third seminar- Methods based on antigen-antibody interaction (ELISA, RIA, IRMA, ELISPOT, immunohistochemistry, Western blot, ELFA, lateral flow tests) Flashcards
Epitope variants
conformational determinants, where denaturation destroys the conformation the epitope had subsequently no more binding occurs.
linear epitope- binding is either allowed in its natural conformation and/or when its denatured
neoantigenic determinant where a new antigen is formed with proteloysis
Different methods use different epitopes recognizing antibodies for the same antigen to detect.
What is a immunogenic carrier?? (not sure about name)
Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. (In general, only large molecules, infectious agents, or insoluble foreign matter can elicit an immune response in the body.) Once the body has generated antibodies to a hapten-carrier adduct, the small-molecule hapten may also be able to bind to the antibody, but it will usually not initiate an immune response; usually only the hapten-carrier adduct can do this.
Generation of polyclonal antibodies with haptan+carrier produces??
Antibodies against the haptan, against the carrier molecules and against the haptan+carrier molecules.
Generation of monoclonal Abs by hybridoma techniques
too lazy to copy, teacher emphasized this as a favorite question in the exam
1) Immunization
2) B cell isolation from spleen
(3) Culture of myeloma cells
(4) Fusion of myeloma cells and B cells
(5) Cloning (separation of the different cell lines) (8) Isolation of antibodies
Immunoglobulins can be conjugated covalently to marker molecules
Marker and its uses?
Not sure how relevent this is but thought it might be nice as a review.
fluorochromeImmunocytochemistry Enzyme Elisa, elispot, immunoblot biotin indirect immunocytochemistry colloid gold electron microscopic immunocytochemistry radioactive isotope RIA, IRMA, RRA
avidin, streptavidin, biotin???
Biotin (B7) is bound to antibody and avidin/ streptavidin bind to biotin,serves as a basis of several test systems.
Statisctical features of diagnostic methods
Sensitivity: is reflects how sensitive the antibody is, what % of the patients samples are recognized as „positive” ones
Specificity: the % of healthy samples tested as „negative”
which methods.. blah blah..
METHODS
Biological body fluid sample – ELISA, RIA, IRMA, LATERAL FLOW
Isolated cells - ELISPOT
Biological solid tissue section– IMMUNOHYSTOCHEMISTRY
Isolated proteins - WESTERN BLOT
Elisa- indirect
TO DETECT ANTIBODY
Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that the secondary antibody may bring about.
- Micro-well plates are incubated with antigens, washed up and blocked with BSA.
- Samples with antibodies are added and washed.
- Enzyme linked secondary antibody are added and washed.
- A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.
***Direct elisa just uses a primary antibody only allowing a fast detection
sandwich elisa
To detect antigen
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen present binds to capture antibody;
(3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody;
(5) substrate is added, and is converted by enzyme to detectable form.
THE DETECTING ANTIBODY IS OF PRIMARY ORIGIN AND NOT SECONDARY LIKE INDIRECT!!!
Competitive ELISA
More sensitive in low concentration range.
The antigen present in the sample, binds to the antibodies, and thus, it inhibits their binding to the purified antigen coated onto a solid surface of the ELISA plate.
1) Primary antibody (unlabeled) is incubated with sample antigen.
2) Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen
A bit more about this step… Serum is mixed with another tube holding a certain amount of known antibodies against HIV antigens for this example.
Then we add this sample to the well (containing bound complexes and unbound complexes incase theres low amount of HIV or none at all)
3) Unbound antibody is removed by washing the plate. (Since they are bound to the antigens from the sample before)
The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence “competition.
This means that if the person is infected he will not have binding antibodies to the well since they will all be occupied before adding them to the well!!
4) The secondary antibody that is specific to the primary antibody and conjugated with an enzyme is added.
A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.
Example for a routine ELISA test result (rheumatoid factor)
Rheumatoid factor (anti IgG antibody) detected by an IGM
generaly elisa can be used in autoimmune diagnostics, endocrinology, microbiology and tumor diagnostics.
Elispot
The Enzyme-Linked ImmunoSpot (ELISPOT) assay is a widely used method for monitoring cellular immune responses.
The ELISPOT technique has proven to be useful means available for monitoring cell-mediated immunity, due to its sensitive and accurate detection of rare antigen-specific T cells (or B cells) and its ability to visualize single positive cells within a population of peripheral blood mononuclear cells (PBMCs).
It is an indirect immunoassay in which we can detect molecules (most often cytokines) secreted by
individual cells.
- The wells of the special 96 well ELISPOT plates have either nitrocellulose or PVDF membrane bottom that is coated under sterile conditions with a capture antibody.
- Next we block the free protein binding surfaces by incubating the surfaces with a buffer containing an indifferent protein (such as bovine serum albumin).
- This is followed by pipetting live cells (e.g. 1-300,000 lymphocytes) into the wells, and incubating them.
4.Then we wash the cells out of the wells, and the
secreted molecules bound to the capture antibody are
visualized by using a labeled secondary antibody and a
substrate that will form an insoluble precipitate.
5.After drying the plate, we analyze the scanned spots
at the bottom of the wells with image analyzer
software.
Example for use of ELISA and ELISPOT in the routine clinical practice
to diagnose for latent tuberculosis infection
Mycobacterium tuberculosis infection induces a cellular immune response
Activated T-lymphocytes release interferon-gamma
During infection memory T-cells arise, tests measure their rapid IFN-gamma release upon antigen contact
ELISA based QuantiFERON test
ELISPOT-based T-Spot test
QuantiFERON, also known as QFT, is the registered trademark of the test for tuberculosis infection or latent tuberculosis. QFT is an interferon-γ release assay (IGRA) used in tuberculosis diagnosis.
Method:
1 ml of blood
Into each of three tubes: incubated with three TB antigen peptides
Released IFN-g amount in harvested plasma is measured by ELISA method.
Result within 24 hours
Requires no specific laboratory background
ELISPOT-based
T-Spot test
Separated PBMC are kept alive under sterile conditions on a coated ELISPOT plate
Incubated with two TB antigens
Released IFN-g amount is measured after washing off cells
Result within 24 hours
Requires specific cell culture conditions
Immunoradiometric assay (IRMA) and Radioimmunoassay (RIA)
RIA is a Competitive method: The radiolabelled antigen competes with the unlabelled one for Ag binding.
More antigen → weaker sign
It is a very sensitive, highly specific and relatively inexpensive method by which we can determine the
concentration of e.g. hormones.
In RIA we most often use I125 labeling of the tyrosines of proteins. The radiolabeled antigen is mixed
with known amounts of an antibody. Next we add biological samples with unknown antigen content. In
this case, the cold (unlabelled) antigen competes for binding to the antibody with the radioactive one.
IRMA
„sandwich” method: it uses radiolabelled antibody
In this method we use monoclonal antibodies bound to the inner surface of polystyrene tubes. The
samples of the patients are incubated with radioactive (125I-labelled) antibodies. Antigens in the
sample bind simultaneously to the immobilized unlabelled antibodies (bound to the wall of the tubes)
and the soluble, radioactive antibodies. This way we ultimately form a solid phase-bound complex.
The unbound radioactive antibodies are removed by washing. Radioactivity of the tubes is proportional
with the antigen contents of the patient samples. Quantitative analysis involves the use of a standard
curve set by measuring known amounts of antigen in the system.
Immunohistochemistry-direct indirect.
Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
We can investigate sections, smears and cultured, adherent cells as well as cell suspensions after centrifuging onto the surface of a glass slide (citospin preparations).
The antigen-antibody interaction may be visualized by fluorescent dyes, metal colloid, radioactive
labeling or an enzyme.
2.2.7.1. Direct method
In a single step we incubate the sample with a labeled antibody (such as FITC-labeled antibody). It is
a rapid procedure; however, it is not too sensitive.
2.2.7.2. Indirect method
The first „layer” in this method is represented by the primary antibody; this is the immunoglobulin that
reacts with the antigen in the tissue. A labeled secondary antibody is also used (representing the
„second layer”) in the method. This secondary antibody reacts with the immunoglobulins of the
species from which the primary antibody is derived. This method is more sensitive than the direct
approach (the signal is amplified, since numerous secondary antibody molecules interact with the
different epitopes of a single primary antibody molecule).
Immunfluorescent diagnostics (IF) examples
Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. Alternatively a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody can be used.
The fluorochromes used for staining cells are detected by the help of a fluorescence microscope. This
is similar to a standard light microscope, except that the illuminating light, from a very powerful source
(halogen lamp).
Detection of Anti-RNP (nuclear riboprotein) autoantibody from blood sample of a patient on cultured hepatocytes.
Detection of insulin secreting pancreatic cells on tissue section
Comparison of traditional and confocal microscopy
normal shitty microscope-
the whole sample is equally exposed by light:
the optical field is excited at the same time
broad, not focused background gets into the image
god damn confocal microscope-
point like exposition is used:
Light produced very close to the focal plane can only be detected
scanning of the sample gives three dimensional images
Western blot analysis
By this method, we can determine the relative amount of a given protein within a biological sample
using an antigen-specific primary antibody. We produce cell- or tissue lysates with a buffer containing
protease inhibitors. Different molecular weight proteins in the sample are separated by SDS
polyacrylamide gel electrophoresis. Next we blot (transfer) the content of the gel onto a nitrocellulose
or PVDF membrane, in such a way that the separated proteins should remain in the same position as
within the gel. Free protein binding capacity of the membrane is blocked by indifferent proteins (such
as with proteins of the fat free milk powder). Next we incubate the membrane with a primary antibody
specific to the desired. This is followed by incubation with a secondary antibody (more precisely with an enzyme-conjugated secondary antibody) that binds to the primary antibodies. Thus, it shows the
site where the primary antibody is bound to.
Lateral flow technology(single step immunochromatography immunoassay/fast test)
The principle of the method is that we immerse a test strip into a biological sample, and fluid migrates
in the strip by capillarity. The sample is mixed with colored, specific antibody-coated microparticles
(e.g. latex); the antigen content of the sample binds to the colored microparticles and migrates along
the test strip. In a given position of the strip, antibodies specific to another epitope of the antigen are died in a zone. Thus, the colored granules accumulate in this part of the strip, if the sample contained the antigen of interest (pl.hCG, LH or HIV). The migrating colored microparticles also accumulate at another location of the strip: in this position anti-immunoglobulin antibody is dried onto the surface.
Thus, it binds the microparticles irrespective of the fact whether it bound antigen or not.
Thus, this second visible band serves as a positive internal control in the test.
important to mention it is used in HIV and Hcg detection.
Summary
Elisa, RIA, IRMA, Lateral flow are used for soluble analyte from body fluids (blood, liqour, urine).
Elispot is used for activity of secretory cells.
western- soluble proteins from lysed cells (or body fluids)
immunocytochemistry- cellular and tissue antigens IN SITU.
