- List the serological techniques used to diagnose disease
- Immunoprecipitation
- Precipitation in solution
- Precipitin ring test
- Flocculation
- Precipitation in gel
- Single (Oudin) Immunodiffusion
- Double Immunodiffusion (Ochterlony assay)
- Radial Immunodiffusion
- Precipitation in solution
- Agglutination
- Direct Agglutination – Lancefield grouping, Rapid streptococcal antigen test
- Indirect (Latex) agglutination – Rheumatoid Factor, Widal test
- Hemagglutination assay – Direct coombs, Indirect coombs, Direct hemagglutination assay, Blood grouping
- Viral Hemagglutination inhibition
- Immunoassay techniques
- Radio-immunoassays (RIA)
- Enzyme Linked Immunosorbent assay (ELISA)
- Fluorescent Immunoassay
- Chemiluminescence Assay
- **Immunoblotting (**Western blot)
- **Immunochromatography (**Lateral Flow Test)
- Immunoprecipitation
- Define the following terms outlining their effects on the tests: Pro zone effect, Zone of equivalence, Post-zone effect
- Pro zone or hook effect
- Excess antibody to the available antigen = no precipitation = false negative results
- Zone of equivalence
- Optimal concentration of both antibody and antigen = precipitation and positive results
- Post zone effect
- Excess antigen to the available antibody = no precipitation = false negative results
- Pro zone or hook effect
In a tabular format, outline the differences between immunoprecipitation and agglutination reactions
| Agglutination | Precipitation | |
|---|---|---|
| Definition | Process of clumping of antigens with their respective antibodies | Process where soluble antigens bind with their specific antibody at an optimum temperature and pH, resulting in the formation of an insoluble precipitate |
| Antigen size | Antigen is relatively smaller | Antigen is relatively larger |
| Solubility | Insoluble antigens are used for agglutination | Soluble antigens are used for precipitation |
| Sensitivity | More sensitive than precipitation reaction | Less sensitive than agglutination reactions |
| Principle | Based on the clumping of particles | Based on the formation of lattices (cross-linking) |
| Nature of reactants | Starting molecules are particles | Starting molecules are ions |
| Requirements | Surface of the antigen has to be exposed for the antibody to bind and form visible clumps | Concentration of the antigen and antibody has to be equal. An any change in this equivalent prevents the formation of precipitins |
| Media | No gel or matrix is required for agglutination | Liquid or semi-solid matrix (gel) is required for precipitation |
| Nature of complex formed | Agglutinins usually settle towards the bottom of the container | Precipitins remains suspended or settle towards the bottom. Flocculants float on the surface of the liquid matrix |
- Using HIV antibody testing as an example; and with illustrations, describe the basic steps of non-competitive direct ELISA (10 marks)
- A buffered solution of anti-gp120 antigen is added to each well of a microtiter plate and given time to adhere to the plastic through charge interactions
- A solution containing non-reactive proteins (bovine serum albumin, casein) is added to each well in order to cover any plastic surface in the well that remain uncoated by the antigen
- The primary antibody with a conjugated enzyme (Horse-radish peroxidase) ****binds specifically to the test antigen coating the wells
- A substrate for the enzyme (OPD [o-phenylenediamine dihydrochloride]), changing color upon reaction with the enzyme (Amber)
- High concentration of primary antibody = higher color change
- Using HIV antibody testing as an example describe the basic steps of Sandwich ELISA
- A surface is prepared with known quantity of capture antibody
- Non-specific binding sites on the surface are blocked (Bovine serum albumin, casein)
- Sample containing antigen (anti-gp120) is applied to the plate and binds to captured antibody
- Plate is washed to remove unbound antigen
- A primary antibody is added and sandwiches the antigen between it and the capture antibody (primary antibody could be in the serum of a donor to be tested for reactivity towards the antigen)
- Enzyme-linked (HRP) secondary antibody is applied as detection antibody, binding non-specifically to the Fc region
- Substrate is added to be converted by the enzyme into a color, fluorescent, or electrochemical signal
- The absorbance, fluorescence, or, electrochemical (current) of the plate’s wells is measured to determine the presence and quantity of the antigen
- Using HIV antibody testing as an example; and with illustrations, describe the basic steps of competitive ELISA
- An Unlabelled antibody is incubated in the presence of its antigen (Sample)
- The bound antibody/antigen complex is added to the antigen-coated well
- The plate is washed so unbound antibodies are removed (the more antigen in the sample, the more Ag-Ab complexes, less unbound antibodies available to bind antigen in the well)
- Secondary Enzyme-linked antibody specific to the primary antibody is added
- A substrate is added and remaining enzymes elicit a chromogenic or fluorescent signal
- Using HBsAg test, describe the basic steps of direct ELISA
- A buffered solution containing HBsAg is added to each well of a microtiter plate and given time to adhere to the plastic through charge interactions
- A solution containing non-reactive proteins (bovine serum albumin, casein) is added to each well in order to cover any plastic surface in the well that remain uncoated by the antigen
- The primary antibody with a conjugated enzyme (Horse-radish peroxidase) ****binds specifically to the test antigen coating the wells
- A substrate for the enzyme (OPD [o-phenylenediamine dihydrochloride]), changing color upon reaction with the enzyme (Amber)
- High concentration of primary antibody = higher color change
- Briefly describe immunoprecipitation under the following: Definition, Types, 2 Clinical Applications
- Definition
- Immunoprecipitation is the formation of an insoluble lattice or precipitate when a soluble antigen binds with their specific antibody, at optimum temperature and pH
- Types
- Precipitation in solution
- Precipitin ring test: Zone of equivalnece appears between the soluble antigens and antibodies in a tube
- Flocculation: Visible clumps appear on the surface of a slide or tube
- Precipitation in gel
- Single (Oudin) Immunodiffusion: Diffusion in one dimension
- Double immunodiffusion (Ouchterlony assay): Proteins diffuse through the gels from wells and form arcs between them at zones of equivalence
- Radial immunodiffusion: Single diffusion in one dimension, Similar to Ouchterlony assay, used to precisely quantify antigen concentration
- Precipitation in gel with an electric field (Immunoelectrophoresis): No passive diffusion, voluntary action by an electric field
- Serum Protein electrophoresis
- Immunoelectrophoresis (IEP)
- Immunofixation electrophoresis (IFE)
- Precipitin ring test – Lancefield grouping of B-hemolytic streptococci
- Precipitation in solution
- 2 clinical applications
- Flocculation – VDRL test for syphilis
- Radial (Mansini) immunodiffusion – C3 and C4 protein assay
- Definition
- Briefly describe Immunoprecipitation
- Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein.
- The reaction between the soluble antigen and the antibody results in the formation of an insoluble lattice or precipitate out of solution at equimolar concentrations.
- Precipitation occurs at the zone of equivalence where the concentration of antigen and antibody is equal, on either side of equivalence, precipitation does not occur if the concentration of either antigen or antibody is in excess or deficient)
- Define the following terms as used in precipitation: Pro-zone effect, Zone of equivalence, Post-zone effect, Precipitin, Flocculation
- Pro-zone effect or Hook effect: excess antibody to the available amount of antigen = no precipitation
- Zone of equivalence: optimal amount of both antibody and antigen = precipitation
- Post-zone effect: excess antigen to the available antibody = no precipitation
- Precipitin: an antibody that produces a visible precipitation when it reacts with its antigen
- Flocculation: Occurs when a lighter precipitate is formed, which floats instead of forming a sediment
- List the precipitation reactions
- Precipitation in solution:
- Precipitin ring test
- Flocculation
- Precipitation in gel
- Single immunodiffusion test
- Double immunodiffusion test
- Radial immunodiffusion test
- Precipitation in gel with an electric field
- Electroimmunodiffusion -SPEP
- Precipitation in solution:
- Briefly describe Precipitation in solution (Simple precipitation)
- Precipitin ring test: zone of equivalence appears between the soluble antigens and antibodies in a tube. Examples: CRP, Lancefield grouping of B-hemolytic streptococci, Ascoli’s thermoprecipitation test
- Flocculation: visible clumps appear on a slide or tube. On tube flocculants will float. Examples: VDRL test (slide test), Kahn test for syphilis (tube test)
- Briefly describe Precipitation in Gel
- Single immunodiffusion (Oudin diffusion): Diffusion in one dimension
- Double immunodiffusion (Ouchterlony assay): agar is highly purifies, wells are formed on agar, proteins diffuse through the gel and precipitin arcs form between the wells at zone of equivalence
- Radial (Mansini) immunodiffusion: Single diffusion in two dimensions, similar to Ouchterlony but is used to precisely quantify antigen concentration, higher diameter = high concentration of antigen, square of diameter directly proportional to concentration of antigen. Example: determining concentration of serum proteins (C3 and C4 complement proteins assay)
- Briefly describe Precipitation in gel with an electric field (Immunoelectrophoresis)
- no passive diffusion – voluntary action by electric current
- Includes:
- Serum protein electrophoresis (SPEP)
- Immunoelectrophoresis (IEP)
- Immunofixation electrophoresis (IFE)
- Describe agglutination, including: direct agglutination, indirect agglutination, hemagglutination assay, viral hemagglutination inhibition
- Agglutination occurs when the interaction between antibodies and a particulate (insoluble) antigen results in visible clumping. It is the process of clumping of antigens with their respective antibodies. Artificial carriers (latex, charcoal particles) or biological carriers (red blood cells – hemagglutination) can be counjugated with antigens and agglutinate upon binding with their specific antibodies.
- Direct agglutination: Bacterial cells agglutinate when exposed to antibodies against their antigenic surface. Example:
- ID serovars of bacteria and viruses (Lancefield grouping of streptococci),
- Rapid streptococcal antigen test against M-protein
- Indirect (Latex) agglutination: Antibodies attached to latex beads are used to improve visualization, usually used when looking for IgM as their structure provides maximum cross linking. Example:
- Rheumatoid factor (RF) – IgM that binds to the Pts IgG,
- Widal test – agglutinates Salmonella enterica serovar typhi in Pt sera
- Hemagglutination assay: detection of antibodies or antigens bound to sialic receptors on RBC surface. Examples**:**
- Direct Coomb’s test (DCT, Direct antihuman globulin test (DAT),
- Indirect Coomb’s test (Indirect antiglobulin test, IAT)
- Direct hemagglutination assays (DHA): detects influenza, mumps and rubella – have viral spikes Neuraminidase (N) and Hemagglutinin (H) that cause hemagglutination when they cross-link RBCs
- Blood typing (grouping)
- Viral Hemagglutination Inhibition: Modified DHA, used to determine the titre of antiviral antibodies, patients serum is mixed with virus, then a standardized amount of RBCs is added and hemagglutination is observed, titre of the patients serum is the highest dilution that blocks agglutination
- Using a labelled diagram, describe the Lateral flow technique
- Lateral flow Assay has 4 parts
- Sample application pad: Where sample and buffer solution is added
- Conjugate pad: Contains the antibody conjugated tag (colloidal gold, latex, fluorophore)
- Nitrocellulose membrane: Contains the test and control strips. Test trips contains antibody against the antigen tested. Control line contains antibody against the conjugate.
- Absorbent pad: Absorbs excess solution
- Mechanism
- Adding the analyte and buffer causes the fluid capillary action
- Analyte binds to conjugate as it migrates
- Analyte-conjugate complex binds to test-line antibodies
- Conjugate also binds to control line for internal quality control, indicating that the analyte has migrated by capillary action
- Positive test show 2 lines at both the test and control
- Lateral flow Assay has 4 parts
Flow cytometry is used in the detection and measurement of physical and chemical characteristics of cell or particles in a heterogeneous fluid mixture
- Describe 5 such characteristics used in cell or particle sorting
- Cell size
- Cell granularity
- Surface receptors
- Intracellular proteins
- Total DNA
- New synthesized
- DNA gene expression
- Transient signals
- List any 5 common applications of flow cytometry in the clinical laboratory
- Protein expression
- Protein post-translational modifications
- RNA (miRNA and mRNA transcripts)
- Cell health status (detect apoptotic cells)
- Cell cycle status (GO/G1 phase, S phase, G2, polyploidy, analysis of proliferation and activation)
- Identification and characterization of distinct subsets of cells within a heterogeneous sample
