Immunology 1 - Exam 2 Flashcards
1
Q
- Which immune assay would BEST able to determine the titer (level) of antigen-specific IgG in serum?
A
ELISA
2
Q
What do you know about heterogenous enzyme immunosorbent assays (EIAs)?
A
Noncompetitive enzyme immunoassays.
- Offer high sensitivity and specificity, simplicity, and low cost
- Enzyme-linked immunosorbent assay (ELISA)
- Used to measure antibody production to infectious agents that are difficult to isolate and for autoantibody testing
> HIV
> Hepatitis B and hepatitis C
- Is easily applied to point-of-care and home testing
3
Q
Describe the steps in heterogenous EIAs (ELISAs).
A
- Antigen is bound to solid phase.
- Patient serum with unknown antibody is added and given time to react.
- After a wash step, an enzyme-labeled antiglobulin (secondary antibody) is added.
- The second antibody reacts with any patient antibody that is bound to the solid phase.
- If no patient antibody is bound to the solid phase, the second labeled antibody will not be bound.
- After a second wash step, enzyme substrate is added.
- The amount of color, fluorescence, or luminescence is measured; this amount is directly proportional to the amount of antibody in the specimen.
4
Q
What is antibody titer? When is it used?
A
- Determines the immune status or exposure of an animal to a particular disease. However, since titer results vary depending on the laboratory and testing methodology as well as the history of the individual itself, there are no definitive answers as to what titer is considered protective against any given disease.
- Antibody titer= a measurement of how muchantibodywas produced that recognizes a particular antigen.
- Antibody titers are often expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
> ELISA and agglutination are a commonmeansof determiningantibody titers.
5
Q
- Which one of the following sequences is appropriate for testing a patient for cytokine levels in serum using an ELISA procedure?
A
antibody against cytokine/patient’s serum/ enzyme-labeled antibody against cytokine/enzyme substrate
6
Q
What are capture assays?
A
- Are best suited to antigens that have multiple determinants.
- Antibodies
- Cytokines
- Proteins
- Tumor markers
> Microorganisms (especially viruses) - Also used in the measurement of immunoglobulins.
7
Q
Describe the steps in heterogeneous EIAs - capture assays?
A
- Excess antibody attached to the solid phase.
- Test sample is added and allowed to react.
- After an appropriate incubation period, the wells are washed and enzyme-labeled antibody is added and allowed to react.
> enzyme labeled antibody typically recognizes a different epitope than the capture antibody. - After a wash step, substrate is added and the reaction is read. Either a colored or chemiluminescent reaction product is detected.
> enzymatic activity is directly proportional to the amount of antigen in the test sample.
8
Q
You are testing samples for recreational drugs using EMIT. This is an example of…
A
Homogenous assay
9
Q
What do you know about homogenous EIAs?
A
- These antigen–antibody systems do not require a washing or separation step.
- Enzyme activity is directly in proportion to the concentration of patient antigen or hapten present in the test solution.
- When antibody binds to specific determinant sites on the antigen, the active site on the enzyme is blocked, resulting in a measurable loss of activity.
- Are less sensitive than heterogenous assays.
- Are rapid and simple to perform.
- Are used to determine low-molecular-weight analytes in serum and urine.
> Hormones
> Therapeutic drugs
> Drugs of abuse
10
Q
What is an Enzyme Multiplied Immunoassay (EMIT)?
A
Enzyme multiplied immunoassay technique (EMIT) is a commonmethodfor qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in serum and urine.
11
Q
What the are components of the EMIT (enzyme multiplied immunoassay) assay method?
A
- Drug
- Antibody
- Substrate
- Enzyme bound to drug
12
Q
What is the general procedure for EMIT (enzyme multiplied immunoassay) assay?
A
- Mix sample containing drug with fixed quantity of enzyme bound drug, and antibody.
- Add substrate
- Measure absorbance at 15 and 45 seconds after substrate addition.
- Quantitate by measuring enzyme-substrate reaction (by UV - visible spectroscopy)
- Δ Absorbance from Reaction rate from Drug concentration
- Non linear relationship between Δ Absorbance and Concentration
- Determine standard curve
13
Q
Point-of-care testing is performed most easily using:
A
Immunochromatographic assay
14
Q
What do you know about rapid immunoassays?
A
- Are membrane-based, single-use, and disposable assays.
- Involve antigen or antibody being coupled to the membrane.
- Are read by looking for a colored reaction.
- May require the separate addition of patient sample, wash reagent, labeled antigen or antibody, and the substrate.
15
Q
What kind of assay is immunochromatography?
A
rapid immunoassay (slide 19)
16
Q
A tissue section is fixed to a microscope slide, then incubated with a fluorescently labeled antibody that binds to an antigen expressed by cells in that tissue. The preparation is then washed and inspected with a fluorescent microscope. This technique is called:
A
direct immunofluorescence.
17
Q
What do you know about fluorescent assays?
A
- Is most commonly associated with qualitative observations involving the use of a fluorescence microscope.
- Involves grading the amount of fluorescence against a dark background.
- Is used for rapid identification of microorganisms in cell culture or infected tissue, tumor-specific antigens on neoplastic tissue, and transplantation antigens.
18
Q
Describe the direct immunofluorescent assay process.
A
- Antibody that is conjugated with a fluorescent tag is added directly to unknown antigen that is fixed to a microscope slide.
- After incubation and a wash step, the slide is read using a fluorescence microscope.
- Technique demonstrates the presence of pathogens in patient samples.
19
Q
Describe the indirect immunofluorescent assay process.
A
- Patient serum is incubated with a known antigen attached to a solid phase.
- The slide is wahsed, and then an anti-human immunoglobulin containing a fluorescent tag is added.
- A sandwich is formed with the first antibody, which localizes the fluorescence.
- Antibody can be identified.
20
Q
Direct vs indirect immunoflourescent assays.
A
Direct: fluorescently-labeled antibody is bound directly to patient antigen.
Indirect: patient antibody binds to selected antigen, and the anti-human immunoglobulin binds to the other end of the patient antibody. The anti-human immunoglobulin has the fluorescent piece attached to it.
21
Q
The standard method for detecting antinuclear antibodies (in certain autoimmune diseases) is:
A
indirect immunofluorescence
22
Q
What do you know about Antinuclear antibodies (ANA) tests?
A
- The antibodies that target “normal” proteins within the nucleus of a cell are called antinuclear antibodies (ANA).
- Most of us have autoantibodies, but typically in small amounts. The presence of large amount of autoantibodies or ANAs can indicate an autoimmune disease.
- An ANA test is performed by exposing the patient’s serum to cells (HEP2 cells). It is then determined whether or not antibodies are present that react to various parts of the nucleus of cells. Thus, the term anti-“nuclear” antibody.
- Fluorescence techniques are frequently used to actually detect the antibodies in the cells, thus ANA testing is sometimes referred to as fluorescent antinuclear antibody test (FANA).
> There are EIA based tests that can also be used for screening or further determination of specific targets in nucleus that antibodies are recognizing. - A positive ANA test means autoantibodies are present. By itself, a positive ANA test does not indicate the presence of an autoimmune disease or the need for therapy.
> Some medications cause a positive ANA. Tell your doctor all prescription, over-the-counter, and street drugs you take.
> ANA testing can produce a positive result without any actual disease process. This typically signals the presence of antinuclear antibodies in a healthy individual.
23
Q
What is the Antinuclear Antibodies (ANA) test procedure?
A
- The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954.
- Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.
- After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein.When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen.This complex can be visualized with the aid of a fluorescent microscope.
- There are automated instruments that can also analyze results.
- In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells.If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence.The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.
- The patient’s serum samples will be typically tested using serial dilutions to determine titer of ANA antibodies.
24
Q
An ANA test shows staining of the periphery (rim) of the cell (left). Which of the following diseases would this most likely be associated with?
A
Lupus (Slide 34)
25
In an assay to determine the presence of a antibody specific for Streptococcus pyogenes in a patient sample, which of the following would be on solid phase?
Antigen
26
What is the major difference between competitive and non-competitive immunoassays?
The amount of bound label is inversely proportional to the concentration of the labeled antigen.
27
What is competitive radio-immunosorbent test (RIST) used for?
Used to detect the total concentration of IgE in patient serum.
28
Define affinity.
Affinity - the initial attraction force between a Fab site on an antibody molecule and an epitope or antigenic determinant site.
> An epitope, also known as antigenic determinant, is the part of an antigen recognized by the immune system, specifically by antibodies, B cells, or T cells.
> For example, the epitope is the specific piece of the antigen to which an antibody binds.
- The strength of attraction depends on the specificity of the antibody for a particular antigen.
- The higher the strength, the higher the affinity
29
Describe cross reactive antibody.
- Cross-reactive antibody - the reaction between an antibody and an antigen that differs from what appears to be an unrelated the immunogen.
> Immunogen – the antigen to which the antibodies were originally generated against.
> The two molecules may be completely different, except for similar epitopes that antibody binds to, perhaps with differing affinities.
- The more the cross-reacting antigen resembles the original antigen, the higher the affinity of the antibody to the cross-reactive antigens.
30
Describe Avidity.
- Avidity - commonly applied to antibody interactions in which multiple antigen-binding sites simultaneously interact with the target antigenic epitopes, often in multimerized structures.
> Involves the combined strength with which a multivalent antibody binds a multivalent antigen
> Individually, each binding interaction may be readily broken; however, when many binding interactions are present at the same time, transient unbinding of a single site does not allow the molecule to diffuse away, and binding of that weak interaction is likely to be restored.
- IgG and IgM could have the same affinity for an antigen, but IgM has 10 binding sites (pentamer), vs. IgG with 2 (monomer). Thus, IgM would have higher avidity.
31
Describe Precipitation reactions.
1. Involve combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.
2. Requires the antigen and antibody to have: multiple binding sites for one another & equal relative concentration of each.
3. The higher the affinity and avidity values for an antibody and the more antigen-antibody complexes that are formed, the more sensitive the test.
32
Describe the Precipitation Curve: zone of equivalence.
- When increasing amounts of soluble antigen are added to fixed amounts of specific antibody, the amount of precipitation increases up to the zone of equivalence.
- When the amount of antigen overwhelms the number of antibody combining sites present, precipitation begins to decline.
- To be detectable, precipitation reactions must be run in the zone of equivalence
33
Describe the Precipitation Curve: prozone.
Prozone phenomenon (antibody excess)
- Antigen combines with only one or two antibody molecules.
> No cross-linkages are formed that allow precipitant to form
- False-negative reactions may occur as a result of high antibody concentration
- If a false-negative reaction is suspected, diluting out the antibody and performing the test again may produce a positive result
34
Describe the Precipitation Curve: Postzone.
Postzone phenomenon (antigen excess)
- Small aggregates are surrounded by excess antigen
> No lattice network is formed
- The presence of a small amount of antibody may be obscured, causing false-negative results
> Test is repeated about a week later with a specimen to give time for further production of antibody
> If the test is negative again, it is unlikely that the patient has the antibody
35
Describe the Ring Test.
- The ring test is a simple serological technique that illustrates the precipitin reaction in solution.
- Antiserum is introduced into a small diameter test tube, and the antigen is then carefully added to form a distinct upper layer. After 4 hours incubation a ring of precipitate forms at the point of contact in the presence of antigen-antibody reaction.
- To detect the precipitin reaction, a series of dilutions of the antigen is used, because both insufficient and excessive antigen amounts of antigen will prevent the formation of a visible precipitate.
36
What are the types of Precipitation Reactions?
- Nephelometry: used for immunoglobulins, complement, C-reactive protein, other serum proteins.
Sensitivity = 1-10 ug AB/ML.
Principle = light that is scatterde at an angle is measured, indicating the amount of antigen or antibody present.
- Radial Immunodiffusion: used for immunoglobulins and complement.
Sensitivity = 10-50 ug AB/ML.
Principle = Antigen diffuses out into gel that is infused with antibody. Measurement of the radius indicates concentration of antigen.
- Ouchterlony Double Diffusion: used for complex antigens (i.e. fungal antigens).
Sensitivity = 20-200 ug AB/ML.
Principle = Both antigen and antibody diffuse out from wells in a gel. The lines of precipitate formed indicate the relationship of antigens.
- Immuno-electrophoresis: used for differentiation of serum proteins.
Sensitivity = 20-200 ug AB/ML.
Principle = electrophoresis of serum is followed by diffusion of antibody from the wells.
- Immunofixation Electrophoresis: used for over or underproduction of antibody.
Sensitivity = variable.
Principle = electrophoresis of serum is followed by direct application of antibody to the gel.
37
What are the steps of Radial Immunodiffusion?
A single-diffusion technique.
1. Antibody is in the support gel, and antigen is applied in a well cut in the gel.
2. Antigen diffuses out until the point of equivalence is reached (end-point method).
> The higher the concentration of antigen, the farther it will diffuse before reaching equivalence (precipitation ring).
3. The square of the diameter is proportional to the antigen concentration.
38
Describe Radial Diffusion reactions.
1. A glass slide coated with agar containing specific antibody.
2. Diffusion disks are formed as antibody is detected in sample.
3. Unknown sample is compared to standard, known concentrations that are graphed. Unknown concentration is predicted using the graph.
39
Describe Ouchterlony Diffusion reactions.
Precipitation reaction that is a double-diffusion technique.
1. Wells are cut in a gel, and both antigen and antibody diffuse out radially.
2. A line of precipitate forms where antigen and antibody meet.
3. 3 possible patterns: identity (common epitopes - arc), partial identity (some common epitopes, but unique epitopes - arc and spur), nonidentity (unique epitopes with no common epitopes - 2 crossing lines).
40
Describe immuno-electrophoresis.
Immuno-electrophoresis combines separation of a complex of antigens by electrophoresis with immunodiffusion of an antisera.
It aids in the diagnosis and evaluation of disease states affecting the immune system. It is usually requested for analysis of serum proteins and can indicate a rise at the immunoglobulin level. For example, immuno-electrophoresis is also used frequently to diagnose multiple myeloma, a disease affecting the bone marrow.
41
Describe the principle of Counter Immuno-electrophoresis (CIE).
The principle of Counter Immuno-electrophoresis method is that at pH 8.4 the immunoglobulins will migrate to the negative end of a capillary system if an electric field has been applied to the capillary system. In the same capillary system, many antigens will migrate towards the positive end at that pH. As the antibody and antigen move towards each other in an electric field, they will soon meet in optimal proportion and visible precipitate is formed.
42
Describe Immunofixation Electrophoresis.
A double-diffusion technique.
1. Unknown antigen is electrophoresed, and then antibody is applied directly to the gel.
2. Precipitates form where antigen–antibody combination has taken place in the gel.
3. Wash away unbound antigen and stain for protein.
4. Technique is used with serum as the antigen to determine over- or underproduction of antibody types.
43
Describe the Agglutination process.
Visible aggregation of particles resulting from combination with specific antibody.
> Similar to precipitation, except instead of soluble antigen, aggregation uses large particles, such as latex beads and RBCs.
- Two step process:
1. Sensitization (initial binding) = antigen and antibody unite through antigenic determinant sites.
2. Lattice formation (formation of large aggregates) = rearrangement of antigen and antibody bonds to form a stable lattice.
- Produces antibodies called agglutinins.
44
Describe types
of direct agglutination reactions.
Direct agglutination:
- The formation of an insoluble network of antigens and their antibodies, when particulate antigen is mixed with specific antiserum.
- Direct agglutination reactions are used, for example, in typing blood or in assessing the presence of antibodies against microorganisms.
> Widal test:
- A rapid screening test used to determine the possibility of typhoid fever
- Uses Salmonella organisms as particulate antigens to detect antibodies against O (somatic) and H (flagellar) antigens
- There are better tests now, but still used in developing countries
> Hemagglutination
- Involves red blood cells
- ABO blood group typing
- Agglutination of RBC’s using IgM specific for different blood group types
45
Describe types of Passive agglutination reactions.
Passive (or indirect) agglutination
- Employs particles that are coated with antigens not normally found on their surfaces
- Examples of particles:
> Erythrocytes, latex beads, gelatin, silicates
> Synthetic beads or particles (provide consistency, uniformity, and stability)
Used to detect:
Rheumatoid factor
Antibodies to Group A Streptococcus antigens
Antibodies to viruses such as rotavirus, cytomegalovirus, rubella, and varicella-zoster
Antibodies to hepatitis viruses and HIV
Process:
- Antigen is attached to the carrier particle
- Agglutination occurs if patient antibody is present
Reverse passive agglutination
- Instead of antigens attached to surface, antibodies with known specificities are
- Process:
> Antibody is attached to the carrier particle
> Agglutination occurs if antigen is present in patient sample
- Common applications:
> Rapid identification of antigens from infectious agents (e.g., Group B Streptococcus, Staphylococcus aureus, streptococcal groups A and B, rotavirus, and Cryptococcus neoformans)
> Detecting soluble antigens in urine, spinal fluid, serum
46
What is agglutination inhibition?
Agglutination inhibition:
- Is based on competition between particulate and soluble antigens for limited antibody-combining sites
- Lack of agglutination = positive reaction
- Used to detect antibodies to certain viruses, such as rubella, mumps, measles, influenza, parainfluenza, HBV, herpes virus, respiratory syncytial virus, and adenovirus
47
Describe instrumentation and quality control and assurance of Particle-counting immunoassay (PACIA).
- Uses nephelometry to increase sensitivity of reactions.
> Nephelometry measures light reflected off of particles.
- Involves a laser in an optical particle counter to measure the number of residual nonagglutinating particles in a specimen.
> Agglutinated particles will not be counted.
> The higher the amount of agglutination, the fewer residual nonagglutinating particles remain.
- Measures serum proteins, therapeutic drugs, tumor markers, and certain viral antigens.
- Avoid cross-reactivity by using a monoclonal antibody directed against an antigenic determinant unique to a particular antigen
- Store reagents properly, and check expiration dates
- Account for sensitivity and specificity of specific test kits used
- Be aware that a negative result does not rule out presence of the disease or antigen
48
A patient’s WBC’s are placed in culture. 3H-thymidine is added to the cultures 48 hours later, and the amount of radioactivity incorporated in DNA (CPM) will be measured. Which of the following should be added to stimulate proliferation of both T cells and B cells?
PWM
49
Lymphocyte stimulation assays are useful to:
- Determine if patient’s lymphocytes are functional
- Determine if patient has previously been exposed to an antigen
- Monitor cell populations related to possible immunodeficiency or immunoproliferative disease
> HIV staging
> Identifying lymphoma or leukemia cells
50
Flow cytometry is commonly used to:
Determine the stage of leukocyte differentiation.
51
Describe flow cytometry.
- Automated process that analyzes cells or beads in fluid suspensions for their light-scattering characteristics.
- Uses fluorochromes to identify cells or particles by size, shape, and antigenic properties.
> Fluoroscein (FITC) and phycoerythrin (PE) are common fluorochromes to be used.
- Allows for rapid and accurate detection of cells found in small numbers
- Uses include:
> Identifying HIV disease stage
> Immunophenotyping of cells
> Diagnosis of leukemia and lymphoma
> Functional assays for various conditions
52
An intrinsic parameter that can be measured by light scatter in a flow cytometer is:
Cell size
53
What is meant by “gating” in flow cytometry?
An electronic window separating subpopulations of cells.
54
As measured in flow cytometry, cells that are the smallest and have the least granules would be identified as:
Lymphocytes
55
A fluorescent signal in flow cytometry is generated by:
Fluorochromes that absorb energy from a laser beam and then release the energy as longer-wavelength light.
56
In flow cytometry, it is possible to detect the expression of 10 different cell membrane proteins simultaneously by:
Staining the cells with 10 different labeled probes that fluoresce at different wavelengths.
57
A mature cytotoxic T cell expresses which markers?
CD8
CD3
CD45
58
Clinical applications of flow cytometry.
- Identifies particular markers for diagnosis and monitoring of leukemias and lymphomas.
- Enumerates peripheral blood CD4+ T cells to classify stages of HIV disease and guide treatment.
- Enumerates CD34+ cells in stem cell transplantation.
- Determines DNA content or ploidy status of tumor cells.
- Helps diagnose inherited diseases.
59
Describe how cytokine production can be detected.
- Flow cytometry can be used to identify cytokine producing cells.
> Intracellular staining - permeabilize cells to allow fluorescently labeled antibody specific for cytokine to enter cytoplasm and interact with cytokines that are being synthesized.
- EIA capture assays can measure cytokines in serum, CSF, culture medium, etc.
> Capture and enzyme labeled detecting antibodies specific for different epitopes on cytokine.
60
What would most likely promote production of IgE?
IL-4
61
Describe the functions of Interleukin 4 (IL-4).
- Produced by Th2 cells, mast cells
- Functions:
> Stimulation of activated B-cell and T-cell proliferation.
> Promotes Th2 cell differentiation, dampens Th1 cell activity and development.
> Differentiation of B cells into plasma cells.
> Induces B-cell class switching to IgE.
> Up-regulates MHC class II production.
> IL-4 decreases the production of IFN-gamma, IL-12, dampens macrophage activation.
> Promotes wound repair.
> Overproduction of IL-4 is associated with allergies.
62
Nitroblue tetrazolium is added to a patient’s WBC’s. Under microscopic examination, the neutrophils, but not lymphocytes, are deep blue color. Which of the following is likely true?
Phagocytes are able to produce reactive oxygen species.
63
Describe Chronic Granulomatous Disease (CGD).
Most common phagocyte deficiency.
- In CGD, phagocytes, e.g. neutrophils, have abnormal respiratory bursts leading to recurrent bacterial and fungal infections.
> Defects in NADPH oxidase
> Common pathogens: Staphylococcus aureus, Burkholderia cepacia, Serratia marcescens, Nocardia species and Aspergillus species.
- Characterized by granulomatous inflammation in areas without clear infection, such as gastrointestinal and genitourinary tract, but also in response to infection.
- The granulomatous inflammation may be severe enough to impede treatment with antibiotics.
> May need to include corticosteroid to reduce inflammatory response
- Treatment:
> Prophylactic treatment with antibiotics significantly decreases frequency of infections.
> Treatment with interferon-gamma may also help reduce the number of severe infections.
> Surgery may be needed to treat some abscesses.
> The only cure for CGD is a bone marrow or stem cell transplant.
64
Describe Nitroblue-tetrazolium (NBT).
- Test checks if neutrophils can change a colorless chemical called nitroblue tetrazolium (NBT) into a deep blue color.
> This test depends upon the direct reduction of NBT to the insoluble blue compound formazan by NADPH oxidase; NADPH is oxidized in the same reaction.
> The cells are then examined under a microscope to see if the chemical has made them turn blue.
> The higher the blue score, the better the cell is at producing reactive oxygen species.
> It is negative in CGD, meaning that it does not turn blue
- Cells can be stimulated with LPS or bacteria to enhance oxidative activity.
- There are versions of this test where you can measure the reaction spectrophotometrically, rather than using a microscope.
65
What process confers specificity in molecular biology methods?
Hybridization
66
Describe process of Southern Blots.
1. Specimen DNA is denatured
2. Treated with restriction enzymes to created DNA fragments
3. Separated by electrophoresis
4. Blotted onto nitrocellulose membrane
5. Membrane hybridized with DNA probe, specific for gene or region of interest
6. Reveal location of fragments that hybridized to probes
67
Describe process of Northern Blots.
1. mRNA is isolated
2. Different sizes of mRNA are separated by electrophoresis
3. Blotted onto nitrocellulose membrane
4. Membrane hybridized with DNA probe, specific for gene of interest
5. Reveal mRNA that hybridized to probes
68
What is PCR?
Polymerase Chain Reaction.
Amplified DNA – Amplimer/Amplicon
Potential for false positives high due to ease of contamination with amplified DNA. Need to be very cautious
69
How would you interpret results of DNA restriction analysis by gel electrophoresis if there was a difference in one band?
The samples are not identical.
70
A patient is being treated for HIV infection. Before treatment, real-time PCR results for viral load had a cycle threshold (CT) of 28, but after 2 months of treatment, a CT of 24 was found. These results indicate that:
The viral load is increasing.
71
A child in a day care has developed pneumonia. A child in a nearby day care also has a respiratory infection, but not as severe. Samples from each child is found to have influenza virus using PCR. Restriction enzyme digests of the amplified samples show identical banding patterns. What conclusion can be drawn from these results?
The children may have been exposed to a common source of the cause of their illness.
72
Which of the following tests can be useful in confirming the specificity of serologic screening assays?
Western Blots.
73
Describe the process of Western Blots.
Western blots often used to confirm specificity of EIA
1. Denature proteins are separated by electrophoresis based on MW
2. Transferred to nitrocellulose membrane
3. React with patient serum (or labeled-antibody specific for antigen)
> Reveal reaction using labeled anti-human antibody
74
What is the difference between Southern Blots, Northern Blots, and Western Blots?
Southern Blots:
- DNA-DNA hybridization
- Examines presence of DNA sequence by hybridization with labeled DNA probe.
Northern Blots:
- RNA-DNA hybridization
- Monitor gene expression by hybridization of labeled DNA probe to mRNA.
Western Blots:
- Protein-antibody reactions.
- Monitor protein expression or antibody specific for protein using enzyme-labeled antibody specific for a protein.
75
Describe the different skin testing for allergies.
A microscopic amount of an allergen is introduced to a patient's skin by various means:
- Skin prick test: pricking the skin with a needle or pin containing a small amount of the allergen.
- Skin scratch test: a deep dermic scratch is performed with help of the blunt bottom of a lancet.
- Intradermic test: a tiny quantity of allergen is injected under the dermis with a hypodermic syringe.
- Skin scrape test: a superficial scrape is performed with help of the bovel of a needle to remove the superficial layer of the epidermis.
- Patch test: applying a patch to the skin, where the patch contains the allergen
If an immuno-response is seen in the form of a rash, urticaria (hives), or (worse) anaphylaxis it can be concluded that the patient has a hypersensitivity (or allergy) to that allergen.
76
Describe the virus neutralization test.
- Serial dilutions of heat inactivated test serum are prepared in a 96 well plate and are incubated with a set amount of infectious virus.
- Following this incubation, virus susceptible cells are added to the virus-serum mixture, and the final virus/serum/cell combination is incubated for a period of 2-3 days.
- After this incubation period the test is read by examining each well of the plate for the presence of viral infection.
77
Describe the complement hemolytic titration assay (CH50).
- CH50 assay uses lysis, the end point of complement activation, as a functional test of overall complement activity.
- Measure the amount of patient serum required to lyse 50% of a standardized concentration of antibody-sensitized sheep red blood cell.
- Diluted serum + Ab-SRBC => lysis if classical pathway complement is active
78
Describe sample preparation.
- Tissue specimens:
> Should be collected and transported in tissue culture medium
> May be at room temperature for imminent analysis; at 4°C if analysis is delayed
> Should be disaggregated via mechanical dissociation or enzymatic digestion
> Should have antibodies (usually monoclonal) added before processing
79
Describe the process of flow cytometry.
1. Automated process that analyzes cells or beads in fluid suspensions for their light-scattering characteristics.
2. Uses fluorochromes to identify cells or particles by size, shape, and antigenic properties.
3. Allows for rapid and accurate detections of cells found in small numbers.
