Analytical Methods in Immunology/Serology
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Explain the principle of direct agglutination.
- Antibody binds to naturally-occuring antigens on particles, linking the particles to form macroscropic agglutinates.
- Febrile agglutin test and Salmonella and Shigella serotyping are examples of direct agglutination.
Explain the principle of passive agglutination.
- Soluble antigen is bound to a carrier such as latex beads, RBCs, bentonite, or charcoal. Carrier particles agglutinate in the presence of the corresponding antibody. The RA factor test is an example of passive agglutination test.
- IgG is bound to latex.
Name a disease that can cause false positive results in latex agglutination tests because of an antibody in the serum that reacts with IgG.
- Rheumatoid factor is an antibody to IgG. It may cause nonspecific agglutination by reacting with the IgG reagent antibody attached to the latex particles.
What is coagglutination?
- It is rapid method used to identify some bacteria
- Antibodies to a specific bacterial antigen are attached to protein A on the surface of S.aureus. When mixed with a colony, if the corresponding bacterial antigen is present, agglutination occurs.
What are hemagglutination tests?
- Agglutination tests in which the antigens are located on RBCs.
- Ex: cold agglutinin test and the heterophile tests.
What type of antibodies can cause false positive reactions in hemagglutination tests?
- Heterophile antibodies.
- They cause nonspecific agglutination of some RBCs
Explain the principle of hemagglutination inhibition.
- Patient serum is incubated with a virus, followed by RBCs known to be agglutinated by the virus.
- If the serum contains the antibody to the virus, the virus is neutralized and agglutination of the RBCs is inhibited. No agglutination is a + reaction.
- Hemagglutination inhibition is the classic method used to detect rubella antibodies.
Explain the principle of precipitation.
Soluble antigen combines with soluble antibody to form an insoluble visible complex. These reactions are usually carried out in agar.
What is the zone of equivalence in precipitation tests?
The point in an antigen-antibody reaction where the number of antigen sites and antibodies are approximately equal, resulting in optimal precipitation.
Is precipitation or agglutination a more sensitive method?
- Fewer antibodies are needed to produce a visible reaction.
What is flocculation?
- A type of precipitation reaction in which soluble antigen reacts with antibody to form fine particles.
- Ex: VDRL, RPR
Rank the following methods in order from lowest to highest sensitivity: agglutination, precipitation, immunoassay.
Precipitation, agglutination, immunoassay
Property of some compounds to absorb light of one wavelength and emit light of a longer wavelength (lower energy).
Explain the principle of the direct fluorescent antibody (DFA) technique.
- Specimen is placed on a slide and overlaid with a fluorescein-labeled antibody.
- If the corresponding antigen is present in the specimen, the antibody binds and fluorescence will be seen under the microscope.
- Ex: DFA tests for Bordetella pertussis and Legionella
Explain the principle of indirect fluorescent antibody (IFA) technique.
- Serum is layered over reagent antigen fixed to a glass slide.
- If the corresponding antibody is present in the serum, it attaches to the antigen. After incubation and washing, fluorescein-labeled anti-human globulin (AHG) is added. It will attach to any antibody bound to the antigen and fluorescence will be seen under the microscope.
- IFA is sometimes referred to as a "sandwich" technique because the patient's antibody is sandwiched between the antigen on the slide and the labeled AHG.
- EX: FTA-ABS and FANA
Explain immunofixation electrophoresis (IFE).
- Specimens are placed on separate tracks in an agarose gel or on cellulose acetate strips.
- The proteins are separated by electrophoresis.
- One track is treated with a fixative that fixes all proteins, creating a reference pattern. The other tracks are treated with monospecific antisera to IgG, IgA, IgM, and kappa and lambda.
- Unprecipitated proteins are washed away and the remaining proteins are stained.
- The locations of the stained proteins are compared to the reference pattern.
- IFE used to identify immunoglobulins and Bence Jones proteins.
Explain the principle of radial immunodiffusion (RID).
- Samples are placed in wells in agar that contains an antibody to the antigen being measured.
- During incubation (18-24 hours at RT), the antigen diffuses out of the well and forms a precipitin ring with the antibody. The ring diameter is proportional to the concentration of the antigen.
- The concentration is determined from a standard curve based on the ring diameters of the standards.
What is the Ouchterlony technique?
- A double-diffusion method that is used to compare antigens in the specimen to antigens in a control. The specimen and controls are placed in adjacent wells on the outside of the agar plate and an antiserum is placed in a central well.
- The plates are incubated for 12-18 hours.
- Precipitin lines form where antigens and antibodies meet and the patterns of precipitation between two wells indicate whether or not the specimen and the control contained related antigens. The technique is most often used to detect fungal exoantigens or serum antibodies.
Describe patterns of identity, non-identity, and partial identity in the Ouchterlony double immunodiffusion test.
- If the antigens in the specimen and in the control are identical, the lines fuse to form an arc (identify).
- If the antigens in the specimen and in the control
What are some sources of error in the Ouchterlony technique?
Irregular hole punching in the agar, overfilling the wells, nonlevel incubation, inadequate time for diffusion, excessive condensation, incorrect labeling of the wells, and drying out or contamination of the plates.
What is a monoclonal antibody?
- An antibody derived from a single clone of B cells.
- Monoclonal antibodies are highly specific.
What is a ligand?
- A molecule that binds to another molecule of complementary configuration.
- In immunoassays, the term refers to the substance being measured.
What is the principle of competitive immunoassays?
- Unlabeled antigen in the patient sample competes with labeled reagent antigen for limited binding sites on a reagent antibody.
- The more unlabeled antigen in the sample, the less labeled antigen will be bound.
- Radioimmunoassay (RIA) is an example of a competitive immunoassay, but it is no longer widely used in clinical labs. This same principle has been applied to enzyme immunoassays (EIA).
What is the most common radioisotope in RIA procedures?
- Emits gamma radiation and has a half life of 60 days.
Why are enzyme immunoassays (EIA) replacing most radioimmunoassays?
- The reagents have a longer shelf-life and the hazards and regulations associated with use of radionucleotides are eliminated.
- EIA is as sensitive and specific as RIA.
Explain a typical EIA procedure for the detection of antigen in a clinical sample.
- Reagent antibody is bound to a solid matrix such as the wells of a microtiter tray, plastic or magnetic beads, or a porous membrane.
- The clinical sample is added to the solid matrix. If the antigen is present in the sample, it binds to the reagent antibody on the solid matrix.
- Unbound sample is removed by washing and a second antibody specific to the antigen is added.
- In the direct method, this second antibody is conjugated to an enzyme. In the indirect method, a third antibody, conjugated to an enzyme and directed against the Fc portion of the unlabeled second antibody is added.
- Following another wash to remove unbound antibody, a substrate specific for the enzyme label is added. The amount of colored end product is directly proportional to the amount of enzyme-bound conjugate which is directly proportional to the amount of antigen in the sample.
What enzyme labels are commonly used in EIA procedures?
- Horseradish peroxidase
- Alkaline phosphatase
What is measured in enzyme immunoassays?
- Light absorbance following reaction of the enzyme-label with a substrate.
- A common source of error in EIA is inadequate washing to remove unbound enzyme-labeled antigen. This can cause false positive reaction.
Explain "flow through" membrane bound EIA.
- Reagent antibody and a positive control are immobolized as dots/lines on the surface of a porous membrane in a disposable plastic cassette. The sample is applied and if the antigen is present, it is captured by the reagent antibody on the membrane.
- An absorbent material beneath pulls the reactants through the membrane, separating the nonreactant components from the antigen-antibody complexes.
- Followed by a wash, application of a signal reagent and another wash.
- A positive reaction is the presence of a colored dot or line at both the test and positive control locations. By immobilizing reagent antigen on the membrane, this method can also be used to detect antibodies.
- These tests have become popular because they are rapid and can be performed as single tests. They are commonly used in physician office labs and clinics and some are approved for home use.
What additional quality control is required for tests with built-in controls?
A positive and negative external control must be tested with each new lot number or different shipment of a given lot number.
What QC is required for antigen-antibody tests that do not have built-in controls?
A positive and negative control must be tested each day of use.
Which methods are most often used on automated immunoassay analyzers?
- Enzyme Immunoassay (EIA)
- Immunofluorescence (IFA)
What is chemiluminescence?
- Production of light energy as a result of a chemical reaction.
- Chemiluminescent molecules such as luminol, acridium ester, and dioxetane phosphate are used as labels for immunoassays.
In fluorescence polarization immunoassays (FPIA), how does the light emission of bound labeled ligand differ from that of free labeled ligand?
- The bound labeled ligand rotates more slowly and emits more polarized fluorscence.
- This difference in light emission between free and bound ligand is why FPIA assays do not require a separation step.
What type of assay is the enzyme-multiplied immunoassay technique (EMIT)?
- It is a competitive, homogenous enzyme immunoassay.
- No separation is required because enzyme activity is inhibited when the enzyme-labeled ligand is bound to antibody. With low concentration of the unlabeled ligand, more enzyme-labeled ligand is bound and there is less enzyme action on the substrate.
What step is required in a heterogenous immunoassay that is not required in a homogenous immunoassay?
- Separation of antigen bound to antibody from unbound antigen.
- The most common method for separation is solid phase attachment.
- Ex: When antibody is attached to the walls of the test tubes, seperation can be achieved by aspiration or decantation.
- Heterogenous assays are more sensitive than homogenous assays.
What is the technique in which immunoglobulins, complement components, and other proteins are measured based on light scatter by immune complexes?
Explain the principle of nephelometry.
- Protein in a patient specimen reacts with reagent antibodies to form insoluble complexes that scatter light.
- The amount of scattered light is proportional to the amount of protein present.
- Quantitation is by comparison of the patine values with standards of known concentration.
Name several analytes that can be measured by nephelometry.
IgG, IgA, IgM, C3, C4, CRP, ceruloplasmin, RA factor, and alpha-1 antitrypsin
With nephelometry, what condition in the specimen could lead to inaccurate results?
Name an instrument that is used to determine CD markers.
- Flow cytometer also known as fluorescence-activated cell sorter (FACS)
- Cells are identified based on the forward and side scattering of light as they flow in single file through a laser beam.
- Gates are set to analyze only lymphs, with fluorescent monoclonal antibodies used as tags.
What are the three most commonly used fluorescent labels used in flow cytometry?
- Fluorescein isothiocyanate
What does the CD45 antigen identify?
What is the clinical significance of the CD34 antigen?
- It is one of the earliest appearing markers on the cell surface and is lost as the cell matures.
- An immunophenotyping panel can be used to determine the proportion and absolute number of CD34 cells in the total white cell population of bone marrow or peripheral blood stem cell preparations. The number is used to determine the amount of stem cell product required for infusion.
What is Western blotting?
- Method to separate and ID proteins in a complex mixture.
- Used to confirm for anti-HIV
What is the first step in HLA testing?
Ficoll-hypaque gradient centrifugation, to obtain mononuclear cells.
What is the name of the test for HLA antigens that is based on uptake of a stain by dead cells that have combined with specific antibody?
The lymphocytotoxicity test.
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