<p>Fundamentals 1: 11:00 - 12:00 Scribe: Lauren Paul Friday, September 25, 2009 Proof: Kristina Hixson Dr. Burrows Antigen-Antibody Reactions Page 1 of 5 I. Antigen-Antibody Reactions [S1]: II. Learning Objectives [S2] a. We’re going to talk about interactions between antigens and antibodies. What makes a good and a weak antigen. b. Well talk about both in vivo and in vitro. III. Structure of an IgG antibody [S3] a. We just looked at our antibody structure. b. Red part is the antigen binding part—variable region. c. Blue part is the constant region. IV. Antigen-Antibody Binding [S4] a. There’s been a lot of actual structures of antibodies and antigens discovered. b. Just looking at these three pictures you can see that antibodies are able to recognize multiple shapes on molecules. c. Here we have a round ball dropping into antigen binding part of antibody molecule. Here’s a situation where the epitope of the antigen looks like a hot dog. Here we have a large epitope covering the entire surface of the CDR3 region of the molecule. d. They can recognize just about anything. V. Features of the Antigen-Antibody Interaction [S5] a. Features of this interaction are pretty typical of any protein-protein interaction. i. Reversible interaction—antigen and antibody can come together and come apart. ii. Non-covalent—no covalent bonds between the molecules. VI. Non-covalent binding [S6] a. Some of the forces involved in protein interactions are listed here: i. Electrostatic forces—positively and negatively charged AAs interacting quite strongly. ii. Hydrogen bonds iii. Van der Waals forces iv. Hydrophobic forces—AAs that like to interact with each other and they don’t like interacting with water. b. H-bonds and Van der Waals are most important for antigen-antibody interactions. They hold them together. c. The antibody is folded up into the immunoglobulin domains and all the hydrophobic AAs are inside the domain and same with electrostatic forces. VII. Features of the Antigen-Antibody Interaction [S7] a. When we talk about interactions between molecules, they have affinity—the strength of binding between the two molecules; the ease of association and dissociation. High affinity—doesn’t come off easily. b. Avidity—have at least bivalent antibodies, so even if there is weak affinity, we have avidity which is summation of two antigen binding arms. It’s basically the summation of multiple affinities. c. For example, IgM have very low affinity but high avidity because they have 10 arms. They are always coming off but there’s always another arm around ready to latch on to the epitope. VIII. Antigen-Antibody Binding [S8] a. Same picture as before except the third cartoon is different (this is what he’s referring to this whole time). b. We have an epitope binding to an antibody and taking up the whole CDR1 through CDR3 surfaces. You can also see that if the epitope was half of what it once was, the affinity would be less because there’s less surface area per contacts mediated by H-bonds that can stabilize an interaction between the antibody and epitope. IX. Terminology[S9] a. Antigen is a very old term and originally meant able to generate antibody. Now it’s defined as a substance that can be recognized by antibody or T cells b. Immunogen is a substance that can actually itself generate an antibody response. c. Usually synonymous terms and used interchangeably but sometimes they aren’t which we’ll talk about in a minute. X. What makes a good antigen[S10] a. Size—large molecules are more immunogenic so they generate better antibodies. b. Chemical composition—proteins more immunogenic because they have complex 3D shapes with bumps and grooves as opposed to carbohydrates which tend to be repetitious (or lipids). c. Similarity to self-antigens—so if we took someone’s albumin and immunized their neighbor with it, they probably won’t make any response, say there was one AA difference. If we took your albumin and immunized a chicken, where there’s a large difference, there would be a large response. Important because we obviously don’t want to make antibodies to our own bodies and sometimes we do and we get auto-immune diseases. So therefore the further distant and less similar antigens will produce a larger, more powerful antibody response. d. The way you give the antigen have some affect on whether its immunogenic. Usually if you’re getting immunized with something you can get an IM or SubQ intermuscular. Oral immunization is weak. Fundamentals 1: 11:00 - 12:00 Scribe: Lauren Paul Friday, September 25, 2009 Proof: Kristina Hixson Dr. Burrows Antigen-Antibody Reactions Page 2 of 5 e. Adjuvants—non-specifically prime up and boost up the immune response. Most vaccines you get, like tetanus, are actually not just tetanus toxide protein; they are complexed with almunimum hydroxide or some other almunimum salt called alum (adjuvant for humans). Stabilizes the protein so it stays around longer and may also stimulate some other receptors that prime up the immune system. f. MHC—we’ll talk about that next week. g. But essentially what makes something immunogenic is it’s complexity—if it has a lot of epitopes you can stimulate a lot of B cells and make a lot of different antibodies, and it’s foreignness—the further distant it is from yours, there are more epitopes you aren’t tolerant to. h. Next week we’ll talk about T cell responses. XI. Some well-known antigens [S11] a. <audio cut out> b. Polio is live vaccine and can replicate in your intestine. One form of vaccination is given orally and is very powerful. XII. Terminology [S12] a. Antigen and immungen are usually synonymous as we talked about before but here is a situation where they aren’t (when you use a hapten). b. Hapten—non-immunogenic substance. So you inject it into as an adjuvant (?), and you’re not going to make antibodies. They are small molecules but you can do tricks to make antibodies—you can couple it to a big molecule (called a carrier). In this case, it’s not an immunogen but it can be an antigen if we trick the immune system into making antibodies for the hapten. XIII. <no title> [S13] a. An example here. This hapten is DNP—dinitrophenol. It’s a benzene ring with two nitrile groups attached. If you were to immunize somebody with DNP, they wouldn’t make antibodies. b. Take a big protein (bovine serum albumin) instead. c. So we’ve covalently attached DNP to that BSA. If we immunized you with DNP, you would make no antibodies; if you immunize with BSA you’ll make antibodies because cow albumin is a lot different than yours. If you immunize with DNP-BSA you’ll make antibodies to DNP, as well as BSA and a few that only recognize DNP when attached to BSA. d. Who cares about DNP though? And that’s true… but there are other haptens that ARE important. XIV. Examples of Haptens [S14] a. Steroid hormones—cholesterol, estradiol and testosterone are structurally similar and are haptens. If you inject a mouse with human steroid hormones, they won’t make antibodies. And you may want antibodies to different hormones to test levels in patients that may have various diseases. b. Thyroid hormones—even more similar, only difference is one iodine. But we can antibodies if we couple them to different proteins and we can make monoclonal antibodies that can distinguish very subtle differences like the presence or absence of an iodine. c. It can be used for various diagnostic assays. You can make antibodies for drugs and see if someone has been smokin’ dope. And you can use it in a clinical type assay. d. There’s examples of haptens that can form hapten carrier complexes in vivo. Penicillin—when you give someone penicillin, it’s a relatively unstable molecule and can be cleaved and that molecule can covalently attach to your self proteins, which happens in everyone but people who make IgE antibodies to penicillin are in trouble then if that person gets penicillin again will have a very serious anaphylactic reaction. e. Haptens have a lot of value in diagnostics. XV. Antigen-Antibody Interactions in vivo [S15] a. Let’s briefly talk about what’s going in vivo cause that’s why we have them. XVI. Direct Inhibition [S16] a. If we have a toxin, they need to bind to a receptor on a cell and get into the cell to do damage. If we have toxin molecule to bind to a host cell, if you can make antibody that block binding, you can inhibit toxic affects on cell. b. That’s how tetanus vaccine works—tetanus toxin, so you get vaccinated against tetanus and make antibodies that neutralize the toxin. XVII. Agglutination [S17] a. Antibodies that agglutinate. For example, bacteria. You make a network of bacteria and it can no longer attach to whatever they are supposed to attach to. If they’re in your mouth you swallow them and get rid of them because it’s just a big mess of bacteria clumped together. XVIII. Inhibit attachment of bacteria to cells [S18] a. E. coli attached to lateral epithelial cells. They can get on there and stay on there and cause serious infections. Specific interactions between molecules on bacteria and epithelial cells. b. If we have antibodies on the molecules to prevent attachment to epithelial cells, they can be secreted by urine and have no infection. In this case it would likely be IgA antibodies doing the protection. Fundamentals 1: 11:00 - 12:00 Scribe: Lauren Paul Friday, September 25, 2009 Proof: Kristina Hixson Dr. Burrows Antigen-Antibody Reactions Page 3 of 5 XIX. Phagocytosis [S19] a. Here’s a macrophage and some bacteria. Macrophage latched onto bacteria and is going to ingest them and destroy them. The way this works is these macrophages have Fc receptors for the constant region of IgG antibodies. We have our bacteria coated with IgG antibodies, they will bind to Fc receptors, macrophage will ingest them and destroy them. XX. Antibodies enhance phagocytosis [S20] a. Bacteria gets attached via Fc receptors on antibodies bound to it. Macrophage zippers up around bacteria, takes it inside where there are lysosomes that can destroy most bacteria. XXI. Opsonization [S21] a. It becomes attached, macrophage zips up around bacteria. XXII. Phagocytosis [S22] a. During this process, lysosome is killing bacteria by things like bleach and enzymes. b. Antigen presentation is going on, too. So some of these broken down parts of the bacteria will be expressed on surfaces of macrophages which will be recognized by T cells. Talk more about that next week. XXIII. Complement Lysis [S23] a. Antibodies are on the surface of some bacteria, you can activate the complement system and the end product is formation of actual holes in the surface of bacteria and it kills them. (Dr. Barnum will talk more about this later). XXIV. Antigen-antibody reactions in vivo [S24] a. We can have neutralization of viruses and toxins. b. Opsonization—coat the bacteria with antibodies and makes them palatable to macrophages. c. Complement activation and prevention of bacterial adherence. d. Unfortunately, it’s not all good because we shouldn’t react to self antigens. In myasthenia gravis, you wind up making antibodies to acetylcholine receptors which is involved in neurotransmission so you get paralysis because acetylcholine doesn’t bind receptor because antibody has made receptor no longer functional. e. Can get serious problems in transfusion—if you give the wrong ABO type blood to a person, you get very serious problems. f. Allergy—in vivo IgE response which can cause a number of problems. XXV. Antigen-Antibody Interactions in vitro Techniques [S25] a. Lots of different assays and techniques. We’ll examine a few. XXVI. Hemagglutination [S26] a. You’re going to look at blood group antigens. Basically they use antibodies to type A and B blood but not for O. The antibodies can agglutinate red cells on whether they are A, B or O. XXVII. Hemagglutination [S27] a. You can visualize it here with a plastic plate with 96 wells. If you add antibodies to red cells, if antibodies are present on red cells, they will form a mesh work. So instead of dropping down to bottom of the well like when there are no antibodies, if they’re agglutinated they will form a mesh work and coats entire surface of bottom of well. XXVIII. ELISA [S28] a. Powerful assay. Testing for HIV so trying to find out if someone has been infected with the virus. Let’s see if we can isolate the virus—a direct test. Not always easy, sometimes the virus is hiding out in a lymph node. So instead of trying to isolate it, see if they have antibodies to the virus. And if they do, it means they have been infected with the virus. b. The first screening of testing for the virus is ELISA. c. What you do is you take one of the 96 little plastic plates with wells in them. You add antigens to the wells. Plastic proteins bind non-specifically and don’t come off very easily. So you put HIV antigens on the well to see if the patient has antibodies to it. You add diluted serum from patient to the well. d. Blue antibody in the second beaker, you can see it binding to the HIV antigen. e. Now the antibody is on there, you have to figure out a way to see if it’s on there so you take a second antibody with an enzyme attached to it. In this case, we have a goat antibody that recognizes human IgG. Take a goat and immunize a goat with human IgG so now that goat has antibody, we can purify it and attach an enzyme to it. Now we have our enzyme conjugated antibody recognizing human IgG. f. We put in a substrate which starts out with no color then turns yellow (last beaker) if the enzyme’s around and that’s how we know there are antibodies to HIV in that sample. If there weren’t any antibodies, and we added our second antibody to human IgG there would be nothing for it to attach to. So you wash between each step to get out stuff that doesn’t bind. There would be no enzyme to attach to and convert the substrate so you wouldn’t get any yellow color. g. You can use ELISA to see any antibody you can stick on the plate. h. Student question: Is it positive if it were a yellow color and negative if it weren’t a color? Answer: Yeah, so add the substrate and it starts out with no color but if there is an enzyme there it becomes a yellow color. Fundamentals 1: 11:00 - 12:00 Scribe: Lauren Paul Friday, September 25, 2009 Proof: Kristina Hixson Dr. Burrows Antigen-Antibody Reactions Page 4 of 5 i. You can tell the titer from this as well—how much you dilute it. If you have a lot of antibodies, you can dilute it far. If you little antibody, you’ll dilute it quicker (low titer). XXIX. <no title> [S29] a. There are false positives in ELISA. Another assay is the Western Blot which is used as a second line assay for HIV testing. b. Gives you much more info than ELISA because you can see which viral proteins the patient has made antibodies to. c. Take HIV viral particle and dissociate it in a strong detergent and destruct virus and now everything is separated in solution. You perform a SDS-PAGE—separating proteins based on size. You have gel like plastic and has wells and put solution on it and put an electric field from top to bottom of the gel and proteins start moving toward positive end of the gel. The bigger ones have a harder time getting there so at the end there is separation of proteins based on size. d. Now what we can do is see if the patient has antibodies to protein. But in a gel, the antibodies can’t get in there so have to transfer proteins to a membrane so they can be incubated with antibody. e. We have proteins, color coded antibodies that then can bind to proteins. and we can once again detect that bound antibody with an enzyme-coupled second antibody. XXX. Western Blot [S30] a. Again, take virus, dissociate the proteins. Electrophorese them. Take gel, lay it on electrode and actually transfer proteins from gel to membrane so now have a replica. b. Now they are accessible to antibodies. XXXI. <no title> [S32] XXXII. <no title> [S33] a. We have membrane strips, all of them have HIV proteins attached to them. So they’ve done the electrophoresis, transferred proteins to membrane, and incubated the membranes with serum from different individuals. b. Here we have positive control (1 column) where we have antibodies to all the different HIV proteins. And a negative control (2 column, someone we know does not have HIV and incubated with a membrane). All have different proteins if you don’t have antibodies to them and put second antibody on there, there is nothing for the second antibody to bind to and no enzymatic reaction will occur (which is what gives us the visible band). c. A patient has no antibodies to HIV, C has a lot of antibodies to the viral proteins, B has mainly antibodies to p24 and little antibodies to p55. B is probably infected but re-tested later because his antibodies would go up. A lot of people have antibodies that cross react with p24 even if not HIV positive. So it would show up positive on ELISA but on a Western Blot let’s say he only had that band, he probably isn’t infected and would do another assay. d. You can see why ELISA is much simpler and a machine can do it all. But you can see this is much more complex and expensive so not used as primary screening for infections. ELISA first. XXXIII. Pregnancy testing [S34] a. Pregnancy testing has a very interesting history. b. What’s being tested for in the assays is human chorionic gonadotropin (hCG)—pregnancy specific hormone. c. Many years ago a bioassay was used; a so-called rabbit test. d. What was done is take a urine sample from a woman who might be pregnant and inject it into a female rabbit. Human hCG is bioactive in rabbit so a few days later you would kill the rabbit and look at ovaries and see if there were changes from hCG. This was not actually a good assay… e. This assay used no antibodies. First antibody-based assay was agglutination inhibition. Remember if you add antibodies to red cells, they will agglutinate. This is an inhibition of agglutination. XXXIV. <no title> [S35] a. You have a kit. And it had small latex beads that have hCG covalently attached and antibody to hCG. b. Test—take urine sample and incubate it with antibodies. Let the antigen-antibodies interact if any antigen there. Then add beads then look for agglutination. c. Two possibilities i. Not pregnant—agglutinates them because no antibodies present to bind with. ii. Pregnant—if hCG is in urine, antibodies bind up hCG and no antibody left. hCG in urine binds up all antibody so none left to bind to beads and agglutinate. d. This was a big improvement although it does have some weaknesses—the difference between a high level of hCG and just a little hCG; so if you take out 50% of antibodies, there will be less agglutination but will still agglutinate and becomes subjective. XXXV. Pregnancy testing [S36] a. One now available in drug store is a variation of an ELISA assay. b. Looked at interactive pictures on website. c. Test strip—semi-permeable membrane is what goes into the urine sample and urine starts moving. Fundamentals 1: 11:00 - 12:00 Scribe: Lauren Paul Friday, September 25, 2009 Proof: Kristina Hixson Dr. Burrows Antigen-Antibody Reactions Page 5 of 5 d. The first chamber, the reaction chamber, has a monoclonal antibody to hCG and this antibody has an enzyme attached to it. This antibody is free to move so as urine moves, antibody moves too. e. T—test chamber, have antibodies here that are stuck on membrane. Polyclonal antibodies to hCG covalently attached to the membrane and won’t move. f. C—control chamber. Make sure the urine gets up this far so that we know it made it to the test chamber. It’s an antibody to mouse immunoglobulin. g. Negative—urine sample with no hCG. Put strip in urine, urine starts moving via capillary action. Here’s our monoclonal mouse antibodies to hCG that start moving. Here’s our antibodies to hCG but no hCG so keeps moving. Here we have antibodies to mouse IgG antibodies, so they’re going to bind up these monoclonal antibodies and they will get trapped there and since substrate is there, it will cleave the substrate so control is positive but test zone is negative. (Did my best here without the diagram… it was a bit scattered.) h. Positive—looking for two bands. Now we have urine with hCG in it. So it moves into reaction chamber and binds to antibody and now have complex of antibody-hCG and now in test chamber the antibodies will bind up that complex. Left over antibodies go to control chamber. Two bands because hCG is here to trap the enzyme- labeled antibody. XXXVI. Summary of antigen-antibody interactions [S37] a. Specificity depends on the variable regions of the immunoglobulin heavy and light chains b. In vivo: i. After antibody binds antigen, the outcome depends on the constant region of the heavy chain ii. Effector functions are determined by antibody isotype XXXVII.Summary of antigen-antibody interactions [S38] a. In vitro: i. All assays depend on exquisite specificity of the antibody ii. Assays differ in the means used to detect the bound antibody or bound antigen iii. Sensitivity: For example, ELISA and Western blot are very sensitive and agglutination is not so sensitive.</p><p>[end 47 min]</p>