Unit 4 Study Guide: Chapter 14
Describe factors that contribute to the re-emergence of vaccine-preventable diseases.
-Vaccination fears -In both the US and UK, a drop in childhood vaccination rates has led to outbreaks of vaccine-preventable diseases
Discuss the Western blot technique and its importance in diagnostic testing.
•A sample of proteins from a bacterial cell or virus is separated via electrical charge within a gel •The proteins distributed throughout the gel are transferred and immobilized on a filter •The filter is incubated with a patient's serum containing antibody •If the serum contains antibodies to the microbe, they will bind to the antigens on the filter paper •A second antibody designed to see the Fc portion of a human antibody as an antigen is applied to the filter paper •Contains a fluorescent or luminescent molecule or enzyme that turns a colorless substrate into a colored product •After incubation, sites of specific antigen-antibody binding appear as a pattern of bands that can be compared with positive and negative controls •Highly specific and sensitive way to identify or verify the presence of microbial-specific antigens in a patient sample •Used as a verification test for HIV screening tests
Describe Immunochromatographic "dipstick" tests and their use in diagnostics.
•Analyze specimens for the presence of antigen-specific antibody •Known antigen is immobilized within a cartridge and the sample is collected on its filter paper tip •As the sample migrates along the cartridge, it may interact with bound antigen, producing a color change in positive samples •Useful for rapid gonorrhea screening and identifying Leishmania and Trypanosoma -used to detect adenoviruses; pregnancy -control line and test line
Discuss how DNA vaccines and recombinant vector vaccines stimulate immunity.
DNA vaccines -Genes encoding highly immunogenic antigens are identified -Target genes are placed into a plasmid -Plasmid is injected into a human host -Human cells take up the plasmid and transcribe and translate the genes -Cells become the antigen producers -Results in a humoral and a cellular immune response RNA vaccines -mRNA injected into cells -translated into encoded protein -protein expressed in cell so dendritic cell recognizes antigens -both T and B cell immunity is produced
Explain why isolating a pathogen through standard culture methods may become an outdated diagnosis strategy.
Downfalls of isolation of a pathogen through standard culture method: -what if the microbe that is isolated is not the cause but just a bystander? -culturing takes 18 hours minimum and many organisms require longer incubation ties -many infections are polymicrobial Example: bloodstream infection (septicemia): - Identifying the cause of septicemia can take 18 - 24 hours, but the critical time frame for appropriate management is less than 6 hours. - If a patient responds to broad-spectrum antibiotics during this time, the prescription is not rewritten for a more appropriate drug. - If the patient does not improve, the delay could be fatal. - More sophisticated diagnostic techniques are needed.
State what adjuvants are and describe their purpose.
Examples of adjuvants are aluminum salts and monophosphoryl lipid A. Subunit vaccines require adjuvants to induce a strong immune response. Aluminum adjuvants are effective at enhancing antibody responses, are well tolerated, do not cause pyrexia and have the strongest safety record of any human adjuvants.
Explain how fluorescent-tagged antibodies may be used in diagnostic testing.
Fluorescent-tagged antibodies can detect antigens in a sample. In Immunoflourescence microscopy: -Utilizes fluorescent-tagged antibodies to recognize a specific antigen in a sample Requires a specialized fluorescent microscope In Immunoflourescence assays (IFAs) -Like ELISA, detects antigens or antibodies in a patient sample -Detection antibody is linked to a fluorescent tag instead of an enzyme -Eliminates the need to add substrate for the detection step In Flow Cytometry: -Allows for enumeration of specific cells -Requires fluorescence-activated cell sorter (FACS)
Describe the advantages of immunological diagnostics over biochemical testing.
Immunological diagnostic tests often rely on antigen-antibody interactions •Immunological diagnostics are essential tools for identifying a variety of pathogens •Immunological tests typically involve serology •Often the goal is to determine if a patient has certain antigens and/or antibodies in their blood Benefit of biochemical tests -Useful for identifying bacteria that are responsible for an infection •Drawbacks of biochemical tests -Can take more than 24 hours to perform -Pathogen must be culturable -Can't identify noncellular pathogens
Describe direct, indirect, and sandwich ELISA techniques.
In general on enzyme-linked immunosorbent assays (ELISAs): -Versatile and rapid diagnostic tests -Rely on: Antibody-antigen interactions-- reporter enzyme attached to a monoclonal detecting antibody; Chemically modifies an added substrate -other wording: Uses an enzyme-linked indicator antibody to visualize antigen-antibody reactions •Relies on a solid support such as a plastic microtiter plate that can adsorb or bind the reactants to its surface Direct ELISA: -Allows for identification of antigens in a sample -Solution (patient sample) possibly containing antigens is added to microtiter plate wells -Antigens stick to the bottom of the wells -Detection antibodies are added and bind if the antigen is present -Unbound antibodies are washed out of wells -Substrate is added -Substrate + antibody-linked reporter enzyme gives a colorimetric or chemiluminescent signal -Microtiter plate is inserted into a plate reader to measure the signal in each well Indirect ELISA: -looks for antibodies not antigens -Requires two antibodies -First antibody from the patient sample recognizes bound antigen on the factory plated microtiter plate -Second antibody is enzyme-linked -Microtiter plate is precoated with antigen -Patient's serum is added to plates -Patient antibodies that recognize the antigens bind -Enzyme-linked detection antibody is added §Binds to specific types of human antibodies -Unbound detection antibodies are washed out -Substrate is added -Signal levels are measured Sandwich ELISA: -measures antigens; plate comes coated with antibodies, not antigens -Requires two antibodies: Capture antibody and detection antibody -Microtiter plate is coated with capture antibody -Sample possibly containing antigen is added -Antigens bind the capture antibody -Detection antibody is added -"Sandwiches" the antigen between two antibodies -Unbound antibodies are washed out Substrate is added and signal is measured
Describe DNA microarrays and explain how they are applied to clinical diagnostics.
Microarrays: absorbent plates or "chips" that contain gene sequences from thousands of infectious agents -selected based on the syndrome being investigated -can be made to contain bacterial, viral, and fungal genes in a single test Patient samples of nucleic acids isolated from them are incubated with labeled gene sequences on the microarray -matching sequences hybridize to the chip -the fluorescent label is detected by a computer program Gene microarrays Gene microarray technology provides a global view of cellular functions - Useful tools for investigating differences between healthy and diseased cells - Utilizes complementary base-pairing between nucleotides -can be used to study the effects of a drug on human cells
Describe the polymerase chain reaction (PCR), real-time PCR, and reverse transcription PCR (RT-PCR) and state the clinical applications of each.
Polymerase chain reaction (PCR): used in many nucleic acid tests; results in production of numerous copies of DNA or RNA molecules within hours; can amplify minute quantities of nucleic acids in a sample, greatly improving the sensitivity of diagnostic tests; can be performed on bacteria, viruses, protozoa, and fungi; necessary for metagenomic analysis of the human body -requires thermocycler -requires reagents including template DNA to be copied, two single-stranded DNA primers, Taq polymerase, deoxynucleotide triphosphates (dNTPs) -requires a small tube with reagents mixed in the proper concentrations is placed in the thermocycler -heating and cooling cycles make new copies of the gene -clinical applications: facilitates gene sequences for genetic disorders; diagnosing infections Real time PCR: - Modified PCR that uses fluorescence imaging to visualize DNA copies as they are made - Computer programs allow technicians to see the data immediately or in "real time" - Sometimes called quantitative PCR (qPCR) -Can measure how many copies of the target gene were initially present in the sample - Reverse transcription PCR (RT-PCR) - Useful for detecting RNA in a sample - Requires reverse transcriptase -Builds DNA that is complementary to target RNA molecules in a sample
Explain herd immunity and describe how it protects nonimmunized people.
Vaccinating a sufficient percentage leads to herd immunity for nonvaccinated individuals •Most pathogens require vaccination of ~85% of the population to have effective herd immunity •Public health immunization initiatives aim to create herd immunity •Vaccines limit the number of susceptible hosts in a population and generate herd immunity by breaking the chain of pathogen transmission. •The fewer disease-susceptible people in a community, the harder it is for a pathogen to be transmitted to a susceptible host •Some vaccines may not be recommended for certain patients (e.g., pregnant women or immune-compromised patients) •Anyone who can be vaccinated should be vaccinated because herd immunity protects the most vulnerable people in the population; Protects premature babies and immune-compromised patients
Describe the various types of vaccine formulations.
1.[One] Active Agent: Attenuated vaccines: active virus or live bacterium rendered nonpathogenic -examples: Varicella-zoster vaccine, MMR (measles, mumps, and rubella) vaccine, rotavirus vaccine, oral polio vaccine (OPV) [Five] Inactivated Agents: 1. Whole-agent vaccines: contain the entire pathogen; inactivated by heat, chemicals, or radiation 2. Subunit Vaccines: Portion of pathogen is used to stimulate an immune response 3. Purified Subunit Vaccines (Natural or Recombinant): made by either purifying parts of the actual pathogen or, in recombinant vaccines, using genetic engineering to make parts of the pathogen. -examples: Hepatitis B (recombinant vaccine), human papillomavirus (HPV; recombinant vaccines), influenza (purified subunit and recombinant subunit vaccines available), acellular pertussis vaccine (purified subunit vaccine) 4. Toxoid Vaccines: inactivated protein toxin -examples: diphtheria and tetanus components of DTap and Tdap vaccines 5. Conjugate (or Polysaccharide) Vaccines: polysaccharides are conjugated or linked to a component that enhances immunogenicity -examples: Haemophilus influenzae type B (Hib vaccine), meningococcal vaccine (Neisseria menigitidis) New vaccines: 1. DNA vaccines are in development for persistent and emerging diseases -genes encoding highly immunogenic antigens are identified, placed into a plasmid, injected into human host where they are transcribed and translated by cells, producing antigen, results in a humoral and cellular immune response 2. RNA vaccines: mRNA injected into cells, translated into encoded protein, protein expressed in cell so dendritic cell recognizes antigens, both T and B cell immunity is produced 3. Recombinant vector vaccines -genetic material from pathogen packed inside harmless virus or bacterium and inserted into body
Discuss the general immunology principles underlying vaccinations.
A vaccine may consist of: -Weakened (attenuated) microbe -Fragments of the microbe -Isolated inactivated toxin -Genetically manufactured portion of the microbe Vaccines stimulate immunological memory -When the vaccinated individual encounters the real microbe at a later time, both T- and B-cell memory cells act quickly to prevent infection
Compare and contrast agglutination and immune precipitation tests.
Agglutination: •Antibodies -Have 2 antigen-binding sites -Can attach to more than one antigen -Can bind antigens into a clump (agglutination) •Agglutination reactions can be seen when antibodies interact with: -Cells that display multiple surface antigens -Tiny synthetic beads coated with antigens •Agglutination reactions are usually used for: -Blood typing -Identify infections -Diagnose noninfectious immune disorders •Testing can identify either antibody or antigen in patient samples (e.g., serum, urine, CSF) Process of agglutination with example: A patient's diluted serum is added to wells in a plate containing antigen-coated beads. If antibodies to T. pallidum antigens are present in the patient's serum, they will cross-link the beads, producing a broad patch of agglutination (a positive result). If antibodies are absent, the beads won't agglutinate and instead they settle at the bottom of the well as a small spot (a negative result). Immunoprecipitation: -Useful for determining what antibodies or antigens are present in a patient's sample -Detects an area where antibodies and antigens interact and form a precipitate in a gel matrix Process of immunoprecipitation tests [two types]: 1. Radial immunodiffusion test (Mancini method) -Quantifies the amount of a particular antigen present in a sample -To perform the test: -Known concentration of antibody is infused in the gel matrix -Test solution is added to a well in the gel -Sample diffuses from the well into the gel -precipitate forms at zone of equivalence 2. Double immunodiffusion test (Ouchterlony test) -Gel diffusion test -Tests a patient sample for diverse antibodies or antigens all at one time -To perform the test: -Both antibody and antigen into wells of a gel matrix -Observe for precipitate formation
