Molecular Diagnostics 2 Flashcards
Structure of Antibodies (ImmunoGlobulin IG)
IgG antibodies consist of four chains, two heavy chains (blue) and two light chains
(red), linked by disulfide bonds. The heavy and light chains come together to form Fab
domain (fragment has the antigen binding site), which have the antigen-binding sites at the
ends. The two heavy chains form the Fc domain (fragment that crystallizes). The Fab
domains are linked to the Fc domain by flexible linkers (hinges).
What is an Epitope?
Epitope is the specific site of the antigenic molecule the Fab region recognizes
antigenic determinant or epitope.
An antibody recognizes a specific group or cluster of
amino acids on the target molecule called an
antigenic determinant or epitope.
multiple myeloma
malignant disorder of antibody-producing
cells –immune B-cells
Generation of Monoclonal Antibody
Large amounts of homogeneous antibody of nearly desired
specificity could be obtained by fusing a short-lived antibodyproducing
cell with an immortal myeloma cell.
ascites fluid
When the hybridoma cells are injected in mice (in the
peritoneal cavity, the gut), they produce tumors containing an
antibody-rich fluid
direct detection
marker molecule can be linked directly to the
primary antibody
indirect detection
stronger signal is
achieved by using an unlabeled primary antibody and then
detecting it with a group of labeled secondary antibodies that bind to it
Western Blotting
- This method is designed to compare protein levels using cell extracts which are
fractionated according to size. - To detect small quantities of a particular protein in the presence of many other
proteins
Western Blotting procedure
A mixture of proteins is
separated by sodium dodecyl
sulfate (SDS) polyacrylamide
gel electrophoresis and then
transferred to a nitrocellulose
membrane. The membrane
is incubated with the first
(primary) antibody directed
against the target protein
followed by a second
(tagged) antibody directed
against the first
enzyme-linked immunosorbent assay (ELISA)
Antibodies can be used to quantify the amount of an antigen in a
sample mixture by the technique
Indirect ELISA
To detect the presence of antibody and is the basis of the
test for HIV infection (an example).
Indirect ELISA procedure
In that test, viral core proteins (the antigen) are absorbed to the bottom of a well. Antibodies from a patient are then added to the coated well and allowed to bind to the antigen.
Finally, enzyme-linked antibodies to human antibodies (for instance, goat antibodies that recognize human antibodies) are allowed to react in the well and unbound antibodies are removed by washing. Substrate is then applied.
An enzyme reaction suggests that the enzyme-linked
antibodies were bound to human antibodies, which in turn
implies that the patient had antibodies to the viral antigen.
ELISA Procedures (A)
A microplate is precoated
with a capture antibody, which is
immobilized to the plate’s
surface. Any analyte (e.g., target
protein) from standard or
biological samples will be
captured (bound) to the antibody
when added to the well.
Unbound/nonspecifically bound
molecules are washed away. A
second antibody conjugated with
horseradish peroxidase (HRP) is
added and binds to the captured
analyte. After the unbound
second antibody is washed away,
the HRP substrate,
tetramethylbenzidine (TMB), is
added to the wells. Blue color is
developed proportionally to the
amount of analyte in the sample.
Indirect ELISA Results
In indirect ELISA,
the production of
color indicates the
amount of an
antibody to a specific
antigen.
Sandwich ELISA Results
In sandwich ELISA, the
production of color
indicates the quantity
of antigen.
