Immunology 1 - Exam 2 Flashcards
- Which immune assay would BEST able to determine the titer (level) of antigen-specific IgG in serum?
ELISA
What do you know about heterogenous enzyme immunosorbent assays (EIAs)?
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
Describe the steps in heterogenous EIAs (ELISAs).
- 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.
What is antibody titer? When is it used?
- 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.
- Which one of the following sequences is appropriate for testing a patient for cytokine levels in serum using an ELISA procedure?
antibody against cytokine/patient’s serum/ enzyme-labeled antibody against cytokine/enzyme substrate
What are capture assays?
- Are best suited to antigens that have multiple determinants.
- Antibodies
- Cytokines
- Proteins
- Tumor markers
> Microorganisms (especially viruses) - Also used in the measurement of immunoglobulins.
Describe the steps in heterogeneous EIAs - capture assays?
- 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.
You are testing samples for recreational drugs using EMIT. This is an example of…
Homogenous assay
What do you know about homogenous EIAs?
- 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
What is an Enzyme Multiplied Immunoassay (EMIT)?
Enzyme multiplied immunoassay technique (EMIT) is a commonmethodfor qualitative and quantitative determination of therapeutic and recreational drugs and certain proteins in serum and urine.
What the are components of the EMIT (enzyme multiplied immunoassay) assay method?
- Drug
- Antibody
- Substrate
- Enzyme bound to drug
What is the general procedure for EMIT (enzyme multiplied immunoassay) assay?
- 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
Point-of-care testing is performed most easily using:
Immunochromatographic assay
What do you know about rapid immunoassays?
- 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.
What kind of assay is immunochromatography?
rapid immunoassay (slide 19)
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:
direct immunofluorescence.
What do you know about fluorescent assays?
- 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.
Describe the direct immunofluorescent assay process.
- 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.
Describe the indirect immunofluorescent assay process.
- 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.
Direct vs indirect immunoflourescent assays.
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.
The standard method for detecting antinuclear antibodies (in certain autoimmune diseases) is:
indirect immunofluorescence
What do you know about Antinuclear antibodies (ANA) tests?
- 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.
What is the Antinuclear Antibodies (ANA) test procedure?
- 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.
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?
Lupus (Slide 34)
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
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.
What is competitive radio-immunosorbent test (RIST) used for?
Used to detect the total concentration of IgE in patient serum.
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
Describe cross reactive antibody.
- Cross-reactive antibody - thereactionbetween anantibodyand 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.
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.
Describe Precipitation reactions.
- Involve combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.
- Requires the antigen and antibody to have: multiple binding sites for one another & equal relative concentration of each.
- The higher the affinity and avidity values for an antibody and the more antigen-antibody complexes that are formed, the more sensitive the test.