antigen-antibody reactions: immunological methods -Vishy Flashcards
What is a titer?
The maximum dilution or the minimum concentration at which an antibody can still bind to an antigen to induce a reaction
newer techniques allow for lower concentrations to form a detectable reaction
What is an agglutination reaction? What is direct agglutination? Indirect agglutination?
an agglutination reaction is when particulate antigens (antigens on inert particles) reacts with specific antibodies and there is aggregation and lattice formation
Direct agglutination is when particles with antibodies belonging to IgM bind to antigens to form a lattice. This is seen in blood group testing. When blood is mixed with anti-A antibodies, there is agglutination when the blood is type A and no agglutination when the blood is another type.
Indirect agglutination is where a non-IgM antibody (such as IgG) is used. Without the pentamer form that IgM has, other antibodies do not aggregate well (because they only have 2 binding sites for the antigen) and require the use of a secondary antibody in order to show form a lattice
What are precipitation reactions? What is the zone of equivalence? Zone of excess antibodies? Zone of excess antibodies?
When the antigen is soluble (liquid), addition of antibodies will result in the binding and formation of a precipitate. These are performed in molten agar
Zone of equivalence= when the concentration of antibody and antigen are optimum and there is precipitation
Zone of excess antibodies= when the antibody is in excess and there is no clear precipitation
Zone of antigen excess= if the antigen is in excess and there is no clear precipitation
What is single diffusion? Is this a qualitative or quantitative process?
antibodies are mixed in a molten agar solution and poured onto a plate. A well is cut into the plate and a soluble antigen is placed in the well. The antigen will diffuse and bind to the antibodies to form a precipitation curve. The further away the curve is, the higher the antigen concentration that was introduced
this process is quantitative
What is a double diffusion (ouchterlony technique)? Is this technique qualitative or quantitative?
similar to simple diffusion, except both the antigen and antibody are in different wells in the agar. Antigen-antibody binding results in precipitation curve formation. If there is no similarity between the antigen-antibody, there will be no binding.
This is a qualitative technique
What is Immunoelectrophosesis?
Protein antigens (such as serum proteins) are loaded into the gel. The presence of electric current is introduced and proteins move according to size and charge (electrophoresis). The gel is then removed and a trough is cut into wells. Antibodies are added to the groups and will diffuse and precipitate with the appropriate serum proteins. Precipitation bands will form where an antibody meets its antigen.
this is used to quantify levels of serum proteins
What is a radio-immunoassay? What is it used to test?
Samples are radio-labeled. Antigen-antibodies are separated and assayed for radioactivity. The test is based off competition between labeled and unlabeled antigens for their antibodies.
It is used to determine levels of hormone in the blood.
To test for a particular hormone (estrogen), you add a known amount of normal, unlabeled antigen (estrogen) to the buffer solution so that it competes for the antibody binding site. Then add a known amount of radioactive antigen to the mixture. Then add a fixed amount of antibody and the radioactive antigen is displaced by the unlabeled antigen
in the serum sample without estrogen, all radio labeled will bind to antibodies producing high radioactivity
if the serum sample has excess estrogen, the serum estrogen that is unlabeled will bind to the antibodies and there will be minimal radioactivity.
can be set up to detect either an antigen or an antibody
What is ELISA?
Enzyme linked immune-sorbant assay
ELISA plates are coated with a potential antigen
Addition of a specific unlabeled antibody (primary antibody) which will bind to the test molecule if it is present.
Washing to remove unbound molecules.
Addition of secondary antibody which will bind to the primary antibody.
The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. (antiantibody conjugated with an enzyme)
Wash to remove unbound antibody.
Addition of a colorless substrate which will react with the secondary antibody to give a color reaction which indicates a positive result.
What are the advantages of ELISA?
Sensitive: nanogram levels or lower
Reproducible
Minimal reagents
Qualitative & Quantitative
Qualitative: Eg HIV testing (detect HIV antibody in a sample where there is known HIV antigen)
quantitative assays: Eg Ther. Drug Monitoring
Greater scope : Wells can be coated with Antigens OR Antibodies
Suitable for automation–>high speed
NO radiation hazards
How does western blotting work? What is it often used to test?
Antigens are run in the gel electrophoresis to separate by weight. then, they are transferred from the gel to nitrocellulose membranes (western blot).
then the membrane is incubated with the patient’s serum. Antibodies in the serum will bind to the antigen. Anti-antibodies are then added which are conjugated with horse radish peroxidase. When a substrate is added, the visualization of color indicates the presence of HIV antibodies, confirming the pt is infected with HIV.
It can be used as a confirmatory test. Ex: to detect HIV antibodies
How does immune-fluorescence work? Is this test qualitative or quantitative?
direct: the antibody against the antigen is covalently linked to fluorescence.
Indirect: the secondary antibody is linked to fluorescence.
both direct and indirect are viewed under fluorescent microscope. the fluorescence is either fluorescent isothiocyanate (FTC) which appears green or Phycoerythrin (PE) which appears red
qualitative not quantitative
What test is used to determine whether an HIV patient has full-blown AIDS? What is the purpose of this test? How does the test work?
FACS- Fluorescent activated cell sorter (aka flow cytometry?)
This is used to quantify the number of CD4+ T cells in HIV patients. In a patient with AIDS, there is a sharp decline in CD4+ T cells, due to viral replication within these cells.
peripheral blood lymphocytes are stained with antibodies against CD4 antigens that are conjugated with fluorescent dye. Cells are passed through a laser beam and scatter based on size and granularity of cells. The fluorescent cells are then quantified.
What is phagocytic efficiency and what is it used for?
To determine if there is a defect in phagocytosis of neutrophils and macrophages.
the monocytes are observed under a microscope for engulfed particles that were presented to them. If there aren’t any engulfed particles, there is a defect in phagocytosis
What can be used to measure proliferation of T cells?
lymphocyte proliferation assay with radioactive 3H thymidine labeling. During cell division, the radioactive thymidine will be incorporated into the DNA.
if there is an increase in radioactivity after some period of time (compared to the control group), the cells are multiplying.
What test can be used to determine the cytotoxic activity of NK cells and cytotoxic T cells?
cytotoxic activity of Nk cells and cytotoxic T cells can be measured using radioactive chromium labeling.
in this test, target cells are labeled with radioactive chromosome and incubated with effector cells. The lysis of these target cells will cause the release of radioactive chromium
