immuno book.
so, instead of getting handouts just read the book. this is exactly from 
the book--chapter 7. this will not be true for the lectures given after 
the first exam. 

Antigen Antibody interaction is the subject of this lecture.
how can we use Ag/Ab interaction for diagnosis? elisa, hemagglutination, 
etc.
so this is a kind of more practical aspect.

difference between antigen and epitope; how Ab recognize complex Ag, etc.

theoretical interactions between Ab/Ag, secondary interactions between 
them as well.

what are consequences of Ab/Ag binding together? three major ones
--AGGLUTINATION: ag/ab bind together - ag is a particle, and it falls out 
of suspension. only occurs w/particulate antigen
--PRECIPITATION: where the Ag is a soluble molecule
--ACTIVATION OF COMPLEMENT- to be discussed friday

for these consequences to occur there must be crosslinking between the 
molecules. If you have an antibody with a particular Ag that's univalent 
(a particle of bacteria, say, with one recognized epitope) you can't 
crosslink it. you need multivalent antigen in order to accomplish 
crosslinking. You almost always have multiple Ag and Ab in serum, so you 
generally do have the ability to crosslink.

[slides]
what holds Ab and Ag together? H bonds, electrostatic forces, van der 
waals forces, and hydrophobic interactions. NOT COVALENT bonds. the 
interaction bet Ag and Ab is actually pretty weak and easily reversible 
by changes in pH, salt concentration, other ion concentrations, etc. and 
certain somethingtropic agents which bind ions.

so how do you measure the degree of interaction, which isn't the same for 
every Ag/Ab interaction? 

high affinity: good fit
low affinity: poor fit

affinity is measured by the association constant equation.

Ab + Ag <===> AgAg

[AbAg]/[Ab][Ag] = K

the concentration of the AgAb complex divided by the product of the 
concentration of antibody and concentration of Ag equals the equilibrium 
constant K. so if you change the Ag or Ab concentration, you change the 
amount of complex. so doubling either Ag or Ab will DOUBLE the complex. 
keep in mind that K is a fixed number. realize this is all 
*concentration* dependent.

so does this affinity measure an AbAg interaction? well, it doesn't 
really reflect what's happening with the whole complex. it's measuring 
the relationship of a specific Ab and a specific binding site on the Ag 
which is called AFFINITY

but AVIDITY is the real interaction between an Ag and Ab molecule. if you 
take the Fab portion of the Ab and you take an Ag, you have a certain K 
measuring the affinity. if you take the whole IgG which has two binding 
sites, and look at a solution with several epitopes in it, you increase 
the interaction, because there are two binding sites on one Ab molecule, 
and because you're looking at all epitopes on the antigen. this is 
AVIDITY and is much greater than the affinity of one Fab portion and one 
of the epitopes of that Ag.

AVIDITY is simply the measure of an Ab and complex Ag and the 
interactions between those two, where affinity is more specific measure 
of interaction between one Fab portion and one epitope.

specificity, cross reactivity, and non reactivity:

so you have the AbAg interactions. some are better than others. 
specificity: a specific Ag and Ab are specific for each other. cross 
reactivity: some shared determinants, such that Ab specific for AgX will 
react mildly with AgY which shares some determinants with AgX. if there 
are not any shared determinants there is no reactivity.

if you make Tb antiserum, you have serum with Ab to multiple Tb epitopes. 
If you put that serum with a similar mycobacterium you would probably see 
some cross reactivity, but if you put it with HIV you probably wouldn't 
see any reactivity.

mycobacterium paratuberculosis is sometimes present in cattle. but there 
is potential for cross reactivity to mycobacterium bovis which is much 
more serious
so you need to evaluate the specificity of the Ab used in the diagnostic 
assay.

say you want to test an animal for Ab to a particular pathogen. you need 
to find out if the patient was exposed to that pathogen, so you need to 
get this info. you do this by putting the pathogen into test tubes, and 
you add the patient's serum into the test tubes in various 
concentrations. if the sample is positive you will see agglutination at 
various concentrations until the sample is too dilute. so you can get an 
idea of how much Ab is in the serum. if you get agglutination at 1:32 but 
not at 1:64 you have a titer of 1:32.

say your patient has a titer of 1:32 of the pathogen you test for. what 
does that mean? well, you have to compare it to a standard for what you 
know to be positive or negative. Eg, if you KNOW an animal is not 
infected, what titer do you get? If THAT titer is 1:32, it could be that 
there are cross reactions that normally occur giving a titer of 1:32. SO 
that would mean that your patient can't be considered exposed....you 
can't say it *isn't* but the test doesn't confirm that it is. Now, if 
normal is 1:4, and your patient is 1:32, then that will be interesting. 
It's now more likely that your patient was infected with the pathogen in 
question. but you'd like to have some kinetics on this exposure. is 1:32 
more or less than what it was last month or next month?

note also that many times at very high concentrations of Ab you don't see 
agglutination: this is called PROZONE phenomenon. a situation where there 
is so much Ab present that there isn't enough Ag for crosslinking. so you 
always need to do several dilutions of the test serum. there could be no 
reaction due to this phenomenon....if you just measured a 1:64 dilution 
and it was negative, dosen't mean animal negative. maybe test didn't even 
work.

also: always need + and - controls. use a known positive and known 
negative.

red cell antigens: important wrt transfusions, autoimmune hemolytic 
anemia, rh factor sensitization, etc. totally can't see slide from here 
at all :(

you can take RBCs and add random Ag to them, and test for it - passive 
hemagglutination. in this way you use the assay and the fact that RBCs 
agglutinate to test for whatever Ag you have put on there. the thing with 
this assay - it's easy to mix rbcs and serum, and you can see the 
agglutination, so it's easy, but the problem in the past was, rbcs and 
abs have charges which tend to repel each other. RBCs are - charged. so 
IgM Abs would give the hemagglutination, but IgG wouldn't, because of the 
zeta potential, which means that the IgG and RBCs repel each other.[note- 
see below. mistake. RBCs repel each other, not RBCs and Ig.] IgM is a 
larger molecule and can overcome the repulsive force. this was a big 
problem. so you use this assay in looking for rh factor antibodies, 
testing for hemolytic anemia, etc. so Coombs improved this assay - he 
used the fact that Ab themselves can be antigens. what he did was - you 
have an RBC and an Ag on there and you think your test serum will 
recognize that Ag. you're looking for IgG in the dog blood. well, because 
of zeta potential you  might not see agglutination. so coombs raised 
antibodies to the dog IgG: he injected dog IgG into a rabbit, and got the 
rabbit to produce anti-dogIgG antibody. so you take those antibodies and 
react them with the dog IgG, it will increase the binding of the dog IgG 
to the red cells and will increase the hemagglutination.

two ways: direct and indirect coombs assay. if you think your patient has 
made Abs to its own RBCs (hemolytic anemia) you take those RBCs (which 
have Ab already attached) and you add anti-Ab Ab, and that should cause 
hemagglutination. that's a direct coombs.

or, take RBCs from an animal, take a test serum, and see if serum 
recognizes an Ag on the RBC as described above  (rabbit thing)- indirect.


take RBCs you know do not have Ab bound, add test serum, and see if it 
binds, by adding anti-patient Ab. - again, indirect (second explanation)

---break---

dr scott remarks that he misspoke about the zeta potential. apparently 
the zeta potential - the problem is the interaction of the RBCs all being 
negatively charged - so the RBCs repel each other, and you need a way to 
bridge that.


you need crosslinking to occur to see precipitation too. you have an AgAb 
complex forming in such a way it is called a lattice, and it will come 
out of solution and can be measured.

PROZONE also occurs with precipitation reactions. If you have Ab excess, 
there is a low amt of precipitation, due to lack of crosslinking. Also if 
you have Ag excess you don't get much precipitation either. When you have 
an appropriate amt of both Ag and Ab, you have "equivalence" and you get 
a lot of precipitation.

immuno-double diffusion

Ag and Ab applied to holes punched in agar gel. you put ag in one hole, 
ab in another. they diffuse through the gel and you see precipitin band 
between the holes. you wash and stain the gel and you can see the line of 
precipitate. note that there may be multiple lines of precipitation if 
there are multiple Ag/Ab interactions. (eg, using complex ab and ag, not 
monoclonal).

in the book it talks about (pp 123-124) different ways of looking at the 
precipitin bands, and using the reaction to see how many Ab may be 
recognizing Ag in a crude Ag prep. they talk about taking Ab to different 
Ag and seeing if there is identity, non-identity, or partial identity. 
THIS IS NOT GOING TO BE ON THE EXAM. p 123-124 NOT ON EXAM. ***NOT ON 
EXAM***** but he's going to tell us about it anyway...but it will NOT BE 
on the exam. (which is like, next friday, btw...) basically superceded by 
other ways of looking at abag interaction: electrophoresis, etc.

another method:
single radial immunodiffusion
you take a gel, cut holes, add different amounts of antigen. the Ag will 
diffuse out from the wells and cause precipitation (this is an Ab 
containing gel) in rings around the holes. you can plot these reactions 
as a standard curve. then if you use an unknown Ag concentration, you can 
match it up with the standard curve. this is used in clinical settings 
due to the simplicity and ease of this method.

immunoelectrophoresis led to outdating of some precipitin rxns. 
basically, you take an Ag mixture and run it on a gel so that the 
components separate out by charge. then you can make a large trough in 
the gel, put in antibodies, and look for a variety of precipitations.

but even that has now been superceded by immunoblotting, where you can 
much MORE specifically look at particular antigens. (western blotting). 
this takes complex Ag, runs them on a gel to separate them out by 
molecular wt. normally when you do this you boil the Ag mix and add SDS 
which makes them all negatively charged, so you get separation by size 
instead of by charge. you take a test antibody and you see if it 
recognizes any of the proteins you have separated out. why is this 
important? you can use it experimentally and diagnostically. he says it 
is used in HIV testing and he's not sure about use in vet med but I know 
it has been used for FIV testing, because we did send out western blot 
FIV tests like 5 yrs ago from berkeley dog and cat and other places.

[missed some stuff- was putting on my coat--it's really cold in here)

ELISAs, competitive radioimmunoassay (cRIA)

cRIA: you have a particular Ag A, and you want to know how much anti A Ab 
there is in the serum. you can radiolabel the Ag, and you have a specific 
amount of A labeled with a particular amt of radioAntiA. 

in a particular test sample, how much A is there?  is the question you 
are answering. 

you add increasing amts of unlabeled Ag and get decreasing amts of 
radiolabeled Ag binding to the Ab. 

If you add NO unlabeled Ag, or "cold" Ag, then 100% of the radiolabeled 
Ag is bound. as you add increasing known amts of cold Ag, the percent of 
labeled Ag will decrease, and you can plot a standard curve. you can then 
come in and ask what is the percent of radiolabeled Ag is not binding 
when you then add an unknown amount of antigen (eg, you add test serum) 
and from that you can extrapolate how much Ag is in the test sample using 
the standard curve.

%bound radiolabled Ag/free Ag = y
amt of unlabeled Ag = x

--

so that's a competitive radioimmunoassay. but what we see more often is 
the elisa assay - enzyme linked immunoabsorbent assay. here you use an 
enzyme with a chromogen which gives a color change.
relatively simple assay - two main types.
one: an antigen is bound to a plate. question is, are there Ab which 
recognize the Ag in your test serum? proteins bind to plastic at 
particular pH levels nonspecifically. so you incubate your antigens 
overnight and they will bind to the plate, so that's how you start. you 
wash off the excess antigen, and then you block the plate (eg, milk wash, 
serum albumen wash) - eg, add irrelevant protein. this means the plate 
can no longer take up any proteins nonspecifically.

ok, so now you add your test serum and incubate. if the test serum has 
antibody for this ag, it will bind. you wash the plate again to wash off 
unbound stuff. then you add a ligand which will bind to the antibody - so 
that would be an anti antibody antibody. and you have that ligand labeled 
such that an enzyme will cause a color change. you're testing whether or 
not the serum has antibodies that recognize a particular antigen. very 
straightforward. you use positive and negative controls, of course. 

sandwich elisa: coat an antibody to the plate, then test for an antigen 
that reacts with it. eg, bind antiA Ab to the plate, then add test 
sample. if A Ag is in the sample, it will bind to the Ab on the plate. 
then you add antiA Ab  to the wells, and if A is there it will bind. then 
you add your third, labelled Ab. you can actually label the second, but 
usually do third, because it amplifies the rxn.


direct and indirect immunofluororassay: IFA
you have an Ab reacting w/specific tissue or cells. the Ab can be 
fluoresceinated (labelled). so you take tissue section and add the 
labelled Ab and see if it reacts. that's direct. or, you have tissue with 
Ab and you add fluoresceinated anti-Ab, and that amplifies it. this is 
indirect. it amplifies bcause you get many anti Ab molecules binding to 
one Ab molecule.


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