Chapter 18 Practical Applications of Immunology Lecture Outline PDF

Chapter 18 Practical Applications of Immunology Antigens & Antibodies 1 Practical Applications of Immunology: An Introduction Test “Applications” = test “uses” Applications will usually involve: Use of substances(Ag’s) that trigger an immune response  Ex - Vaccines l ooking at patients bl ood...

Chapter 18 Practical Applications of Immunology Antigens & Antibodies 1 Practical Applications of Immunology: An Introduction Test “Applications” = test “uses” Applications will usually involve: Use of substances(Ag’s) that trigger an immune response  Ex - Vaccines l ooking at patients bl ood sampl es Testing/analysis of a patient’s blood (serum) sample  Ex - Serology What are some common practical applications (uses) of immunology? High IgM= current disease, Force you make Ab High IgG you had disease 1. Vaccination inject Ag as a resultyou can make Ab Fordisease prevention ou can l ookforAg (pathogen)orAb (IgM specifical l y)in the bl ood fortimel yand accurate 2. Serological testing ydiagnosis (Dx=diagnosis) bl ood testing 2 Immunity and Vaccination Edward Jenner Historically: inoculation of Cowpox virus to prevent Smallpox Vaccine - injection of substances (antigens) into the body  induce pathogen immune response Vaccination (“vacca” = cow): not verystrong immune response 1. Provokes a Primary immune response  leads to formation of memory B cells and antibodies you al readymade memoryB 2. Produces a rapid & intense Secondary immune response bc cel l s;you can skip cl ono-sel ection tive-vaccine -inj ect pathogen and cl ono-expansion Natural Immunity vs Artificial Immunity ac passive -inj ect Ab Immunogen - a substance (Ag) that stimulates the immune system Herd immunity - protection for most of the population strong immunogens made from protein weakimmunogens made from Lipids 3 Desirable Criteria for An Ideal Vaccine characteristics 1. Vaccine safety must not cause disease oril l ness must not be poisonous  Vaccine must never be pathogenic or toxigenic 2. Vaccine effectiveness it must work,have a high success rate  Vaccine must be a strong immunogen and stimulate the immune system 3. Vaccine with oral administration strong immunogen wil lstimul ate strong immune response Protein Painl ess  Vaccine taken orally is preferred over injectable vaccine people dont like pain/needles 4. Vaccine that requires only one injection DTaP,MMR etc  Vaccine that requires 1 injection or combination vaccine is preferred 5. Vaccine is affordable & easy access peopl e dont l ike coming back formore vaccines  Vaccine is available to most people at a reasonable cost 4 Types of Vaccines Bcit is al ive it wil lcause MILD side effects 1. Live, attenuated vaccines Weakened pathogens weak,Live pathogen Closely mimic an actual infection EX:MMR and Smal l poxvaccines 2. Inactivated, killed vaccines Killed pathogens Safer than live vaccines! EX:Infl uenza and Pol io 3. Toxoid vaccines inactive toxin;non poisonous, Inactivated toxinbut willproduce immune response ->you make Ab to Toxin Immune response will target the toxin EX:Td vaccine 5 Types of Vaccines means combined 4. Conjugated Vaccines get inj ected with Main component and attached Protein to it.Protein promotes strong immune response! Main vaccine component is combined with a strong immunogen (protein) EX:BacterialPnemonia vaccine 5. Subunit vaccines smal lpiece enough to give you immune response Use antigenic fragments (Ex - pieces of virus or bacterium) to stimulate an immune response EX:Hep B vaccine and Pertussis vaccine (DTaP orTDaP) a-acel l ul ar,a piece 6. mRNA (nucleic acid) vaccines RNA You do not inject the live pathogen! (inject only bacterial/viral nucleic transl ation acid) Inject mRNA into host cells  make protein Ag’s; these Ag’s stimulate host immune response! EX:Covid Vaccines 6 Common Vaccines and Preventable Diseases Vaccine Type Disease Vaccine Characteristics Disease prevented byVaccines PREVENTED Prevent Bact Diseases Prevent ViralDz Toxoid vaccine Diphtheria & Tetanus -Antibodies will target the toxins Conjugated vaccine Pneumococcal Pneumonia -Capsular polysaccharides combined with proteins Subunit vaccine Pertussis -Use of bacterial fragments (use piece of the cell wall) Subunit vaccine Hepatitis B -Use of viral fragments (use Hep B surface antigens) Live, attenuated vaccine Measles, Mumps, Rubella -Inject live, attenuated virus Inactivated, killed vaccine Poliomyelitis (Salk vaccine) & Influenza -Inject inactivated, killed virus Live vaccinia vaccine Smallpox -Inject live vaccinia virus mRNA (Nucleic Acid) Vaccine COVID-19 (Pfizer-BioNTech & Moderna vaccines) -Inject viral mRNA 7 Diagnostictesting = ID the disease Immunity and Serological Testing Serological testing involves testing of a patient blood sample What part of the blood sample is being = the cl earpart ofbl ood (not pl asma but anal ogous to it) tested? SERUM Serum has NO cl otting factors;Pl asma HAS Cl otting factors What are we looking for in the serum? Looking forAgs Abs in patient serum What is the purpose of a serological test? to give an accurate and timel yDx Serology = study or diagnostic examination of blood serum (to see how the immune system responds to pathogens or substances introduced into the body) CF,ions,gasses,Abs,Ags l ookforwhat' s in the serum;are there Abs and Ags present forDzbeing tested 8 Serological Test Results Positive Test:  Positive for the Ag (pathogen) or Ab (usually IgM)  Person has the disease! /OUTCOME Negative Test: Negative for the Ag (pathogen) or Ab (usually IgM) Person does NOT have the disease! Ifyou have Ag/pathogen = DZ Ifyou have Ag/pathogen --> makes Ab/IgM against pathogen --> DZ 9 Desirable Criteria for An Ideal Serological Test 1. High specificity/accuracy can onl yhave 1 resul t/interpretation onl y1 possibl e outcome -positive ornegative  Test must be unambiguous (can only have 1 interpretation)  Test must be reliable no false pos orneg results  Ex - pregnancy test, HIV test, athlete drug doping test 2. High sensitivity  Test uses a small sample size  Test can detect small quantities Ag’s or Ab’s 3. Easy set-up & interpretation  Test construction is simple  Test results are easy to read EX: test shows col orchange,show symbol s (+-)orl ines etc 10 Desirable Criteria for a Good Serological Test 4. High throughput  Throughput - ability to simultaneously test hundreds or thousands of samples  Many tests are carried out at one time due to machine automation 5. Affordable cost & easy access  Test is available to most people at a reasonable cost 11 Serological Tests Direct Testing Methods vs Indirect Testing Methods Two types of serological tests: 1. Direct serological test - What are you looking for? Antigen/pathogen 2. Indirect serological test - What are you looking for? Ab orIgM For all serological tests: you are looking for either the presence or absence of the antigen (pathogen) or the antibody (IgM) in the patient serum sample Serological testing of patient serum sample involves: Antigen - provided by either the patient sample or the lab test Antibody - provided by either patient sample or the lab test serol ogicaltest from l ab + patient serum sampl e (is there Ag orAb in patient serum?) 12 Serological Tests: Let’s Re-Cap Two ways to know if patient has a disease: 1. Patient has the Ag (pathogen), or..... 2. Patient has the Ab (IgM) - made in response to the pathogen! bl ood sampl e Need to perform a serological test to look for the Ag or Ab in the patient’s blood serum The serological test (lab) provides either the Ag or Ab, and the patient would provide the the counterpart of that lab test component 1. If lab test provides the Ag, then looking to see if patient provides the Ab This is called an INDIRECT test 2. If lab test provides the Ab, then looking to see if patient provides the Ag This is called a DIRECT test 13 Common Examples of Serological Tests 1. 2. 3. 4. 5. Precipitation Reactions & Agglutination Reactions Neutralization Reactions Fluorescent-Antibody Techniques Compliment-Fixation Reactions ELISA 14 Precipitation Reactions Invol ves Ag that is shed/not attached to main pathogen Inol ves antigen The Ag is attached to the main pathogen Involve soluble antigens......(vs particulate antigens) This one forPrecipitation RXN This one is forAgl uttanation RXN Soluble antigens react with antibodies (IgM or IgG) Immune complexes form at the “equivalence zone”  formation forms at the center,forming precipitin ring of “precipitin ring” Precipitin ring = cloudy line formation where there is a 1:1 ratio of Ag to Ab Test interpretation: if immune complexes formed, then either Ag’s or Ab’s were present in the patient’s serum sample  patient has the disease! positive forAg positive Direct What does a positive-precipitation reaction mean? positive forAb positive Indirect immunocompl exformed, What does a negative-precipitation reaction mean? no negative test. 15 Sol ubl e surface markers shedding offAg IgG is orIgM is formed Ag Particul ate Sol ubl e: Sampl e can come from patient orthe l ab Ag Ab comes to Ag and form immunocompl ex 16 Precipitation Reactions at the equival ence zone Figure 18.3 Precipitation Curve Figure 18.4 Precipitin Ring Test 17 percipitant ring at equil ence zone percepitataion ring at equivel ance zone no percepitation ring 18 Precipitation Reactions Applications/uses: 1. VDRL Test (Venereal Disease Research Laboratory) - blood test IgM for Syphilis; looking for patient antibodies against Ag treponema Is VDRL Test a direct or indirect serological test? Indirect,bclooking forAb's used to distinguish between different species ofGenus Streptococcus 2. Lancefield Classification of Streptococcal species Classifies Streptococci into groups from A - K and H -U  Ex - GAS (Group A Streptococci) = Streptococcus pyogenes GBS GCS, etc. 19 Cl umping Agglutination Reactions not shed,stays on the main antigen Involve particulate antigens Involves IgM EXCLUSIVELY Particulate antigens linked to antibodies (IgM)  form immune complexes particul ar antigen l eads to cl umping (Aggl utination)of pathogens in one l ocation Figure 18.5 Adapted 20 Agglutination Reactions Acting as Ag (from l ab) patients serum sampl e added;patient provided Ab' s IgM --> Positive Indirect Aggl utination Test a) Latex beads coated with an antigen will agglutinate when mixed with patient serum if the serum contains IgM antibodies against the antigen. Patient provided Ag' s Lab test had Ab' s IgM Form immune compl ex, cl umping.Positive Direct Aggl utination Test b) Latex beads coated with antibodies will agglutinate when mixed with patient serum if the serum contains antigens specific to the antibodies. 21 patient sampl e patient sampl e Lab test Ifimmune compl ex forms --> Cl umping Indirect Aggl utination Positive Test form immune compl ex --> Cl umping.Test is Direct Aggl utination Positive Test 22 Agglutination Reactions Applications/uses: ifyou have a RBC,there is onl yanti B ab' s can bind. W hen anti B binds to RBC it l eads to destruction ofRBC EX:Cl umping Add Add 1. Hemagglutination - for blood typing Anti A Cl ump bind to Agglutination of RBC surface A Anti B antigens and complementary Cl ump binds antibodies B Anti A Anti B 2. Antibody Quantitation - measuring Cl ump Cl ump bind to binds AB AB antibody titer Used to differentiate between primary vs secondary exposure O is universalDonorbcit doesnt have anysurface markers IgG IgM Blood Typing 23 Neutralization Reactions Goalto neutral ize a toxin ora virus Trying to determine if antibodies to the toxin or virus are present in the patient serum sample Is this a direct test or indirect test? Indirect bclooking forAb's Toxin neutralization test: Cel l = test indicator comes from the l ab Does the patient have the disease? behaving l ike Ag comes from the Lab No,#1 bcpatients sampl e didnt have Ab' s to toxin;#2 Al so bcindicatorcel lgot damaged patients sampl e containing Ab' s Does the patient have the disease? Behaving l ike Ag comes from l ab Yes,#1 bcpatient sampl e has Ab' s to toxin; #2 Indicatorcel ldidnt get damaged. Indicatorcel l Figure 18.9 a) The effects of a toxin on a susceptible cell and neutralization of the toxin by antitoxin 24 Neutralization Reactions Viral hemagglutination inhibition test: patient sampl e Does the patient have the disease? No,#1 bcpatient sampl e doesnt have Ab' s present;#2 There was cl umping ofRBCs; theywere cl umping because viruses cl umped RBCs together. Does the patient have the disease? Yes.#1 Patient' s sampl e has Ab' s forthe virus;#2 No cl umping,bc viruses were neutral ized bypatients Ab' s Figure 18.9 b) Viral hemagglutination inhibition test to detect antibodies to a virus. These viruses will normally cause hemagglutination when mixed with red blood cells. If antibodies to the virus are present, as shown in bottom diagram, they neutralize the virus and inhibit hemagglutination. 25 Neutralization Reactions Applications/uses: 1. Diagnosis of bacterial diseases (toxin production) 2. Diagnosis of viral diseases 26 Fluorescent-Antibody Techniques Combine fluorescent dyes with antibodies Two types: Fl uorescent Antibody 1. Direct Fluorescent-Antibody (FA) Tests 2. Indirect Fluorescent-Antibody (FA) Tests 27 l ooking forpathogen Direct Fluorescent-Antibody (FA) Test streptococcus pyogenes Looking for a specific microorganism in a clinical sample Application/use - to identify GAS from patient’s throat sample A diagnostic test for which disease? strept throat orscarlet fever Pathogen from patient from tube Resul t:Direct Positive Fl uorescent chain pattern Figure 18.11 a) 28 Looking forAb' s (IgM) Indirect Fluorescent-Antibody (FA) Test Looking for a specific antibody in serum Fluorescent dye-labeled Anti-human antibody is added and will react with any human antibody in serum if the result is positive Application/use - to determine if T. pallidum from patient’s blood sample is producing Ab’s from Lab (F.A) Patient serum sampl e Ag from Lab A diagnostic test for which disease? onl ybind forhuman Ab' s cal lit anti human Ab Figure 18.11 b) 29 Direct FA Test vs Indirect FA Test Figure 18.11 30 invol ve compl ement proteins Complement-Fixation (CF) Reactions circul ate in the bl ood to the STEM REGION ofAb AKA immune compl ex Complement serum protein binds (“is fixed to”) to the Ag-Ab orthe rest ofthe complex; no more circulating complement proteins available! ftest! from the patient Detects small amounts of antibodies SENSITIVE Therefore, this is a highly ________________ test! Application/use - diagnosis of bacterial & viral diseases Two possible results:  Positive (indirect) CF test  Negative (indirect) CF test bcl ooking forpatients Abs not pathogen COMPLEMENT FIXATION IS INDIRECT TEST ONLY! ANOTHER ONE IS NUTRALIZATION TEST INDIRECT TEST ONLY! 31 Positive CF Test vs Negative CF Test ANIBODY BELONGS TO PATIENT (INDRECT TEST) ANTIGEN PROVIDED BY LAB LEFT COLUM IS POSITIVE TEST LAB LAB RIGHT COLLUM IS NEGATIVE Two stages for the CF test: PATIENT PATIENT 1. Compliment fixation stage What does this stage tell us? whetheral lcompl iment proteins got used and made immune compl ex patient has Abs (IgM) 2. Indicator stage bcyou cant see Ab' s, need to do Indicatorstage What does this stage tell us? IgM Figure 18.10 form immune compl ex no immune compl ex ALL compl iment proteins binds to stem region of patient' s Ab ALL compl iment proteins used up in forming immune32compl ex Positive CF Test vs Negative CF Test Lab test indicator Lab Has 2 immune compl exes al lcompl ement Ps is used up so no compl ements are avail to bind to sheep Ab' s. Figure 18.10 Lab test indicator Lab 1 immune compl ex al lcompl ements avail abl e to bind ship Ab' s,sheep RBC' s l ysed,test tube turns red -NEGATIVE TEST. 33 Positive CF Test vs Negative CF Test Two possible results: 1. Positive test: bccompl ements bind patient Ab' s -No hemolysis bcal ll ab compl ements bind patient Ab' s -Yes Ab No hemol ysis -Yes disease 2. Negative test: -Yes hemolysis -No Ab -No disease 1. 1. 2. 2. 3. 4. Figure 18.10 3. 4. 34 Positive Complement Fixation Test 1. An antigen and inactivated compliment are provided by the lab. These are added to a serum sample containing the patient’s Abs which are specific for the Ag. An immune complex will form consisting of the lab Ag and the patient’s Ab. 2. As a result of immune complex formation, compliment gets activated and compliment fixation occurs (all available compliment binds to Ab stem region of the immune complex). This is part of the “Compliment Fixation Stage” of the test. Since all the compliment got used-up, no more compliment will be available to bind to future immune complex formations. 35 Positive Complement Fixation Test 3. As a test indicator, sheep RBCs are introduced into the patient’s serum sample. Abs against sheep RBC are also introduced into patient’s serum sample. An immune complex will form between sheep RBC and Ab against sheep RBC. 4. No hemolysis of sheep RBC occurs because compliment did not bind to the Ab against sheep RBC. This is part of the “Indicator Stage” of the test. Compliment did not bind because it was all used-up and already fixed (compliment fixation) in the earlier step to the 1st immune complex. Thus, compliment was no longer available. No hemolysis of sheep RBC indicates a positive test the patient’s Ab, and thus, positive for the disease. 36 Negative Complement Fixation Test 1. An antigen and inactivated compliment are provided by the lab. These are added to a serum sample that does NOT contain the patient’s Abs which would have been specific for the Ag. NO immune complex forms because the patient’s serum sample has no Abs against the lab Ag. 2. As a result of NO immune complex formation, compliment does NOT get activated and compliment fixation does NOT occur (compliment does not bind to Ab stem region of the immune complex). This is part of the “Compliment Fixation Stage” of the test. Since no compliment got used-up because it did not bind to Ab stem region, all of the compliment will be available to bind to future immune complex formations. 37 Negative Complement Fixation Test 3. As a test indicator, sheep RBCs are introduced into the patient’s serum sample. Abs against sheep RBC are also introduced into patient’s serum sample. An immune complex will form between sheep RBC and Ab against sheep RBC. 4. Hemolysis of sheep RBC occurs because compliment gets activated and compliment fixation now occurs (all available compliment binds to Ab stem region of the immune complex). Compliment binds to the Ab against sheep RBC. This is part of the “Indicator Stage” of the test. Compliment can bind because it was not used-up and not fixed (no compliment fixation) in the earlier step because there was no immune complex formation during the “Compliment Fixation Stage” of the test. Thus, compliment was available. Hemolysis of sheep RBC indicates a 38 negative test the patient’s Ab, and thus, negative for the disease. Enzyme-Linked Immunosorbent Assay (ELISA) can detect smal lamount ofantigens High specificity High sensitivity Application/use: Home pregnancy test Drug testing HIV testing 39 The ELISA Method for HIV Testing: Direct ELISA vs Indirect ELISA enzymes immune compl ex formed have to rinse between steps Sandwich is onl y formed in DIRECT ELISA test ONLY! added enzyme-l inked Ab (l ab) ADSORBED/not Absorbed provided byLab provided by patient (antigen) product forms and produces col orchange l ooking forpathogen immune compl ex forms Antigen provided byLab l ooking forAb' s Figure 18.14 No sandwich formed,and have Enzyme l inked anti-human Ab' s Ab' s provided by inked Anti-human Ab product forms and produces visibl e col or patient (Ab' s) Enzyme-l bcit binds to human Ab' s change 40 The ELISA Method for HIV Testing: Direct ELISA vs Indirect ELISA Sandwich forms no sandwich form Antibody(Lab) Anti-human Ab' s (Lab) Enzyme-l inked Ab (Lab) Substrate (Lab) Substrate (Lab) Pathogen (Lab) Pathogen (Patient) Antibody(Patient) 41 The ELISA Method for HIV Testing Direct ELISA Indirect ELISA 1. What are you looking for? Pathogen from patient' s serum 2. What are the components of this test & which components are provided by the lab & which by ,Enzyme-l inked Ab the patient? Lab=Antibody Substrate. Patient=Pathogen. 3. Applications/uses? 1. What are you looking for? Antibody 2. What are the components of this test & which components are provided by the lab & which by Enzyme-l inked Anti-human Ab, the patient? Lab=Pathogen, Substrate.Patient=Antibody 3. Applications/uses? HIV BEFORE Seroconversion,Pregnancy,Drug testing HIV AFTER seroconversion (bcthats when Abs appear) 42 ELISA: The Use of Monoclonal Antibodies in a Home Pregnancy Test hCG is present in urine veryspecificAbs made forhCG Figure 18.13 43 Relative Sensitivities of Serological Tests High sensitivity most sensitive ofal ltests! Radioimmunoassay Antigen that radioactivelymarked PCR Compliment-Fixation Tests ELISA Intermediate sensitivity Agglutination reactions Neutralization reactions Low sensitivity Precipitation reactions 44