What are the 2 Simple Agarose Gel-based Immunoassays?
Which Simple Agarose Gel-based Immunoassays can be used with simple antigen mixtures only?
Describe how Immuno-double diffusion assays are used
agar 1-2% at pH 7-8.5) is poured onto slides and allowed to set.
Wells are punched into the agar, and antigen and antibody are added to the different wells.
Over time, the antigen and antibody diffuse through the agar, and when the antigen and antibody diffuse into each other, the antibody can bind to the antigen.
If the antibody has specificity for the antigen, it will bind to and cross-link the antigen causing it to precipitate (leaving a line of precipitation) (Panel A).
If there are two antigens that can both be recognized by the antibody (polyclonal), two precipitin lines will form independently (Panel B).
How is the immuno double diffusion assay used?
used qualitatively to determine if an antibody can bind to a specific antigen,
but it cannot be used as a quantitative assay (can not be used to determine concentration of antigen or antibody).
What immunoassay can be used to analyze complex mixture of antigens?
Describe the process of immunoelectrophoresis
a 1-2% agarose gel is poured, allowed to set with a central trough and a single well on each side of trough.
An antigen mixture can be added to one or both of wells
antigens are separated by applying an electric charge across the gel;
the (+)-charged proteins will migrate toward (-) electrode while the (-)proteins will migrate toward (+)electrode.
Once proteins separated, Ab is added to trough and both the Ab and antigen are allowed to diffuse into agar.
When Ab and antigen have diffused into same area, Ab will bind to its specific antigen, crosslink, and a precipitin line will form.
What is a common application of immunoelectrophoresis?
One common application for this technique is to evaluate patient sera for immunoglobulin content
What is the result of immunoelectrophoresis?
a qualitative but not quantitative assay
it cannot be used to determine antibody/antigen concentration
Describe the sensitivity of simple agarose gel-based immunoassays
neither of these assays is terribly sensitive; they both operate in the range of 20 μg/ml to 2 mg/ml.
What assay can be used to detect and measure Abs that are specific for a particular antigen? Describe this assays sensitivity wrt Simple Agarose Gel-based Immunoassays
procedure is more sensitive than the gel-based assays
Describe how you perform the Hemagglutination assay
Ab sample(s) is serially diluted (usually a 2-fold dilution series) in physiological saline and is pipetted into wells of an agglutination plate
RBCs are prepared by covalently or non-covalently binding antigen of interest to surface of RBC (and by addition of a protein that will prevent non-specific agglutination).
These modified RBCs are added to each well of plate.
each individual well that contains a sufficient number of antigen-specific Abs to agglutinate (crosslink) RBCs, they will sink as a “mat” to bottom of well.
If insufficient antigen-specific Ab is present, the RBCs will fall individually to the bottom of well to form a red pellet (easily visible).
There are a number of disease syndromes that involve the production of auto-antibodies that bind to a patient’s own RBCs. Abs bind to what? complement proteins bind to what? what is the result?
“self”-reactive antibodies can bind the patient’s RBCs
complement proteins may subsequently bind to the bound antibodies.
This can result is destruction of RBCs (hemolytic anemia).
What are the Specific Clinical uses of Hemagglutination assays? Describe the sensitivity of these assays
Direct Coomb’s test:
Indirect Coomb’s test
Monospot test (Paul Brunnel test)
• these assays are fairly sensitive; can detect antibodies at less than 1μg/ml
When is a Complement Fixation Assay used and why?
used clinically to identify the presence of antibodies specific for a variety of common human pathogens
a series of serum proteins that become activated by antigen:antibody complexes or microbial surfaces.
Activation of complement results in what?
the formation of a membrane attack complex that inserts into the cell membrane resulting in the lysis of the cell
Describe how you perform the complement fixation assay
1) the test antiserum is titered (2-fold dilution series), and then a fixed amount of antigen is added to each well;
2) complement is then added to each well, and if antigen:antibody complexes are present (meaning that the patient sera contained antibodies to the antigen), they will fix the complement and consume it;
3) the final step is to add sheep red blood cells (sRBCs; indicator cells) together with a sub-agglutinating quantity of sRBC-specific Abs. If there is any remaining complement, the indicator cells will be lysed
WRT complement fixation assay, there will be no lysis of sRBCs if the patient has what?
antibodies specific for the test antigen in their serum
Describe the complement fixation assay wrt sensitivity
• these assays are fairly sensitive; can detect antibodies at less than 1μg/ml
What are monoclonal antibodies? What produces them and what is their antigen specificity and isotype?
monoclonal antibodies are Abs that were produced by a population of B cells that all
descended from a single cell;
monoclonal antibodies are produced by a cloned B cell line
They all have exact same antigen specificity (for a single epitope) and they are all of the same isotype
Can monoclonal antibodies be collected from the serum of an individual?
no, under no circumstances.
By definition, antibodies collected from the serum of any individual are polyclonal antibodies (meaning that they are derived from multiple B cell “lines” and have many different specificities and have different isotypes)
Why should we be interested in monoclonal antibodies?
they are extremely useful as diagnostic reagents, as research tools,
potentially therapeutic agents.
specificity of an Ab for its antigen make it a useful reagent for detecting or purifying the antigen.
very useful in a variety of clinical and laboratory tests and as reagents for biological research.
For such applications, large quantities of identical antibodies are required
Define antiserum (pleural antisera) and tell what does it contain?
the fluid component of clotted blood from an immune individual that contains antibodies against an antigen;
an antiserum contains a heterologous collection of antibodies that bind to the antigen
T/F B cells are not self-renewing in vitro (in culture)
Production of Monoclonal Antibodies is done in 7 steps. Give a brief outline of these
immunize mouse w/ antigen of interest and monitor to ensure proper Ab for antigen
remove spleen/lymph node, prepare a single cell suspension, and count the cells
mix the splenocytes/lymph node cells with a fusion partner cell line; myeloma:splenocyte ratio should be 2:1
add PEG to mix for cell fusion to occur and hybridomas result
after selection, culture screened for Ab production and hybridomas for specific antigen are kept
hybridomas for desired Ab are cloned via limiting dilution
hybridoma cultured on large scale and Abs produced recovered from culture supernatants
Why can myeloma cells be grown indefinitely in vitro?
myeloma cells are immortal because of their malignant properties
How can the production of monoclonal Abs occur naturally as a myeloma (cancer of plasma cell)?
myeloma cell would have to be cloned before the antibodies produced by this cell “line” would be considered monoclonal.
This is an unusual event, and it typically does not result in the production of monoclonal antibodies that would be used as a specific tool.
•• B cell:B cell fusions will only survive in culture for how long
around 5 days
Why is it necessary to introduce myeloma:myeloma fusions to a HAT medium?
myeloma:myeloma fusions are immortal cells and must be selected against;
myeloma cells are sensitive to hypoxanthine-aminopterin-thymidine (HAT) (they do not have a functional hypoxanthine:guanine phosphoribosyl transferase (HGPRT) gene), they cannot live in the HAT medium and will die within a 5 day period
What type of myeloma fusions will survive in HAT medium? How?
B cell:myeloma fusions will survive in HAT medium
B cells have a functional HGPRT gene and therefore are not sensitive to HAT medium
What is a hybridoma? Why is it important?
hybridomas, are a B cell that been fused to an immortal myeloma cell, so it can live and replicate in vitro indefinitely;
they constitutively (all the time) produce the antibody that the particular B cell produced
hybridomas are screened for antibody production using what?
ELISA or Western blotting
What is the process of limiting dilution? what is the result of it?
process insures that the hybridoma culture of interest was started from a single cell;
therefore, every cell in the culture is identical and each produce antibodies of identical isotype and specificity (monoclonal antibodies)
What is a popular method for purifying Abs? How does it accomplish this?
protein A (a component of staphylococcal cell walls) is a naturally produced molecule that has affinity for the Fc region of IgG molecule. Which part does it bind to?
binds to the Fc region of IgG1, IgG2, and IgG4.
Describe the process of affinity chromatography wrt protein A
protein A can be covalently linked to agarose beads (sepharose);
these beads can be used to load a column.
Once the column is formed, a sample containing antibodies can be passed through the column.
The antibody molecules will bind to the protein A-sepharose while all other proteins pass through the column.
After the column is washed thoroughly to remove all unbound proteins, the antibodies can be eluted from the column using a high salt elution buffer
Why is it important to easily modify Ab molecules for use in immunoassays?
modifications make it possible to easily detect the antibody;
these modifications do not affect the antibody’s ability to bind to its specific ligand
what are the 4 ways in which Ab are modified via immuno assays?
•• labeled with radionuclides
•• labeled with enzymes so can be easily detected by addition of a metabolizable substrate with a color reaction
•• can be covalently coupled to fluorochromes => fluorochromes are molecules that can be excited by light of a specific wavelength, device can measure emitted light can be used to detect the presence of fluorochrome
•• can be labeled with proteins that can be used as an amplification system
What are antibody conjugates?
antibodies that have been labeled with either a radionuclide, an enzyme, a fluorochrome, or with a component of an amplification system
What are secondary antibodies?
What must they have in order to be this?
Abs used to indirectly detect the presence of an antigen by binding to an antigen:antibody (primary antibody)
a secondary antibody should have specificity for the primary antibody
T/F secondary antibodies are polyclonal preparations of antibodies
false, •• secondary antibodies can also be monoclonal antibody preparations
What is a non-quantitative method of determining whether an Ab sample can bind to a protein antigen?
Western blot analysis
Describe the process of western blot analysis
a mixture of proteins including the antigen of interest are separated by polyacrylamide electrophoresis
electrophoretically transferred to a solid support (nitrocellulose).
The membrane is then probed with the Ab sample (Ab binds to its specific antigen on the membrane).
Bound Ab is then detected using a secondary Ab bound to an enzyme.
The membrane is then immersed in an appropriate substrate to detect secondary antibodies that are bound to the primary antibody:antigen complex
What are the benefits and limitations to western blot analysis?
benefits: purified antigen or antibody are not needed; highly sensitive
What is ELISA?
Enzyme-linked immunosorbant assay (ELISA)
• ELISA is a quantitative method of measuring antigen-specific antibody
Describe the process of a simple ELISA
wells are coated with purified antigen and unbound antigen is washed away.
To eliminate non-specific Ab binding, all wells are pre-blocked by addition of a non-competing protein-containing solution (blocking solution).
Serially diluted Ab (diluted in blocking solutions) are then pipetted into appropriate wells and are allowed to reach a binding equil. (usually 1-2 hrs).
unbound Ab are washed away, and then a saturating amount of secondary Ab (usually enzyme conjugated) is added to each well and allowed to reach a binding equilibrium (1-2 hrs).
Unbound secondary Ab is washed away, and then a colorimetric metabolizable substrate is added to each well and the plate is incubated until the reaction (indicated by color-change) is complete.
The results are determined by quantitating the color change spectrophotometrically (using a microplate reader).
How are the results of a simple ELISA made?
Describe the process of a capture ELISA
can be used to quantitate a particular antigen in a sample.
The basic procedure is the same as described for the simple ELISA, however, the microtiter plate is initially coated with a monoclonal antibody specific for the antigen.
After washing, the antigen solution is added to the wells and allowed to bind to the capture Ab.
After washing, a secondary antibody specific for the same antigen (different epitope) is added and the assay is developed in the same was as described above.
What are the benefits and limitations of ELISA?
benefits: very sensitive, quantitative, high throughput (many samples can be tested at once), relatively inexpensive
limitations: must have either purified antigen or monoclonal Ab (or both) depending on what type of assay is being performed
When an animal is immunized with an appropriate antigen and produces an antibody response, the most common site to find antibodies is where?
in the blood serum
a serum sample from an immunized animal is commonly referred to as a polyclonal antiserum. Why?
Since most immunogens have multiple epitopes, and antibodies are made by many different B cells, a complex mixture of antibodies can be elicited by one immunogen (also, antibodies with identical specificity, but different isotype are generated).
a flow cytometer is a very powerful tool for what? What 2 criteria does it function by?
analysis of cell preparations;
this instrument can be used
to rapidly analyze individual cells based on: 1) size and granularity, and 2) surface marker expression
What does a basic flow cytometer look like?
A basic unit has a fluidics system, a single laser and 4 detectors (forward scatter detector, side-scatter detector, a green photomultiplier tube and a red photomultiplier tube
Describe how the fluidics system works within a flow cytometer
the fluidics system carries cells at a very rapid rate and in a “single-file” fashion through the laser stream; as the cells move through the laser stream, the detectors collect two types of data:
as the cells move through the laser stream, the detectors collect two types of data. Describe them
1) measurements of light scattering gives information on the physical characteristics of the
particle (size and granularity) => depending on the size, refractive index, and granularity of the cell. Two types of scattered light are measured by the flow cytometer.
2) measurements of fluorescence emissions gives information regarding the association of each cell with fluorochrome-labeled antibodies; if the cell moving through the laser stream is conjugated to a fluorochrome, it will emit light following excitation by the illuminating beam; these emissions will be detected by either the green or red photomultiplier tube (depending on the color of the emission). FITC emits green light upon excitation, while PE emits red light
Describe the 2 types of scattered light measured within a flow cytometer
Forward Scatter- One of the photomultiplier tubes sits directly in front of the illuminating beam with a metal bar in front of it to prevent the beam from striking it directly. Light that has hit a particle will be bent and go around the bar to hit the photomultiplier tube. The forward scatter is influenced by the size and refractive index of the particle.
Side Scatter- A second photomultiplier tube sits at right angles to the illuminating light with a filter that is the same color as the illuminating light in front of it. This tube will measure light that has been scattered to the side of a rough or irregular particle. The rougher, more irregular, or more granular a particle is- the more it will scatter the illuminating beam to the side
WRT forward and side scatter, how does data analysis help further Ab evaluation?
forward scatter and side scatter data can be used to select the population of cells that can then be further evaluated for marker expression;
once the cell population has been selected, those cells can be “gated”, and only the gated cells will be further analyzed for marker expression
What happens if a single marker or two makers are stained wrt data analyis using the flow cytometer?
if only a single marker was stained, the data is usually analyzed in a histogram
If two markers have been stained, the analysis of each marker staining can be done separately using histogram plots, or can be performed simultaneously using a dot- plot or a contour plot
What is cell sorting wrt flow cytometry?
a basic flow cytometer simply measures the size, shape, and fluorescent emissions as cells are passed through one (or more) lasers.
How is a cell sorter used in clinical applications? What does it measure?
a cell sorter is a special flow cytometer that can actually collect cells that have the desired surface phenotype after they have passed through the laser.
These cells can be collected in sterile condition for use in clinical applications
the collection of CD34+ cells from bone marrow for performing a bone marrow transplant would be an example of what?
What is the purpose of Immunohistochemistry / Immunofluorescence?
• antibodies can be used to visualize the anatomic distribution of an antigen within a tissue or cell.
involves the use of antibodies that are conjugated to visible particles (e.g. colloidal gold) or enzymes that can metabolize a chromogenic substrate;
electron microscopy or conventional light microscopy, respectively, can be used to visualize tissue sections stained in this way
involves the use of antibodies that are conjugated to fluorochromes;
a fluorescent microscope can be used to visualize tissue sections stained in this way
What techniques are clinically useful in diagnosis of autoimmune disorders of the connective tissue system?
Immunohistochemistry / Immunofluorescence