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Flashcards in I11 Deck (24)
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1
Q

T or F. when an antibody binds to its antigen, the immune complex precipitates

A

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.

2
Q

What is an Ouchterlony double immunodiffusion assay?

A

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.

3
Q

How does immunoelectrophoresis work?

A

agarose-based assayThis 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.

4
Q

How to perform a two-fold dilution series in triplicate.

A

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.

5
Q

Hemagglutination assays

A

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.

6
Q

Direct Coomb’s test (or direct antiglobulin test/DAT)

A

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 anemiaTo 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).

7
Q

Indirect Coomb’s Assay

A

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.`

8
Q

Monospot test

A

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.

9
Q

Complement fixation assay

A

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

10
Q

What is a “primary antibody“?

A

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.

11
Q

What is a “secondary antibody”?

A

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.

12
Q

What is “convalescent serum”?

A

refers to serum from a patient that has recovered from an infection.

13
Q

What types of antibodies are usually present in convalescent serum?

A

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.

14
Q

What is “acute-phase serum”?

A

refers to serum collected from a patient that is actively infected with a pathogen.

15
Q

Most of the antibodies in acute-phase serum are ___.

A

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.

16
Q

ELISA

A

is typically based on usage of secondary antibodies that have been conjugated to either an enzyme or to an amplification system such as avidin:biotin enzyme conjugates.(Top-Left Panel): The 1st step is to bind the antigen of interest to the wells of a microtiter plate (in this example, two different antigens have been bound to different wells of the plate, and then antibodies (that have been covalently conjugated to an enzyme) that are specific for one of the two antigens is added to both wells and allowed to reach a binding equilibrium. You can see that the antibodies bind to antigen A but not antigen B.(Bottom-Left Panel): The second step is to wash all unbound materials from the well of the microtiter plate. This will leave only the antigen that is bound to the well, and the antibodies that are bound to the antigen.(Right-Hand Panel): The last step is to add a chromogenic substrate that will undergo a color change in the presence of the enzyme that was conjugated to the antibody. You can see in the figure that the color change occurred in the well in which the enzyme-conjugated antibodies bound to antigen A. This color change can be measured with a spectrophotometer or using a microplate absorbance reader.(All Panels): Because this assay can be performed in microtiter plates, it can be quantitative. It is highly sensitive, it is high throughput, and it can be performed using either a mixture of antigen or a mixture of antibodies, but not both.

17
Q

RAST

A

During the second block of CMOD we will be talking about hypersensitivity responses, or allergies. There are four classifications of allergies that include the type 1, or immediate-type hypersensitivities. These allergic responses are mediated by mast cells, and they are initiated when allergen-specific IgE that is bound to IgE receptors on the surface of mast cells become cross-linked by allergen, causing the mast cells to degranulate. You will learn much more about this later. For now, you need to know that when a patient presents with symptoms that are consistent with immediate-type hypersensitivity, it is essential that the the offending allergen be identified.Until recently (around 2010), an assay known as RAST was used to identify the offending allergen…or more precisely, to determine which allergen(s) the patient has made an IgE response to. As you know, IgE is found at very low levels in the serum, so this assay requires very high sensitivity. RAST is essentially an ELISA-type assay. The key modification is that the detection antibody (anti-human IgE) has been radiolabeled. Methods to measure the radiolabel are extremely sensitive, giving this assay a huge dynamic range and making it possible to detect very small quantities of allergen-specific antibodies in the blood of a patient.

18
Q

ImmunoCAP

A

Since 2010, a new assay known as ImmunoCAP has been used to measure allergen-specific IgE in the serum of patients suffering from type 1 hypersensitivity. This assay is also essentially an ELISA that utilizes an anti-human IgE secondary antibody that has been conjugated to a fluorescent enzyme, making the assay extremely sensitive. This assay is a big advance, because the use of radionuclides requires a lot of oversight headaches.

19
Q

51Cr-release assays.

A

This is historically the most commonly used assay to measure antigen-specific CTLs. As you should know now, CTLs are the key effector cells for killing of virus-infected cells and they play an important role in control of infections with intracellular bacteria or parasites. As we will discuss in the second half of the course, CTLs are also one of the effector cells that kill tumor cells (the other is the NK cell).A 51Cr-release assay measures the ability of T cells or NK cells to kill target cells. For instance, if you want to know if a patient has produced cytomegalovirus-specific CTLs, you can infect cells with CMV to use as target cells. These CMV-infected cells would then need to be labeled with 51Cr by adding it to their cell culture medium, allowing the cells to take up 51Cr. The labeled target cells would then need to be washed to remove exogenous 51Cr. Once the target cells have been labeled and washed, a known number of them are then added to the wells of a microtiter plate in culture medium.To determine if the test effector cells contain CTLs specific for CMV antigens, effector cells are added to the labeled target cells at several known effector cell:target cell ratios and allowed to incubate for a while. To measure cytotoxicity, the microtiter plate is subjected to centrifugation to pellet the target cells (and effector cells). Supernatants are collected from control wells (non-immune effector cells) to determine how much 51Cr was released naturally or spontaneously, and from test wells to determine how much 51Cr was released due to cytotoxicity of effector cells. Finally, the cell pellets are lysed from control wells to determine how much 51Cr the cells were labeled with. 51Cr is measured using a technique known as scintillation (you do not need to know how this works).A formula is then used to calculate the cytoxicity of the effector cells

20
Q

Western blot.

A

The first step is to separate a complex mixture of proteins (usually) in what is known as a polyacrylamide gel. This is done by performing electrophoresis, and typically the separation is based on the size of each protein in the mixture (proteins can also be separated based on charge). Once the proteins have been electrophoretically separated in the gel, they are transferred to a solid nitrocellulose membrane via a second round of electrophoresis. Now, the membrane is incubated with the antibody of interest for long enough to reach a binding equilibrium. The membrane is then washed to remove any unbound antibodies, and then a secondary antibody that has been conjugated to an enzyme or a radionuclide is added and incubated. The bound secondary antibody is then either visualized via addition of a chromogenic substrate or via radiography, respectively (autoradiography is shown in this example).purified Ag and/or Ab not required, very sensitive, non-quantitivative, low throughput

21
Q

Immunohistochemistry

A

a method that can be used to visualize the location of specific antigens within a tissue or even a single cell. Antibodies that have been modified by covalently binding them to either fluorochromes or colloidal gold particles (among other reporter substances) can be used for these studies.

22
Q

Flow cytometric analysis or fluorescence activated cell sorting is a very important technique that is used for more-and-more clinical studies.

A

This technique enables the user to identify important surface markers on the surface of host cells by using monoclonal antibodies (conjugated to fluorochromes) that are specific for those proteins. The instrument has a fluidics systems that funnels a mixture of cells that have been labeled with antibodies through a series of lasers in single file. As the cells pass through the lasers, a series of detectors measure the light that is emitted from each cell.The forward scatter detector is directly in line with the laser as the cells pass through, allowing that detector to determine the relative size of each cell, based on the amount of light scatter that occurs as the cell passes through the laser.The side scatter detector measures the regularity of the surface of each cell (based again on light scatter as the cell passes through the laser).The more granular or irregular the surface of the cell is, the more light will be scattered and the higher the side scatter will be.

23
Q

This is a dotplot (one type of graph for looking at flow cytometric data) that shows the forward scatter (cell size) on the X-axis and the side- scatter (granularity/irregularity) along the Y-axis. Nothing on this forward scatter/side scatter plot tells you anything about the antibodies that were bound to the cells as they passed through the laser.In this example, most of the cells in the preparation were lymphocytes. Lymphocytes are small and they have very few obvious irregularities on their surface or in their cytoplasm. So, they have very low forward-scatter and relatively low side-scatter. The lymphocytes have been circled in that “gate”.

A

This is a dotplot (one type of graph for looking at flow cytometric data) that shows the forward scatter (cell size) on the X-axis and the side- scatter (granularity/irregularity) along the Y-axis. Nothing on this forward scatter/side scatter plot tells you anything about the antibodies that were bound to the cells as they passed through the laser.In this example, most of the cells in the preparation were lymphocytes. Lymphocytes are small and they have very few obvious irregularities on their surface or in their cytoplasm. So, they have very low forward-scatter and relatively low side-scatter. The lymphocytes have been circled in that “gate”.

24
Q

What is nephelometry?

A

a technique to measure IgG, IgA, and IgM concentrations in serum using light-scattering