ANTIGEN AND ANTIBODY REACTIONS I, II Immunobiology, BIOIMI 494G Joe McGillis, Ph.D. Sept. 17, 19 MN-380/MN-375 323-6721 jpmcgiO [e-mail] Office Hours: I am here most days from about 9:30 A.M. to about 7:00 P.M. I will make it point to be in my office 30 minutes before and after each lecture. However, you are welcome, and I encourage you to come by any time that you have a question. The best thing to do is to call my office or lab at 323-6721/323-6689 before you come over to insure that I will be available. If not, we can easily schedule a time that is convenient. If you have questions, please come see me as soon as possible. After all, it is what I get paid for! The Ten Most Important Reasons to Pay Attention, Ask Questions About Things You Don't Understand, Study Immunology All Weekend and Learn All About B Cells and Antibodies!!!! 10. By 1:00 P.M. there is no edible food left on campus. 9. Getting an A in Immunobiology will increase your chances of getting into graduate/dental/veterinary/medical school, but probably not clown school. 8. Life sucks without antibodies. (Life can also suck with antibodies!!) 7. To understand what Dr. McGillis willlo~. you with about complement it will be essential to understand antibodies. (teach) 6. There is generally no good live Jazz to be heard at 1:00 P.M. on weekdays. 5. Western Kentucky is going to kick the snot of UK next weekend so there is no reason to go the football game. You might as well save 20 bucks, stay home and study Immunology! 4. Dr. Roszman, who is right most of the time, and is himself a pretty hip cat, thinks it's important. 3. During the last 60 years more fundamental advances in biology and medicine have resulted from research in, related to, or dependent on Immunology than any other SIngle biomedical discipline. 2. Dr. McGillis, who is always right and is the hippest cat around, thinks it's important. 1. You are here because you love science and want to learn everything you can before tuition goes up again! !!! ANTIGEN AND ANTIBODY REACTIONS I, II Immunobiology, BIOIMI 494G Sept. 12, 14 I. ConceptslReview for next 7 lectures 1. Clonality 2. Ig Forms a. CDRs b. soluble forms c. cell membrane bound forms II. Ig Functions 1. 2. 3. 4. Opsonization ADCC Sequestration of toxins Prevention of parasite entry or colonization III. Theoretical and practical considerations of antigen-antibody interactions 1. Affinity of antigen-antibody binding a. non-covalent forces b. affinity c. avidity 2. Precipitin Formation - soluble antigens a. Precipitin formation in solution b. Precipitin formation in gels - immunodiffusion 3. Agglutination - particulate antigens IV. Practical applications of antibodies: 1. General Considerations a. Polyclonal vs. Monoclonal b. Methods of detection c. Direct vs. Indirect labeling s-s s-s Heavy chains Light chains Variable domains Constant domains Immunoglobulin domain Disulfide bonds Antigen binding site Hinge region Amino and Carboxy terminus (a) ,, (b) .-1 Loops ___ COO \ . Y I VAlUABLE DOMAIN CONSTANT DOMAIN 56 SECTION&quot; ANTIGEN RECOGNITION AND LYMPHOCYTE ACTIVATION Macrophage Bacteria --- + ~ ---f ~ ---f ~ .~ R Inefficient phagocytosis Antibacterial antibody Opsonization Enhanced phago~ytosis Binding of opsonized bacteria to macrophage Fc receptors 58 SECTION&quot; ANTIGEN RECOGNITION AND LYMPHOCYTE ACTIVATION A Antibody-dependent cell-medi~ted cytotoxicity (ADCC) ~--(gG f. Low-affinity /FCYRIII - -.. ~ TARGET CELL LYSIS &quot; ' &quot; Surface Precoated antigen TAAGETGELL NKCELL NH z CHz-OH 0 =C-CHz-CHz I The interaction between an antibody and an antigen depends on four types of noncovalent forces: (1) . ionic bonds between oppositely charged residues, (2) hy drogen bonds in which a hydrogen atom is shared be tween two electronegative atoms, (3) hydrophobic inter actions in which water forces hydrophobic groups together to maximize hydrogen bonding of water molecules, and (4) van der Waals interactions between the outer electron clouds of two atoms. In an aqueous environment noncova lent interactions are extremely weak and depend upon close structural complementarity between antibody and antigen. FIGURE 6-1 13-782 500 SHEETS. FILLER ~ SOUARE '&amp;NIJtftJrl./$fJflnd ~ 42 399 M........ V.S ..... :~J:~ ,~~m~~~~~~~~~ :~~ j'gi :f,~EEJE~~r U8'd~~ 200 RECYCLED ~Il F. 5 SOuAAE ~ + rr -t ~ D () ~ ~:-J7 0 ?1 ff o ~ 1\ n II ?' i!'~ ~~ ;- ~ oJ .k&gt; ' '-. ~ -., \J \1 ~ tc\ p r~ - rr ,,~ ~ 7\ ~ ~ .1&gt; 1\ ~. ~ 7\ 1 -b '&quot; ? 7\ . 7&quot;&quot;&quot;~ ~~ n . ~ -, ~ t&quot; r1lf ]:&gt; , J ~ J &gt; 11' \1' () rt:t&gt; 3&gt; r-, r--,n &quot; LJ 0- () s {\. ~ 7\ 17&quot;&quot; l!5 . n . ~ .. r ,I J:P riP W~ \&gt;' :t&gt; ~ ~ } \' .~ _. &gt; , } f\ /. ~ U ( V' w U ( ~ ~ (&gt; ~ '&quot; + \' -+ -J -t \ I r -+ 13-1t2 5l'1OSHT:eS,FlllER 5SOUA~ ~Hltfon&quot;eg,.nd ,... '2-369 .2 )92 &quot;2..:399 200 SHEE1S EYE-EAS&quot; 5 SQUARE 100 AECVClEC WhITE. ~ SQUARE 2QO ACYCLE0 &quot;,&quot;:-lITE 5 SOUARE I:. &quot;&quot;'-&quot;nU.s.... ~~::1 1~~m~~~t~~~~~ () ~ &quot;t () ~ ~ ~ o D ,,&quot;-~ J&gt; ) c: It [ Ab .4.5:7 ~ t\) &quot; &quot;) .&gt; .. ~ &quot;I (t) c.; o (0 ~ ~ ~ IJ ~ ~} i r ;' \&gt; . + ~ Lt:~ f\ ..... J__ ~~\ J'. \Y.,-;l-l' \.; r+;; \ ~ 1.. () } B&quot;..., D ~ 9 ~ ~ ~ .. -{\)-, ~ r- ex !t 1 r~ v,- ~ , t l tk 1 I I ~ 1~ I C' &gt;' :A p ~ 0- h i- .. \ 1J ~ lA ri ~ ~ _. t -5 ........ r C \ ~ 4 ~ t}t ~t&quot; \ e ~ ~ lA -\ '&quot; f ., &quot; ~ .......... I .&gt; r ' ~ '5 '- ~ c ..lo-- ~ d f b.\'-' Itf a-- . ~ -. ~ t- &gt; P J It 5' ) l&gt; J \.... C 1 -...t\ VI 0 - ~ 3s ~ ~ - FIGURE 6-4 A precipitin curve for a system of one an tibody and its antigen. This plot of the amount of anti body predpitatedversus increasing antigen concentra .tions (at constant total anti body) reveals.three zones: a zone of antibody excess in which precipitation is inhib ited and excess antibody can be detected in the superna tant; an equivalence zone of maximal precipitation In which antibody-and antigen form iarge insoluble com plexes (purpie) and neither antibOdy nor antigen can be detected in the supernatant; and a zone of antigen excess In Which precipitation is in hibited and excess antigen can be detected in the super natant. V . ~. ~. + Antibody-excess zone eXCeSSAb A excess g Equivalence zone Antigen-excess zone ) Supernatants { + + + + + &quot; + + Antibody precipitated Antigen added ( A B c o FIgUft.. ...-5. Single radial diffusion in agar (radial immu nodiffusion). A: Petri dish is filled with semisolid agar so Iulion containing antibody to antigen S After agar hard ens. the center well is filled with a precisely measured amount of material containing antigen S. B: Antigen S is allowed to diffuse radially from the center well for 24-48 hours. C: Where antigen S meets corresponding anti IJOdy to S in the agar, precipitation results. After reaction proceeds to completion or at a timed interval. a sharp IXlrder or a ring is formed. D: By serial dilution of a known standard quantity of antigen S-S/1, Sl2, S/4, ~'Il rings of progressively decreasing size are formed Ihe amount of antigen S in unknown specimens can be lillculaled and compared with standard in the timed in ~val (Fahey) method (Fig 18--{)). 2 liFe.. V 3 FIGURE 6-7 Determination of antigen concentration by radial immUnodiffu sion. The area of the ring of precipitation is proponional to the concentration of antigen. A standard curve can be ob tained from the results with known con centrations of antigen (wells 1 - 3). From the standard curve, the antigen concen tration can be determined in samples of unknown concentration (wells UI' U2 , and U~). [Photograph from D. M. Weir (ed.), 1986, Handbook ofExperlmental Immunology, Blackwell SCientific Publi cations.] S c: u c: o u ~ o c: Antigen (unknown concentration) v v c: ~ Area of precipitin ring (a) (bl agglutinaled tube or well l nonagglutinated , ~ (c) serum r--------- ~ blood type anti-A anti-A normal serum control serum dilution @@@ @ @ @ @ 112 114 118 1116 1132 1164 (i~~~ @ A 8 A or 8 2 3 4 5 6 7 8 9 10 0 Cat,ie&lt; ParlOCle + ~~ 0 AnlOQef1 Bound to Carr~ () =&gt;-+ ~ kUibody ~--. do ~hna(llJll RG. 2. Pas.o;ivc ilgglutinalion h.-chniqU&lt;:. v , Polyclonal vs. <a href="/keyword/monoclonal-antibodies/" >monoclonal antibodies</a> Polyclonal Antibodies 1. Produced by immunization of an animal followed by preparation of 'antiserum' from blood or isolation of immunoglobulins from serum. 2. Antiserum or purified polyclonal immunoglobulin contains a collection of distinct antibody proteins that recognize distinct epitopes on the target antigen. The antibodies are derived from distinct B cells that respond to the same antigen. 3. Most common animal sources are rabbit, goat, donkey or sheep. 4. Polyclonal antibodies have higher avidity and are more efficient at crosslinking an antigen. 5. Polyclonal antibodies are more prone to crossreactivity and overall may be less specific than monoclonals. One reason is that a polyclonal antiserum may contain clones that recognize an epitope that is common to multiple related proteins. <a href="/keyword/monoclonal-antibodies/" >monoclonal antibodies</a> 1. Production. Individual antibody producing cells are isolated from and immunized animal and are 'immortalized' in culture. Antibodies are isolated from culture media. Alternatively, the immortalized, tumor, monoclonal cell line can be injected into the peritoneum of a host mouse. The peritoneal fluid, call 'ascites', can then be collected. The ascites fluid contains very high levels of the <a href="/keyword/monoclonal-antibody/" >monoclonal antibody</a> . 2. <a href="/keyword/monoclonal-antibodies/" >monoclonal antibodies</a> are derived from a single antigen specific B cell, are therefore identical in primary structure (amino acid sequence in the variable domains) and therefore all recognize a single distinct epitope on an antigen. 3. Most common animal sources are mouse and rat. 4. <a href="/keyword/monoclonal-antibodies/" >monoclonal antibodies</a> have lower avidities and are less efficient at crosslinking antigens. 5. <a href="/keyword/monoclonal-antibodies/" >monoclonal antibodies</a> are less prone to non specific artifacts by selection of clones that recognize epitopes that are unique to the target antigen. Common Methods of Detection in Antibody Based Assays 1. Fluorescent labels - small fluorescent molecules can be covalently crosslinked to immunoglobulins. Examples: Abbrev. FITC PE APC Chemical Name fluoresceine isothiocyanate phycoerythrin Color Green Red Blue ? 2. Enzymes - enzymes can be covalently linked to immunoglobulin proteins Examples: Horse Radish Peroxidase Glucose Oxidase a. colorless substrates converted to colored substrate b. chemiluminescence - non luminescent subtrate converted to luminescent substrate that can be detected in a fluorimeter or on x-ray film 3. Radioisotopes - radioactive moieties can be covalently linked to immunoglobulins. Most common is 1251. Can be detected on x-ray film or by coating antibody bound specimen&quot; with a photosensitive emulsion. Direct and Indirect Methods of Detection 1. Direct - the primary antibody is labeled. Advantages Can use more colors More Specific -lower background and non specific staining 2. Indirect Methods Disadvantages Less sensitive a. Second Antibody - uses a labeled 2 nd antibody specific for the primary antibody. Advantages More sensitive - amplification of primary signal Disadvantages More difficult to use with multiple colors More prone to high background and non specific staining b. Avidin-Biotin - Avidin (streptavidin, SA) and biotin bind with a very high affinity. Biotin is a small molecule that can be covalently crosslinked to proteins. Avidin is a large molecule to which multiple fluorescent molecules can be attached. Primary antibodies can be tagged with biotin and then detected with SA. Advantages More sensitive - amplification of primary signal Can be in multiple color systems Disadvantages More prone to high background and non specific staining