Immuno 11 Flashcards
T or F. when an antibody binds to its antigen, the immune complex precipitates
T. There are several agarose based antibody assays (known as immunodiffusion assays) that take advantage of this because when immune complexes precipitate in agarose, a visible line forms (a precipitin line). These techniques have limited utility and are typically used with simple antigen mixtures.
What is an Ouchterlony double immunodiffusion assay?
A gel plate (or slide) is cut to form a series of holes (“wells”) in the gel. A sample extract of interest (for example human cells harvested from tonsil tissue) is placed in one well, and sera or purified antibodies are placed in another well and the plate left for 48 hours to develop. During this time the antigens in the sample extract and the antibodies each diffuse out of their respective wells. Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form an immune complex. The immune complex precipitates in the gel to give a thin white line, which is a visual signature of antigen recognition.
These assays are not very sensitive, they cannot be used to determine antibody concentration, and they cannot be used with complex mixtures of antigens.
How does immunoelectrophoresis work?
agarose-based assay
This assay involves an electrophoretic step to separate either the antibodies or the antigens within the agarose prior to the immnodiffusion step.
This method can be used with much more complex antigen mixtures than simple immunodiffusion.
This assay uses an agar plate that has cutouts of a simple trough in the middle with a single hole on each side above and below the midline area of the trough. In this example, a patient sera is being tested to determine which isotypes of antibody it contains. A sample of the patient’s serum is pipetted into the hole on top while serum from a normal patient is added to the hole on the bottom.
(Middle Panel): The proteins in both of the serum samples are then separated by electrophoresis. When applying a current through the agarose gel, the positively charged proteins migrate toward the negative pole while the negatively charged proteins migrate toward the positive pole.
(Right-Hand Panel): The final step is to add rabbit anti-human antiserum into the central trough. Once it has been added, the proteins from the serum are allowed to diffuse into the gel. As the anti-immunoglobulin antibodies from the rabbit serum meet and bind to their cognate antigens, precipitin lines form.
How to perform a two-fold dilution series in triplicate.
the top three rows of cells (column 2-12) have been filled with 100 μl of vehicle (or dilution solution: this is a solution that will have no impact on the measurement but can be used for dilutions).
(Step 2; Upper Right-Hand panel): add 200 μl the test sample into column 1 (1st three rows).
(Step 3; Lower Left-Hand panel): perform serial dilutions by pipetting 100 μl from column 1 into column 2 and mix well, then pipette 100 μl from column 2 to column 3 and mix well, and so on until column 12 has been done. Finally, 100 μl is removed from column 12 to leave each well with a final volume of 100 μl.
(Lower Right-Hand Panel): The plate shown in the lower right demonstrates what has occurred in the plate. With each transfer of 100 μl from one column into the next column, a two-fold dilution has occurred, and moving from left to right, the wells in each column have one-half as much of the text sample as the previous well. As you can see, a two-fold dilution series can result in a large dilution factor on a single 96-well plate. The wells in column 12 have been diluted by a factor of 2048.
Dilution factors are typically reported as the inverse of the dilution as follows: column 2 contains sample that has been diluted 1⁄2, but the dilution factor is reported as 2; column 2 was then diluted by a factor of 2 to leave column three with 1⁄4 of the original quantity of the test sample for a dilution factor of 4. Column 4 resulted from dilution of column 3 (which was 1⁄4), resulting in a sample that has 1/8 of the original concentration of the test sample for a dilution factor of 8, and so on.
Immunological assays are typically performed in triplicate to ensure that the results of the assay are consistent.
Hemagglutination assays
Hemagglutination assays were a real innovation when they were developed because the enabled the use of 96 well plates, making it possible to perform relatively high-throughput and quantitative measurements of antigen-specific antibodies without needing to have expensive plate readers. Because of their vivid color, red blood cells can be visualized with the naked eye in these assays.
Hemmagglutination assays take advantage of the visibility of RBCs as well as with the multivalent binding capacity of antibody molecules. When antibodies bind with specificity to RBC surface molecules, it causes crosslinking of the RBCs When this occurs, it can be easily visualized because the crosslinked RBCs fall to the bottom of the well in what could be described as a diffuse mat. When there is no crosslinking of the RBCs, they fall to the bottom of the cone-shaped wells to form a small red dot.
These assays can be used to identify RBC-specific antibodies in a patient’s serum, or they can be used in a more general way by complexing an antigen of interest to the surface of the RBC to use the RBCs as an indicator (because the result can be scored visually). Today, the latter option is not used very commonly.
The most important tests you will need to know about are the direct Coomb’s and the indirect Coomb’s assays. I strongly recommend that you know how and why these two assays are performed.
Direct Coomb’s test (or direct antiglobulin test/DAT)
Performed to look for antibodies or complement proteins bound to the surface of RBCs collected directly from a patient. Antibodies or complement proteins bound to the surface of RBC is indicative of autoimmune hemolytic anemia
To perform a DAT, RBCs are collected from the patient and then washed to remove serum containing unbound antibody and complement proteins. The next step is to add the Coomb’s reagent to the patient’s RBCs and incubate for a short period of time. The Coomb’s reagent is a preparation of antihuman antibodies that have specificity for human immunoglobulins and complement proteins.
If the RBCs have human anti-RBC antibodies and/or complement proteins bound to their surface, an agglutination reaction will occur resulting in crosslinking of the RBCs via the added antihuman antibodies
This assay is highly sensitive and can be performed in a microtiter plate (so it can be used quantitatively).
Indirect Coomb’s Assay
The indirect Coomb’s assay (or the indirect antiglobulin test/IAT) is routinely performed to cross-match blood (prior to blood transfusions) and for prenatal testing of pregnant women.
This test is used to detect anti-RBC-specific antibodies in the serum of a patient.
To perform crossmatching of a patient’s blood prior to a transfusion:
A serum sample with antibodies collected from a patient who needs a transfusion is mixed with RBC collected from the donor blood sample.
Any existing blood group-specific antibodies from the recipient patient’s serum will bind to the RBCs from the donor.
The next step is to add the Coomb’s reagent (anti- human immunoglobulin/complement) to the mixture. If the recipient patient’s serum contains antibodies that are specific for the donor RBCs, a hemagglutination reaction will occur. If a hemagglutination reaction occurs, the donor blood should not be transfused into the recipient patient.
We will talk about the use of the IAT for prenatal screening in the second half of the course.
Like the DAT, this assay is highly sensitive, and it can be done quantitatively.`
Monospot test
Used for diagnosis of Epstein-Barr virus.
This test detects antibodies that are known as heterophile antibodies. Heterophile antibodies are produced by people infected with EBV. These antibodies will agglutinate horse RBCs (for some reason).
The test is done just like an indirect Coomb’s except that horse RBCs are used as the indicator cells.
Complement fixation assay
also uses RBCs as an indicator cell, but it is very different from a hemagglutination assay.
For this assay, an antibody sample is added to a tube with the antigen (can be a mixture or purified) and allowed to incubate long enough for an equilibrium of binding to occur.
The next step is to add a mixture of complement to the antigen:antibody mixture. If antibodies bound to the antigen, the classical complement cascade will occur, resulting in depletion of the complement components that were added to the reaction.
The final step is to add indicator cells into the reaction. These indicator cells already have been incubated with antibodies that have specificity for an RBC surface determinant, so if any complement components remain in the reaction mixture, the indicator red cells will activate the complement cascade, ultimately resulting in formation of membrane attack complex that will destroy the RBCs.
So, if the initial antibody sample had specificity for the antigen of interest, the complement components added to the mixture would be depleted before the indicator cells are added. Therefore, the indicator cells would not be destroyed and will be visible.
If there was no antigen:antibody binding in step 1, the complement proteins would not have been used up prior to addition of the indicator RBCs. In that case, the indicator cells would activate the classical pathway, resulting in formation of membrane attack complex that would destroy the indicator RBCs.
This assay is very sensitive and it can be performed in a 96-well plate (can be used quantitatively) and can be used with complex antigen mixtures.n
What is a “primary antibody“?
typically refers to a test sample that may or may not contain antibodies that are specific for some antigen of interest. A serum sample from a patient would be a nice example.
What is a “secondary antibody”?
refers to an antibody that will be used to detect the primary antibody. Secondary antibodies are typically conjugated to an indicator molecule such as an enzyme or a fluorochrome.
What is “convalescent serum”?
refers to serum from a patient that has recovered from an infection.
What types of antibodies are usually present in convalescent serum?
Convalescent serum typically has low levels of IgM and high levels of IgG that are specific for antigens from the pathogen that caused the infection that they have recuperated from.
What is “acute-phase serum”?
refers to serum collected from a patient that is actively infected with a pathogen.
Most of the antibodies in acute-phase serum are ___.
Because the acquired immune response is being generated, most of the antibodies that are being produced are IgM during this stage of infection. Therefore, acute-phase serum usually has higher levels of IgM and low levels of IgG. The serum should also contain elevated levels of mannose-binding protein, C-reactive protein and fibrinogen.
