IMM 4.1 (PT 1) TRANSCRIPT PDF

Let's do lecture from my office today because at least I can guarantee with so many of you guys not in the lecture hall at least this time I can guarantee that audio everything else works because I'm in my office and connected to everything versus down in the lecture hall. So since there were techni...

Let's do lecture from my office today because at least I can guarantee with so many of you guys not in the lecture hall at least this time I can guarantee that audio everything else works because I'm in my office and connected to everything versus down in the lecture hall. So since there were technically only three people in the lecture waiting I do appreciate the understanding they had with allowing me to do this instead. And so this is where we'd all rather be snuggling Noah and Thor right now. Office hours this week will be normal because your exam is Friday and so tomorrow Tuesday it'll be four to five p.m. virtual which does work out really well so I don't share any of my cooties with y'all. Thankfully it's not viral which makes it easier but still don't get anyone else to get sick before the exams. Thursday I should be good everything will be fine by then I'm getting lots of sleep doing all the homeopathic **** I'm supposed to to feel better. So Thursday will be four to five in person and that should be in 3 16 j where it normally is. Now for the rest of the semester just so you guys are aware this lecture is indeed on module four and it's immunological techniques and here's the structure for the rest of it. Wednesday will be a vaccine lecture which is one of my favorites overall too. I'd say these two are probably my top two favorite lectures. And then your exam is Friday so we do have office hours that Thursday night. I fly out that morning for a wedding and so I will be reachable by email but that's probably the best way to reach me is like campus messaging basically which does come to my email too. And then that Monday because I will still be out of town at the wedding and visiting family. Monday's lecture is an asynchronous documentary and so your review paper is based on this documentary and so all information will be posted. You guys have a little bit of a week to do it. It's basically like a two-page double spaced paper and it's a lot of fun because it's you know supporting evidence for your beliefs. It's a pretty good project overall. And then we go into primary and secondary immunodeficiencies. Again notice that next Friday the 15th is also asynchronous because research days is from one to four and so all your BMS classes should be asynchronous that day. This is also the second attempt that you can get that one bonus point for this class and again you only get one total. So I do encourage you to go if you haven't already. It's a great chance to check out what research is happening at Marion as well as meet faculty, other med students, things like that and there's usually like snacks and coffee and stuff. Then we dive into transplantation immune pharmacotherapy and cancer. It kind of falls under like these three days depending on how much we get through. Then we dive into hypersensitivity and that's that Monday before Thanksgiving. So this is an asynchronous recording from one of our former student doctors Nate Pham who's doing his current residency in anesthesiology but he's a phenomenal lecturer and so we have a recording of that and that's intentional so that way if you do decide to head out for that week the recording will already be posted so you technically don't need to be there for my class. So if you want to disappear for a week to go be with family for Thanksgiving I encourage it. Please go have a wonderful time for me. Then we have Thanksgiving break that Wednesday and that Friday and we come back to autoimmune disorders. We'll do some what's wrong with new case studies which is interactive and usually including a lot of these different diseases that we learned about within all of these other modules. Then we have our final debrief when we talk about kind of synthesizing all the information and putting it together kind of for the end of the semester. So a lot of fun. I think students really enjoy this section just because we get a chance to talk about you know the clinical presentations of these things that we've talked about like what does CD40 deficiency look like and how does the immune system play a role in all these different disorders and diseases and so it's a lot of fun to kind of put this all together and kind of see what's going on. This is the one kind of example where we will have some math on the exam but you guys do have access to a calculator and I do always include easy numbers typically a whole number so it's you know an easy division like it's 100 divided by 10 it's 10 on purpose because I would like it to be you guys understand the concepts more than how well can you do basic math and so you know there will be math on it because we will talk about sensitivity and specificity and the equations to calculate this like what a true positive or true negative is. We'll talk about antibody antigen interactions which you guys should already know based on our foundation for you know most of the other semester plus especially module three with antibodies overall and we'll talk about affinity too specifically we'll talk about the precipitant reaction the quantitative curve in three zones involved in that which is a part of antigen antibody interactions. We'll talk about the different laboratory tests used and I do it does it say describe the protocols and the circumstances in which they would be used I'm talking about a broad overview I'm not going to nitpick on silly things like how many washes are you supposed to do you know the more important high yield information from that is like what what is the purpose of a wash a wash is usually to remove excess binding that's not necessary or excess particles that are there and so you know I don't care if it's three by ten minutes or three by five minutes I care about that you understand what a wash is and why we would do it at certain point so I'm not going to be super nitpicking about every single step but I want you to be able to apply the big picture premises and concepts and why we do the different steps. There will be a lot more clinical questions and you can see these especially incorporated into later lectures especially when we talk about the different diseases and which tests you would use to identify the diseases and so you'll see this kind of lecture intermixed with a lot of the other lectures we'll talk about the disorders we'll talk about the rest of this module too and so there's a lot of synthesis overall I do expect you to interpret results so tell me what this means you know if we see these certain values you know what does this mean for the patient is this test positive or negative things like that just very basically we will cover what the difference between a qualitative and a quantitative tests are and so I don't want you to be super nitpicking on like which one's which but in just very basics a qualitative test is usually a visual kind of yes or no there's no hard data we don't have concrete values it's not like blood work quantitative would be like if we had specific blood values that you know if you have greater than 10 cells it means you have this you know if we have 11 cells it's a quantitative number versus you know qualitative is just telling you if something is present or not and so typically anytime you see a color change but we're not measuring the actual quantitative values from that color change it's considered a qualitative test versus a quantitative is if we're like okay we need 3.095 before we can even diagnose a patient you know with a specific disorder that's what I mean by quantitative and so I want a loose overview and understanding of that now we talk about sensitivity and specificity they mean two different things even though you know the general public sometimes uses them interchangeably you know or uses them in place of each other unfortunately but basically you need to understand the premises behind a test because it determines you know how good that test is and what we should use that test for no test is perfect we frequently don't have anything you know worth 100 but we can get pretty pretty close in a lot of them here and so we kind of weigh out the sensitivity and specificity as we're making calculations of various things here and so for example how much emphasis should be placed on a particular test depends on these values so sensitivity by definition is the probability that the test will be positive in a patient who has the disease in question so sensitivity is basically measuring like hey if we have a covid test you know what is the probability that the covid test would get it right in a patient who has covid it's pretty simple like pregnancy test you know is the patient pregnant or not and we talk about specificity though specificity is the probability that the result will be negative in a patient who does not have the disease there is a minor distinction there because you know there are let's use the a pregnancy test for example there are some conditions and disorders where you have elevated hormones that can sometimes look like it's you know pregnancy but it's not there are other conditions that can cause this and so specificity is basically saying the probability that you know we can we distinguish um you know that the patient is not pregnant that's something else going on and so the sensitivity and specificity of the test can be determined by using a two by two table and so you will see some questions on the exam where you are given this table and so i won't include the values of true positive or false positive but i will include the x and y axes to say you know they have the disease or they don't test as positive or negative so you have to be able to determine you know which of these quadrants is true positive or true negative first and then you have to be able to calculate the equations for each so sensitivity is basically taking your true positives divided by the sum of your true positives and your false negatives versus specificity is taking your true negative and dividing it by the sum of your false positive and your true negative and so you have a couple different consequences when we go through this so a true positive is an outcome where the model correctly predicts the positive outcome a true negative by definition is an outcome where the model correctly predicts there shouldn't be an outcome or the negative outcome a false positive which sounds as when we say it you know there's a positive when there isn't so it's an incorrect prediction of a positive outcome and a false negative is an outcome where the model incorrectly predicts the negative outcome and this kind of contributes to error like type one and type two error if you ever talked about that with psychology and so if we're going through the actual examples here so this is a made-up disease known as examineous paralysis as you guys can gather it's a scientific term for the disease where students freeze up on an exam or brain freeze and so this is data from a previous experience and so we have the graph here in this column we have students who freeze in this column we have students who function now in this row the test that we've created diagnosed examineous paralysis shows that the test is positive and in this case it's showing over here the students where the test was negative and so this is students who freeze where the test was correctly positive this is tests where the students who function but the test was positive which means it's a false positive and then down here we have students who freeze and the test is negative so it's a false negative and over here we have that false positive here or students who function but it is negative i'm sorry true negative i said true negative i said it was a false positive it's true negative over here and so it does line up with these markers here overall and so we calculate the sensitivity we're taking that true positive value here and dividing it by your true positive plus your false negative so these guys over here a and c and then your specificity is taking that true negative which is this guy down here and then dividing it by the sum of your false positive and your true negative over here and so what we end up getting with that is and here's the actual breakdown of the math overall again it's this plus a plus c and that gives you the value if we were trying to convert your decimals into a percentage you always multiply by 100 and that gives you 77 percent versus down here you know when we divide up the math and actually put it together we get 0.8 we multiply it by 100 to get a percentage it's 80 overall so you will be excited to do stuff like this however when i give you these tables it will be given to you like this without the designation of true positive or true negative or anything like that so it's up to you to figure that part out but again i will pick whole numbers and things that are easy to divide by like six divided by two because i don't want it to be oh can you actually do math like can you get the numbers right i care more about do you know the difference between these two and can you actually define them and calculate it properly and so it's not going to trip you up on the math it is considered at that point easy math you do still have access to a calculator in exam soft in case you panic but again i choose intentionally easy numbers because i want you to show that you understand this more than you know can you divide 1593 divided by 1593 plus 473 that's a lot of now we talk about antigen antibody interactions this is the foundation of pretty much all of these different tests because as you guys know antigen binds to antibody and it's some of them are some of the most specific non-covalent biochemical reactions known to man basically they can be represented by this equation here antigen binds to antibody giving us antigen antibody complexes and for the most part the formation is skewed heavily in this direction that's why there's a longer arrow because binding is easier than breaking apart so we do have a smaller arrow pointing in the other direction because it is possible various different chemicals and proteases and things like that to break these down and separate them back in this direction but again the equation is heavily skewed to form these complexes here and so we talk about affinity a definition overall it's the strength of the interactions overall so you have this antigen antibody association over the dissociation of it means you know basically it's skewed more towards you have stronger affinity or likeness towards the binding together and so different immunoglobulin molecules within an individual actually have a wide range of affinity and we frequently talk about like we talked about affinity maturation as well but you can also think about affinity like with the fact you know that certain ones are better primed to bind each other so that affinity maturation overall especially as we go through additional training and you know point mutations with that somatic hypermutation to make the binding more efficient overall so the valence is the number of epitope binding sites in an immunoglobulin molecule and it can vary from two which is in all those monomerics think about that Y this is one binding site that's the other on the Y molecule versus and so that's the monomeric form because they have two arms unless it's functionally monovalent like that IgG4 where each arm can only bind one separate item and so it can also have four valences which is like secretory IgA that dimeric form because it's two of those monomeric molecules stuck together so two plus two with those binding sites gives you four versus when we talk about the pentameric IgM because it has five of those monomeric things stuck together by the j chain we get 10 total binding sites and we talk about the avidity of a molecule the avidity is the sum of the affinity with the binding sites of the multiple binding sites so it's basically affinity you know with valence overall to give you this total amount and so if you have really good affinity and it's an IgM you have phenomenal affinity because you have 10 binding sites for this high affinity you know antibody at this point and so that's the overall term for that and so that affects overall the binding that we see in the precipitation basically and so we call this the precipitin reaction it's you know term based on precipitation precipitin here by definition is the re-interaction of soluble antigen with soluble antibody that results in the formation of those antigen antibody complexes and in this reaction you'll see that this form is the lattices and so it's kind of like that clumping in agglutination that we see this is the foundation behind agglutination assays which is large enough to precipitate from solution so again we looked at like that covid test a couple classes ago with effector functions that when we have binding of that antigen to that antibody you saw that you know that clear blue solid like liquid where everything is uniformly blue across to this area where so white liquid but clumps of blue throughout and those are the precipitation that is forming within that test specifically here so it's one of the first quantitative assays because we can actually measure it overall so technically it could be considered qualitative too because if we visualize and just see clumping just off the bat we can say hey yes like we have antigen present it's binding onto the antibodies or you know antibodies and antigen binds on we can see there's antigen present and so anytime you just visually see clumping it's qualitative but we were able to do this to create it quantitatively and how we did this is by doing testing so we do dilutions i don't know if you guys have ever done dilutions in a lab but basically we create a lot of different variables overall and so we are diluting it so it's more you know dilute overall basically means there's less of a solute within it basically here and so when we have kind of an even dilution in the middle where it's equally dispersed between antigen and antibody it's like our peak of it and that's the zone of equivalence here too but because of this depending on what results we see we've taken a lot of computers and algorithms and taught them what you know these these values will visually look like in order to quantify and so if you guys have ever seen um you know like in the Liza test which we will talk about you know we have standards we actually do the dilution and we can measure exactly what the color looks like and tell the computer that like if it's this shade of yellow it means this much you know antigen antibody is present and so we could actually get quantitative values from qualitative tests we have these standards in place in this case the various dilutions from the bottom in order you know to educate a computer and like these testing devices for reading on what is present here and so we talk about the quantitative precipitant curve that's when we created by mixing and incubating varying amounts of antigen which we in this case kept in a constant volume with equal and constant volumes of anti serum which contains antibodies so we get anti in front here and so a similar curve can be generated if we flipped it or we kept antigen constant and varied the amount of antibody again we're trying to bind antigen with antibody you can either start with antibodies then add antigen or start with antigen and add antibodies it doesn't really matter and so it will end up forming three separate areas within the curve and so we're able to describe you know qualitatively what's present and so on this left side here where we have a lot more antigen but not enough antibody overall so that's what's kind of shown in this graph here it's known as the zone of antigen excess we have too much antigen and not enough antibodies to bind it and so it's a consequence you get these small soluble complexes which can't really bind onto each other and as a consequence we really don't see precipitation occurring just because we have too much antigen and it's affecting our ability to test versus when we talk about this zone of equivalence it's equal amounts basically here in equal ratios of antigen with antibody and that diluent overall and so we see equal amounts also known as equivalence which leads to these large insoluble complexes and that's what actually causes that clumping that we can visualize kind of on those like blood ***** tests or that covid antibody antigen test we looked at earlier with the coloring on the other end and the extreme other end of things we have the zone of antibody excess and in this case we have too much antibody compared to antigen and the same thing occurs because we have too much antibody enough not enough antigen we still don't get those complexes instead we get antibody excess leading to those small soluble complexes which we don't see precipitation overall and so it is critical overall because we can have some false positives and false negatives due to the fact that we have too much antigen or too much antibody and so that is kind of critical and that's actually kind of the premise behind why they told us and why they still tell us to do multiple covid testing because again depending on you know how sick you are at certain points say you have a ton of antigen in your body right now and not enough antibodies and you overwhelm the antibodies you know those tests are going to be negative falsely because you're in that zone of antibody or antigen excess versus you know if we're looking for something with antibodies and we don't have enough antigen to test for it oops we're going to have another false negative so it is important overall kind of that we have a good ratio that's again why they tell us to test multiple times if you think you have covid so the amount of precipitate formed depends on the ratio of antigen to antibody and is also affected by the of the antibody and that's why we frequently use IgM when we're creating these different tests just because again it's a vidity it's a valence plus the affinity we pick a high affinity IgM and because it has 10 total sites for binding it has high valence too and so we end up having a high ovidity overall that's why we frequently use IgM IgG is kind of the second best one that we have but again we do need a lot of that because as we talked about with you know precipitation and things like that and agglutination you need like two or more IgG molecules for that to occur and so again continuing this is just the definitions of what we just talked about here but with the zone of antigen access or AGX excess it's insufficient antibody to form large lattices and so the antigen antibody complexes are too small to precipitate leading to the formation of these soluble complexes so you don't see clumping or precipitation that equivalence zone by definition is where you have optimal precipitation and that occurs in that area here and you'll see large lattices again do the ovidity of IgM and you'll see visible precipitating complexes that's when we start to see the clumping on the test like when you knew what blood type you had because you saw the blood clumping on the little paper then on the opposite side again you have the zone of antibody excess where you don't have enough antigen present to form large lattices and it leads to again the formation of these soluble complexes so you don't see clumping you're not able to tell that it's positive and so again this pretty much applies to all the tests we're going to be talking about because you kind of want this ideal zone of equivalence as we're going through these various tests and that's why we frequently use standards and to create these ratios as we're actually going through and creating these tests overall and so this is the overview of kind of all the different testing we're going to go through today basically we have epitope detection where we're looking for specific molecules with antibodies here and so we can use something as particulate antigens which is bound onto something or soluble and so when we talk about particulate you have two different types direct agglutination or indirect or passive agglutination and we'll talk about those in a little bit but you guys know direct is the most direct path indirect is a few more steps with soluble antigens we can do radioimmuno diffusion or double diffusion or even immuno electrophoresis are the different techniques we'll talk about there we do epitope quantitation by antibodies this is again where we are actually telling a computer you know what the different color changes means in terms of quantitative value so we can determine an actual lab value overall and so the three ones we'll talk about are radioimmunoassays and there's a direct which you guys know is the most direct quickest test and indirect which has a few more steps involved then we have fluorescent immunosorbent assay or FIA which we will talk about and as you guys know there's fluorescence involved you also have enzyme linked immunosorbent assay ELISA's frequently done in various lab tests and again in case you've never done one before i do have like a link you can click on if you want to do kind of like a fake online ELISA test just kind of go through and teach yourself how to do it say hypothetically we want to look for epitope detection in and on cells so this is kind of looking at like serum saliva other things like that but if we want to do specific like cells like we want to do blood screening for cancer or we take a biopsy of a tissue we want to stain for stuff too we can do either immunofluorescence which is known as IF and we'll use monoclonal antibodies for that we used actually monoclonal antibodies for a lot of these guys here too and then flow cytometry which you've talked about before so you will see again similar results on this test here too you can also see assessment of immune function so this is slightly different this is more so looking at the functions of the test so all these guys are just looking for specific you know epitopes present or antibodies present this is specifically making sure the cells can function and so some of the different tests that we'll do is a phagocytic function test to make sure your phagocytes are phagocytizing you have a proliferation test to make sure your cells are properly proliferating or in like the case of cancer if they're proliferating too much we also have a cytotoxic T lymphocyte assay where we can actually test our cytotoxic CD8 T cells to make sure they are indeed killing because we have some disorders where they don't kill enough due to whatever reason which we will talk about a couple of those and so that can make sure that the T cells are indeed killing what they're supposed to and then we have assessment of hypersensitivity and this is frequently looking for IgE or IgG antibodies since those are heavily implicated with allergies overall and so we'll talk about allergy skin testing which is just basically looking at the body's response to these different antigens and so we first move in and discuss epitope detection by antibodies again these are the different subtypes we're going to talk about but again heavily based on that precipitin test that we just talked about here and so many clinical lab tests are based on the specificity of antibodies for antigen and their ability to recognize these epitopes on the antigen and so they are epitope detecting tools and most are based on the quantitative precipitin curve actually most of us we'll talk about today are based on this and so again you know with particulate antigens we're looking at something bound on something like an antigen on a piece of bacteria versus soluble you guys know these are tiny pieces of something that we're looking for and so again based on all of this that we talked about before you know again when we talk about particulate antigens these are things like so they're whole molecules so like erythrocytes bacteria even antigen coated coated latex beads so they're like thicker larger molecules where antigen is bound onto it that is what a particulate antigen is and they are typically normally evenly dispersed in suspension so this is again what we would see if we had particulate antigens evenly dispersed when we have cross-linking in that clumping together of these different things of the antigen bearing particles by antibodies will disrupt the homogeneity of the liquid thus producing the clumping of particles so basically it's the premise of agglutination but we're talking about again that precipitin reaction heavily synonymous with agglutination overall and so what we're seeing here you can have what's known as a direct agglutination test where you have iGM antibodies already directly ready to bind onto the antigen and if they efficiently cross-link the particles you'll get this clumping here you have what's known as an indirect or passive where you first need to use an anti-immunoglobulin molecule so basically you're connecting two immunoglobulin molecules to each other in order for the clumping to occur so there's an extra step involved in that and so that'd be like if we added a solution first and then needed to add a second solution and then we saw clumping that is indirect or passive agglutination here and so we're still using it to cross-link antigens there's just an extra step in the process here there's another distinction you need to make too especially with laboratory tests if you ever see the term hemagglutination this is specifically referring to agglutination and clumping seen with red blood cells and so it's a specific if you see the term hemagglutination test we're just looking to see if we can have clumping of those red blood cells and in this case we were just trying to do a simple blood test to determine what the patient's blood is and so here you know they added antibodies to type a blood and here they added antibodies to type b blood that's why it's called anti-a and anti-b here you can see clumping here because there's no type b present there's no antigen on those erythrocytes versus here we had antibody to a antigen and the blood cells just happen to have an antigen on the surface thus leaning to that clumping overall and giving us the result that the patient does have with the hemagglutination test but they do have type a blood overall and again that direct agglutination that we talked about usually uses that IgM antibodies to cross-link those epitopes because again it has high avidity with the number of affinity overall again it is large semi-unoglobulin it is very good at cross-linking so we do have other isotopes that are a little less efficient because they're a smaller binding site but we can technically do this with IgG especially with a few more molecules of that overall and so again very similar to that quantitative precipitin reaction we talked about before if you have too much antibodies obviously it inhibits agglutination if you have too much antigen it inhibits agglutination too and so there's something else we want to talk about with this too and it kind of ties into disease that we will talk about especially in micro you have this thing known as prozone which is a you know period typically of non-specific symptoms and usually at that point so like kind of what I was experiencing last week where I knew I was feeling crummy I was feverish had a headache just felt bleh but I wasn't showing any specific disease like symptoms yet it's a prodrome disease basically where you're early on and it's linked to prozone because it means I'm kind of early enough in infection where I probably don't have you know enough antibodies present to show up you know in this case overall to show up for a test so kind of that non-specific area overall and so it's kind of before I'm sorry it's before you have enough antigen overall because you have enough antibodies for this testing so say hypothetically I did have COVID which I don't have tested like three or four times at this point because I was kind of worried when I was in this period here but again if I'm early enough in infection where I don't have enough antigen you know replicating in my body and I'm still taking the COVID test and the swabbing if there's too much antibody on the COVID test and the antigen that I'm you know getting from my nose is not enough to bind I won't see it because there's antibody excess and so it's that prozone period when I'm kind of too early in infection which is why again they do recommend testing multiple times because again you know as you start to get a little bit more infectious there's more antigen present in your body you know you are more likely to test here and so that's frequently too why we do dilutions of antibody and that's again when you're at home why they recommend doing multiple COVID testing overalls to kind of rule out that effective prozone where early enough in infection you're in that prodrome period where you're not really showing specific disease yet you may not have enough antigen to test positive yet and so in a lab what we can do with that is actually use dilutions of antibody overall and we'll add it to identical concentrations of particular antigen and do that dilution curve you can actually do it in the lab to make sure and so say hypothetically you know you do get a patient sample and you have the access and the ability in a lab in order to test if you think a patient is early enough in infection you can actually dilute the antibody solution and if you see binding at that point when you're kind of getting rid of that excess and you're kind of more in that you know equal balance and you see it positive then you know that the patient was probably in prozone and you just didn't have enough sample yet and so it is important to know that we typically do dilutions of antibody or again if it's an at-home example that we do repeat tests and so that eventually hopefully we will catch it like say you tested too early and that's why again they do recommend with pregnancy you know waiting two weeks until after your missed period because usually by that point you have enough hcg in your blood where it would be detectable really in pregnancy you don't always see that and so that's important and why we have the premise of kind of waiting a little bit longer to test or again you know say you tested two weeks after your period and miss period and you know still shows negative but you wait another week and test again you can still be pregnant and so that's why we do have those you know repeat at home concepts. Another definition here too titer which you guys have all heard like vaccination or if you've ever had to do measurement of your immune function like I think you get to get your MMR titers to make sure you guys are immune to MMR. It is by definition the lowest concentration of antibody that causes agglutination so we could actually you know tell you know how much antibody you guys actually have in your body to tell you kind of how immune you are to certain things you know by taking blood samples and adding it to cultures with antigen to like measles, mumps, and rubella and seeing how much binding occurs and if there's a low titer it's that lowest concentration of antibody and so that causes agglutination we can actually test to determine what your titer is to antibodies to see how immune you are to certain things which is super awesome overall and so we frequently report these guys as a reciprocal of the dilution so we'll see it as a ratio like 1 to 16, 1 to 32 things like that. If you've done an MMR titer recently they do report in slightly different values but they will tell you kind of if you're within the normal range or not. If you don't have enough antibodies I usually encourage you to get another vaccination to kind of boost your experience and increase those antibody numbers overall. In the same kind of concept of this direct indirectors has an extra step added again we talked about how you need a secondary antibody with this and so you have this particulate antigen here we have this primary antibody which is specific in this case they're using IgG it is specific for the specific particle antigens particulate antigens here but in order to cause those clumpings you need a secondary antibody to cause this clumping and so those antibodies are going to bind onto the FC region like their fab fragments are recommended to bind onto the FC fragments of everything else thus leading to this clumping and so again we frequently use this with non-IGM antibodies like IgG we'll frequently use primary and secondary steps with this and so the sensitivity may be enhanced by the addition on this anti-immunoglobulin reagent like in this case we could use a rabbit anti-human immunoglobulin which is kind of cool and these are known as second step antibodies or secondary antibodies overall because they increase the binding over a greater span and they increase the valence because again you have more binding sites they attach on to each other by their ability to bind the primary antibodies this is a technique you will use with frequently non-IGM antibodies which already have phenomenal acidity but this kind of increases that validity overall validity yes uh avidity overall not validity vidity overall because it increases their valence and you guys know vidity is the combination of that valence plus the affinity overall now when we talk about soluble antigens these can be things like found in serum or just tiny pieces found throughout and so they can still precipitate from solution on reaction with the right amount of antibody so we still talk about the precipitum curve with it too and so we use it to prepare several different antigen antibody samples and so oftentimes it is too difficult and time consuming to find application in clinical laboratory though and so that's why we have simple modifications involved like this radioimmuno diffusion here where you actually see the precipitants in agar which is this kind of like jelly-like substance in a petri dish here it's a semi-solid growth medium kind of looks like clear jello they can smell like different things depending on which type of jelly you make overall and so there is a short video on it we're not going to watch the whole thing but i do want to point out that it is useful overall i'm going to watch the visualization agar well this is not a very useful video but you actually measure the precipitin ring which we will talk about a lot with micro and so it is a useful visualization for that aspect here but when we're talking about it basically what happens is we'll take this agar gel with a uniform distribution of you know antigen in it so we'll take soluble antigen with an agar gel and so we have that uniform concentration then kind of pour it in this plate we also call this the mancini technique if you've ever heard of that but basically it allows us for the rapid and precise determination of the quantity of antigen loaded into the wells based on how wide the precipitin ring is and so again if you put antibody in this case they're using antibody in the gel you could use it's flip-flop so if you put antigen in the gel obviously your test sample would have antibody or if you put antigen in the gel or antibody in the gel you put antigen in the solution so you can use those interchangeably here but say for this example we are using antibody in the gel we want to see how much covid is present in the patient's sample what we can do then is take that solution and add it into a hole in the middle of the agar gel and those antibodies and solutions will diffuse out into the gel depending on you know they'll keep going until they weren't out of stuff to bind with and based on how far that curve goes and that ring goes that precipitin ring which is the space around it we can determine kind of what the binding site is and so if you have a lot more antigen present it will spread farther out because it has to bind onto a lot more antibodies and so the ring will be larger as a consequence of this and so the ring diameter is directly proportional to antigen concentration you know if we flipped it we had antigen in the gel and we added antibodies here we could measure how far out they go here too and it allows for the rapid and precise determination of the quantity because we can actually use a ruler to measure how far out you know this ring is on the exam I would do something very easy where it's a very very clear cut one is a larger ring and a larger precipitin ring than the other so it's just a quick visualization of which you know has more in solution and so again this could also be qualitative for just reporting on like you know this ring is bigger we use this test a lot when we're looking at whether antibiotics are effective because we also see that zone of clearance with it and again we see you know if the bacteria can keep growing despite the antibiotics you know you won't have any kind of precipitin ring versus if you do have um you know antibody I'm sorry antibiotics that work really well that don't let that bacteria grow um you will see kind of a zone of clearance around it which is kind of cool and so I may have flip-flopped that I am tired you guys uh no if it grows right up to the thing it means uh it is susceptible or resistant to the antibiotic but if you see this clearance it is means it's susceptible and you can use that antibiotic um overall and so we frequently again take that petri dish we use that molten agar which in this case we can use antibody in the gel for this example here too when it cools and solidifies we cut a hole in it or well is another term that we use for that and then we pour that antibody solution our antigen solution inside and then we measure how far out it diffuses basically here and so we can determine again using standard curves if we know a specific amount of antigens and we have standards saying that like if it moves out this far there's 10 pieces present or if it moves out this far there's 100 pieces present and so that's why standards are important to be able to turn a qualitative test or yes or no into a quantitative where we can actually directly compare and measure specifically overall now we do epitope quantitation by antibodies here so this is again especially if we're using things um so basically we're using things bound on moving into kind of soluble stuff here too uh we will talk about these guys here uh and so actually hold on I just realized I skipped so sorry you guys I did realize I cut this stuff out so I would basically just define that we know that these guys can be used with soluble antigens overall and so we did talk about the radial immunodefusion but for the other guys I don't expect you to know more than this double diffusion is a slightly different test there's two steps to it and so just know that these fall under your soluble antigen test but again I forgot for shortness of time I did cut it shorter and so just know those guys are there and now I'm going to move into this second section here of epitope quantitation by antibodies overall and so here the specificity of the antibody molecules makes them ideal for a wide detection of different epitopes and so antigens or the antibodies can be labeled with radioactive molecules or fluorescent molecules enzymes or even heavy metals in order to produce a color change that we can see and then we can again tell a computer that like if it looks like this shade of yellow this is how much is present using those standard curves we can actually quantify this here too and so the antigen or antibody binding can then be readily detectable and quantifiable again using those standards and so here's a good video on the radio amino acid if you'd like to watch I will also I think briefly touch on direct or indirect examples again direct is faster indirect has a few more steps and then you have your fluorescent immunosorbent acid and your enzyme linked immunosorbent acid so again these are going to be more so for your soluble molecules and fluids and things like that overall here and things that are bound so we talk about a radio amino acid it is sensitive but risky because radio as it sounds there is an exposure to radioactivity and radioactive waste and so this is something that we do not recommend with pregnant individuals because you don't want to be exposed to radiation they are moving more away from this now that other testing methods are more effective but it's something that you can see in certain places here so when you use a direct it's shown on this side it's a little bit faster again the antibodies bind directly on and in this case little stars here show that this primary antibody has been labeled with a radioactive fluorophore and so if there's binding that occurs we use a computer or other device to measure how much radioactive is present and so here it does use radio labeled antibody or its ligand and antigen in this case and then antibody is incubated with the ligand and unbound reactants are removed so this is when we use a wash basically or phase separation we're removing excess unbound things and measuring the quantity of bound present so that's known as phase separation specifically we use an indirect this uses radio labeled secondary so in this case we have primaries that bind onto our antigen of interest but then we need that radio labeled secondary to bind on to our primaries to cause that clumping and be able to measure it at this point here too and so we do use phase separation methods to remove first any unbound antigen here and then any unbound secondary too and so there's usually more washes and phase separations with this too so that's one example again very similar concepts but using radioactive now when we move into fluorescent immunosorbent acid this is one that is preferred same with elises because again there's less risk so you're not using radioactive nuclides of any variety present here but it's relying on antibodies or their ligands labeled with various fluorescent dyes so we put fluorophores on there they're basically labeled to glow under you know exposure to light and things like that we can use lasers and we can use leds and so we have various dyes if you guys have ever done staining you know fitsy is fluorescein isothiocyanate which is a green color or you have phycoerythrin PE which can be like uh you know pinkish purplish and so it is specific and relatively sensitive overall it does not have hazards associated with radio nuclides like RIA the phase separation of antibody and ligand in this case antigen by immobilization of one reactant onto a polystyrene prior to addition of fluorocorium labeled reaction so what this means is we'll add the antigen solution and get it to bind onto the bottom of a well and so we'll typically use like a 96 well plate which basically means it's a plastic plate with 96 little holes in it it's like a petri dish book a plate square plate or rectangular plate with 96 little circular wells that you can put samples in here too we'll wash off anything excess that didn't bind so it's kind of that phase separation here and in this case they're doing an indirect where we add that unlabeled primary antibody so it'll bind onto any of the antigen down to the bottom of this plate will wash off any excess because again excess can produce false binding and things like that and in this case we'll add that fluorochrome labeled secondary antibody and so it has that color shown to it it will bind onto those primary antibodies here again we wash off any excess so there's not additional fluorescence showing up and then by exposing it to a light if there is binding that fluorophore will glow and produce a color and we end up reading the fluorescence off of that so it's really really cool fluorescence means binding and so we can actually measure how fluorescent something is from this test overall so this is specifically with fluorescence overall when we talk about ELISA notice again it is a very similar picture to what's here with the exception of we're using an enzyme labeled secondary antibody so we don't see fluorescence and the cool thing about this is we frequently don't need a light source in this case to activate it instead we need a chromogenic substrate to trigger that enzymatic action and produce color and usually with ELISA you know we see more of less of a fluorescence color more so like a blue or a yellow like it's very clear cut you don't need any additional things to be able to see it the enzymatic reaction produces a color visible to the naked eye which is nice so you don't need all these extra devices you know to be able to measure it like with a fluorescent um immunosorbent assay here it is specific and quantitative and it's replaced RIA in several tests because again no radioactive um and it is you know very safe it's pretty quick and efficient and very speedy overall and no radioactive decay reagents are relatively stable pretty cheap overall and so the sensitivity is actually greater than or equal to RIA of fluorescent immunosorbent assays because it's an enzyme labeled reaction to turn a chromogenic or colored basically shrubs substrate to go from clear to showing color and we can actually measure how much is there based on how dark that color is you'll frequently see it in like blue or yellow blue is easiest to see frequently with a bright blue and so the darker the blue you know there's more binding present so if you increase the substrate incubation time overall it does allow or lower concentrations of enzyme to convert more substrate to enhance tense test sensitivity so within limits like you don't want to leave it you know for four days in hopes something binds but this is kind of our way of dealing with the fact that like if you have too much antibody or too much antigen it kind of addresses that factor by you know allowing it and the enzymes to stay on a little bit longer it's like within one or two hours you can actually then you know make it a little bit more specific for low concentrations which is pretty nice overall if you've never done it before you've never done an ELISA here's a really great resource online to go through a virtual lab in practice you don't need to do it for the test but it is useful if you've never kind of seen the premise of it you're kind of curious of what it looks like i will do some practice exam questions with you guys where you will actually see you know color changes i can show you what it means overall now moving into my favorite test is if we're using detection in and on cells this is what my lab uses quite frequently because i'm in immunology so i care about the immune cells and that's what i'm trying to you know focus on and so we're looking at epitopes expressed both in and on the surface of cells may be detected using either radio labeled enzyme labeled or fluorochrome so again the fluorescent labeled antibodies overall and so the main one we'll talk about is immunofluorescence you'll also see this term as immunohistochemistry for kind of using a enzyme labeled you know color change like DAB i showed you guys with that germinal center i was at brown and i was like you could do one saying that's kind of it i prefer fluorescence because we can do actually multiple stains and we can actually for multiple different epitopes on one sample which is really nice and so the premise with this you'll have cells or tissue kind of on a microscope slide a glass slide basically overall and what we could do is a direct which is the fastest or an indirect which means longer steps here in order to stain and see if whatever markers we're looking for are present so with me i'm looking for specific markers of different brain cells and so i can actually pick like if i know a neuron marker looks like this and an astrocyte marker is this i can add those and see which cells in the brain are neurons or astrocytes which is super super cool overall and so again we use those fluorescent dyes because it's immunofluorescence in this case here too and so we couple them to an antibody and so if it's a direct coupling here where it's bound onto that first primary antibody it's in its direct fluorescence and direct immunofluorescence and we'll see a color present when we shine a light on it using a fluorescent microscope versus an indirect we have this unlabeled primary which does bind our antigen of interest but then we need a fluorescent labeled secondary antibody to bind onto our primary antibody to produce color change when it binds and we shine light on it here and so this would be like with fitsy we're looking for a green stain basically here and so you know we do washes in between each you know incubation to make sure we wash off any excess so there's not inappropriate binding of any variety here and then we will measure if the epitope is present by visualizing it and looking at it under a microscope overall which is pretty cool and i'll show you guys some pictures kind of next class since i don't think we're going to be able to get through all of this here so this is a little bit more of what we talked about with flow cytometry with last class but it does fall under you know detection in and on cells because we can use this you know to measure different types of blood cells and especially if you want to differentiate different types of small leukocytes between B cells and T cells and what subtypes of that we can use this overall and so again we're mixing a cocktail of different fluorescent antibodies to bind on different targets in a mixture of cells usually from you know patients blood either we can then see you know which are present because each one will glow a different color depending on what we've you know labeled for each marker and so we can label you know markers for T cells blue and markers for B cells green and then we actually measure kind of the colors that go through determine like how many blue T cells having green B cells whatever we're going to do in that case and so again it does a tiny dot for each cell that's present and allows us to measure it and so we're adding a little bit more on here too because we do gating strategies with flow cytometry you can do cocktails i think about to like 15 different antibodies now and so obviously a graph is x and y there's only two things you can look at so what we'll do with the gating strategies we can actually pick different types of graphs we're picking like what the x and y axis is based on the different you know antibodies and things we've added in there to select for more precise cells and so again like going through this one it's showing we're looking for CD45 and CD3 obviously cells with high levels of CD3 are going to be T cells and so again we're looking at this graph and you know CD45 is expressed on T cells too we would select that population and then of this population we then tell the computer that in the next graph we want to take these CD3 positive cells and further split them into ones that stand for either CD4 or CD8 and then we can go through and actually split out the populations to make your CD4 T cells or your CD8 or the ones that are kind of weird and in between and expressing maybe you know either or or ones that are expressing none of those and so it is a pretty cool test overall so we'll stop here and pick up a lot of the stuff actually nice class this actually works out really well that we are doing technically probably both of these i recorded in my office just because you guys are studying for exam three right now but we will add a lot more examples of these tests as we kind of go through the semester so if you're not able to visualize it let me know there's a lot of resources out there to help you picture kind of what's going on and i will start to include more results of what these tests look like so you guys can get a little bit more familiar with that as we go on but good luck studying and then again obviously i'll pick up whatever i didn't get to today with some of the lecture for wednesday as well all right thank you guys so much