255 Chapter 17 Immunization and Immune Testing Brief History of Immunization 12th century: smallpox; variolation (process of infection with smallpox scab to induce immunity) 1721: Lady Mary Montagu ---> England 1796: Edward Jenner
255 Principles of Vaccine Preparation: A vaccine must be effective, safe and easy to administer. It must also provide a long-lasting protection. Most vaccines are prepared as: 1. Killed whole cells or inactivated viruses. 2. Live, attenuated cells or viruses. 3. As parts of cells or viruses (these are called
subcellular, subunit or acellular vaccines). 256 Live vaccines can be prepared by a process called attenuation. 1. Attenuation can be achieved by long-term cultivation. 2. Selection of mutant strains that grow at colder temperatures (cold mutants).
3. Continued passage of microbes through their non-natural hosts or tissue culture. 4. Removal of virulence genes. 256 Types of Vaccines: Subunit vaccines: Subcellular vaccines: Acellular vaccines:
Polyvalent vaccine: Polio: type 1: Leon type 2: Lansing type 3: Brundhilde Genetically Engineered Vaccines Veggie vaccine: 1. The gene responsible for the surface protein of a pathogen is isolated.
2. The gene is inserted into plant cells (banana, potato, corn, tomato). 3. Plant cells are then able to produce the surface protein of the pathogen. 4. When the plant along with the surface protein is ingested, the protein will stimulate the immune system to produce antibody against it. This antibody should be able to react with the surface protein of the pathogen to provide protection.
257 Subunit Vaccines: A gene that codes for a surface protein of a pathogen is inserted into a plasmid which is introduced into a cloning host. The host is able to produce the surface protein. This protein is the subunit vaccine.
258 DNA vaccine: 1. The gene coding for a surface protein (epitope) of a pathogen is isolated and inserted into a plasmid vector. 2. The plasmid is introduced into an appropriate host cell, which can then be cloned. 3. The plasmids can be harvested from the cloning host.
4. The plasmids suspended in saline solution are used as a DNA vaccine. 5. When the plasmids are injected into a host, they are incorporated in the host genome. 258 6. The host is able to produce the surface protein (epitope) of the pathogen, which will be recognized as a foreign antigen by the host
immune system. 7. Some surface proteins are secreted and taken up by phagocytes, which then present them to the B lymphocytes, activating the B lymphocytes to produce antibodies against the surface protein. 258 8. Some surface proteins are attached to
the surface of the host cells and activate the T lymphocytes. Thus, the DNA vaccine provides both humoral immunity and cell-mediated immunity to the host against the pathogen. 258 Trojan horse vaccine: 1. The genes coding for the surface proteins of
HIV, herpes simplex type 2, Mycobacterium tuberculosis, and M. leprae have been incorporated into the genome of vaccinia (cowpox virus) which is used as a carrier. 2. When the vaccinia with these surface proteins is injected into a host, it will stimulate the immune system to produce antibodies against the pathogens with these surface proteins.
258 Adjuvant: Herd immunity: Immunotherapy: Immunization Passive Immunotherapy Administration of antiserum that contains preformed antibodies
Provides immediate protection against a recent infection or ongoing disease Antisera have several limitations Can trigger allergic reactions called serum sickness Antibodies of antisera are degraded relatively quickly Individual not protected from subsequent infections Limitations are overcome through development of
hybridomas 259 Monoclonal Antibodies: 1. Immunize an animal with a specific antigen. 2. Harvest B cells from the spleen. 3. Mix B cells and myeloma cells (cancer cells of the bone marrow), and add
polyethylene glycol to facilitate cell fusion. 4. Some cells fuse to form hybridoma cells. 20 259 5. They are transferred to a selective medium, which contains no histidine (an amino acid).
6. Cancer cells need histidine to grow. So, they are eliminated. B lymphocytes will automatically die off as they cannot propagate in vitro. 7. Hybridoma cells can survive in the medium as they can synthesize the amino acid. 259
8. The hybridoma cells can be cloned in vitro or in vivo. Applications: 1. Diagnostic tests 2. Treatment of diseases and cancers 259 Titer of Antibody: 1. The serum from a person is diluted (2-fold
dilution) in a series of test tubes or microtiter plate. 2. An equal amount of an antigen is added to each tube. 3. The tubes are mixed well and incubated. 4. The most diluted test tube that shows a positive test (such as agglutination) is taken as the titer of antibody. For example, if the last tube that shows a positive result is 1:4, it is expressed as
the reciprocal of which is 4. That is, the titer is 4. 260 To Confirm A Disease Using The Titers of Paired Sera: 1. Acute phase serum is taken from a patient. 2. A 2-fold dilution is made in a series of test tubes or a microtiter plate.
3. An equal amount of an antigen is added to each tube. 4. The titer is read from the most diluted test tube. For example, the titer is 4. 5. Convalescent serum is taken from the same patient. 260 6. An equal amount of the same antigen is added to
each tube. 7. A 2-fold dilution is made in a series of test tubes or a microtiter plate. 8. The titer is read from the most diluted test tube. For example, the titer is 16. 9. If there is a 4 folds or more increase in titer, it confirms that the patient has been infected with the disease. If not, it means that the patient was not infected with the disease under testing. The
patient was infected with other disease. 260 Serologic Tests: Agglutination Test: Particulate antigen + Serum (agglutinin?) Agglutination: Positive No agglutination: Negative
Agglutination Test 260 Rapid Plasma Reagin Card (RPR) Test: 0.05 ml serum + 1 drop of charcoal-bound cardiolipin Rotate at 100 rpm for 8 minutes Agglutination: Reactive
No agglutination: Non-reactive (Negative) Agglutination Tests Widal Test: Typhoid fever (Salmonella typhi) Weil-Felix test: Rickettsial diseases 260 Latex Agglutination Test
Pregnancy Test: If a woman is pregnant she produces human chorionic gonadotropin (HCG). Purificed HCG is injected into an animal, which then produces antibody against HCG. This antibody is harvested and used as a reagent in the test. Latex particles are coated with HCG. It is used as a second reagent.
Latex Agglutination Test Urine + Antibody vs HCG Mix well Latex particles coated with HCH are added to the mixture. If agglutination occurs, the test is negative
If no agglutination occurs, the test is positive. 260 If agglutination of latex particles occurs, it means that the urine does not contain HCG. Antibody vs HCG reacts with latex particles and causes agglutination. If no agglutination of latex particles occurs, it means that the urine does contain HCG.
The HCG in the urine reacts with the antibody vs HCG. No antibody is available to react with latex particles. That is why there is no agglutination. It means that the woman is pregnant. 260 Viral Hemagglutination Some viruses have the ability to cause
agglutination, called hemagglutination, of red blood cells. In the presence of antibody, the virus cannot agglutinate the RBC. The antibody connects to the virus, preventing it from clumping the RBC. 260 Viral Hemagglutination
Viruses + RBC Hemagglutination (influenza, rubella, measles, mumps, vaccinia, arboviruses, adenoviruses, reoviruses and some enteroviruses) 261
Hemagglutination-Inhibition (HI) Test: Virus + Serum + RBC Agglutination: No agglutination: + Positive (+) means the serum must contain antibody. That means the patient has been infected with the virus. 261 Precipitin Test
Precipitation: + Soluble Antigen + Serum No precipitation: Zone of antigen excess: Zone of antibody excess: Zone of optimum concentration: Characteristics of precipitation reactions
Figure 17.6 261 Precipitin Test Ring Test: Antigen solution is slowly poured into a tube containing serum (antibody?) A ring of precipitation: + No ring of precipitation: -
261 Precipitin Test VDRL (Venereal disease research laboratory) Test: 0.05 ml of heated serum + one drop of cardiolipin rotate at 180 rpm for 4 minutes Precipitation: Reactive No precipitation: Non-reactive
261-262 Precipitin Test Immunodiffusion: If both antigen and antibody are homologous, a line or band of precipitation is formed.
Single Diffusion Technique (Oudin Technique): A tube of antibody in agar is prepared. Antigen solution is added. If a band of precipitation occurs the test is positive. If no precipitation occurs, the test is negative. 262 Double Diffusion Technique:
A. Oakley and Fulthorpe Technique: A tube of antibody in agar with a layer of plain agar on top is prepared. A solution of antigen is added. If a band of precipitation in the plain agar occurs, the test is positive. If no precipitation occurs, the test is negative.
263 B. Ouchterlony Technique (Plate Method): Wells are cut from an agar plate. An antibody is placed in the central well, and different antigens are separately placed in the wells around the central well. If a line of precipitation occurs between the antigen and the antibody, the test is positive for the antigen.
263 Immunodiffusion Reaction of identity: Reaction of partial identity: Reaction of non-identity: 263 Immunoelectrophoresis:
It is a combination of immunodiffusion and electrophoresis. Positively charged proteins migrate to the cathode, and the negatively charged proteins migrate to the anode. 264 Complement-Fixation (CF) Test: (Antigen + Complement + Serum)
+ (RBC + Hemolysin) Mix, Incubate No hemolysis: Positive test Hemolysis: Negative test Wassermann test: for syphilis 264
Treponema pallidum immobilization (TPI) Test: Live Treponema + Serum + Complement Immobilized: Positive test Motile: Negative test 264 Neutralization Test (NT): Antigen (Virus or Toxin) + Serum
Inoculate into animals or tissue culture Animals die: Negative test Animals survive: Positive test CPE (cytopathic effect) in tissue culture: Negative test No CPE: Positive test
264 Quellung Test: It is used to identify different serotypes of pneumococci. Unknown pneumococcus + Specific antiserum Capsule swelling (due to light effect): + No capsule swelling: - 265
Fluorescent-Antibody Technique: 1. Direct method: to detect an unknown antigen by using a known antibody. Unknown antigen + Labeled antibody Presence of brilliant greenish color: + Absence of brilliant greenish color: - Fig. 17.15
265 2. Indirect method: to detect an unknown antibody by using a known antigen. Antigen + Serum + Labeled anti-antibody Presence of brilliant greenish color: + Absence of brilliant greenish color: - 265
Enzyme-Linked Immunoabsorbent Assay (ELISA): 1. Direct method (sandwich technique): to detect an unknown antigen by using a known antibody. Known antibody + Unknown antigen + Enzyme-linked antibody + Substrate Color change: + No color change: -
Fig. 17.16.c 266 2. Indirect method: to detect an unknown antibody by using a known antigen. Known antigen + Unknown antibody + Enzyme-linked anti-antibody + Substrate
Color change: + No color change: - Immune Testing Recent Developments in Immune Testing Immunochromatography Very rapid and easy to read ELISAs Antigen solution flows through a porous strip, where it encounters labeled antibody
Visible line produced when antigen-antibody immune complexes encounter antibody against them Used in pregnancy testing and for rapid identification of some infections Figure 17.15 Immunochromatographic dipstick. Zone of antibodies
linked to colloidal metal, color too diffuse to see Line of fixed anti-antibody Strep A
Dip Strep A Anti-antibodies stop movement of antibodyantigen complexes. Color becomes visible because of density of complexes. Dip
Movement of fluid containing complexes of antibodies bound to antigen Prepared antigen
extract from patients nasal sample 266 Western Blot (to detect a specific protein): 1. A sample is separated by electrophoresis. 2. It is transferred (transblotted) to a nitrocellulose paper. 3. The transblot is cut into strips, which are
incubated with a solution of antibody vs the test agent to allow antigen-antibody reaction. 4. The strips are washed to remove the unbound antibody molecules. 266 5. The strips are treated with an enzyme linked antihuman immunoglobulin
(enzyme-labeled anti-antibody). The strips are washed. 6. Substrate of the enzyme is added. 7. If a visible color change occurs, the test is positive. If not, the test is negative. 266 Southern Blot (to detect a specific DNA sequence): It was devised by Ed. Southern in 1975.
1. DNA is cleaved by restriction endonuclease. 2. The DNA fragments are separated by electrophoresis. 3. The DNA fragments are denatured. 4. The single-stranded DNA fragments are transferred (transblotted) to a nitrocellulose or nylon membrane. 266
5. A labeled DNA probe or RNA probe is added. The probe can be labeled with radioactive isotope, fluorescent dye or enzyme. 6. Rinse thoroughly to remove the unbound labeled probe. 7. The DNA sequence of interest can be detected. Autoradiography is used for radioactive isotope-labeled probe, ultraviolet light is used for fluorescent dye-labeled probe, and
substrate is used for enzyme-labeled probe. Northern blot is used to detect a specific RNA sequence using a labeled DNA probe or RNA probe. 267 In Vivo Testing: Skin Test
Tuberculin Lepromin Schick Dick Disease Tuberculosis Leprosy Diphtheria
Agent M. tuberculosis M. leprae Corynebacterium diphtheriae Scarlet fever Streptococcus pyrogens
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