Immunology and vaccinology Q&A
Havard University ID 503
Clinical immunology and vaccinology
Immunological and vaccinology Take away Exam
1. Describe the ELISA technique, immunoprecipitation, agglutination, and their applications.
ELISA
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed
for detecting and quantifying peptides, proteins, antibodies, and hormones. In ELISA, an antigen
must be immobilized to a solid surface and then complexed with an antibody linked to an
enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation
with a substrate to produce a measurable product. The most crucial element of the detection
strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will
passively bind antibodies and proteins. The binding and immobilization of reagents makes
ELISAs simple to design and perform. Having the reactants of the ELISA immobilized to the
microplate surface enables easy separation of bound from non-bound material during the assay.
This ability to wash away non-specifically bound materials makes the ELISA a powerful tool for
measuring specific analytes within a crude preparation.
How ELISA Works?
, Enzyme-linked immunosorbent assays (ELISA) principles are very similar to other
immunoassay technologies. ELISAs rely on specific antibodies to bind the target antigen, and a
detection system to indicate the presence and quantity of antigen binding. In order to maximize
the sensitivity and precision of the assay, the plate must be carefully coated with high-affinity
antibodies. Here are the list of our most popular ELISA kit.
Insulin
Cortisol
TNF alpha
IL-1 Beta
IL-6
IL-10
IFN Gamma
Adiponectin
VEGF
4 TYPES OF ELISA
Comparisons on Direct, Indirect, Sandwich, Competitive ELISA
ELISAs can be performed with a number of modifications to the basic procedure: direct,
indirect, sandwich or competitive. The key step, immobilization of the antigen of interest, can be
accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has
been attached to the plate. The antigen is then detected either directly (enzyme-labeled primary
,antibody) or indirectly (enzyme-labeled secondary antibody). The detection antibodies are
usually labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). A large
selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The
choice of substrate depends upon the required assay sensitivity and the instrumentation available
for signal-detection (spectrophotometer, fluorometer or luminometer).
Among the standard assay formats discussed and illustrated below, where differences in
both capture and detection were the concern, it is important to differentiate between the
particular strategies that exist specifically for the detection step. However, an antigen is captured
to the plate (by direct adsorption to the surface or through a pre-coated "capture" antibody, as in
a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely
determines the sensitivity of an ELISA.
DIRECT ELISA
For direct detection, an antigen coated to a multi-well plate is detected by an antibody
that has been directly conjugated to an enzyme. This detection method is a good option if there is
no commercially available ELISA kits for your target protein.
Advantages:
Quick because only one antibody and fewer steps are used.
Cross-reactivity of secondary antibody is eliminated.
Disadvantages:
Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
, Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes
or tags.
Labeling primary antibodies for each specific ELISA system is time-consuming and expensive.
No flexibility in choice of primary antibody label from one experiment to another.
Minimal signal amplification.
INDIRECT ELISA
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an
enzyme-labeled secondary antibody is bound to the first antibody
Advantages:
A wide variety of labeled secondary antibodies are available commercially.
Versatile because many primary antibodies can be made in one species and the same labeled
secondary antibody can be used for detection.
Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Sensitivity is increased because each primary antibody contains several epitopes that can be
bound by the labeled secondary antibody, allowing for signal amplification.
Disadvantages:
Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
Havard University ID 503
Clinical immunology and vaccinology
Immunological and vaccinology Take away Exam
1. Describe the ELISA technique, immunoprecipitation, agglutination, and their applications.
ELISA
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed
for detecting and quantifying peptides, proteins, antibodies, and hormones. In ELISA, an antigen
must be immobilized to a solid surface and then complexed with an antibody linked to an
enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation
with a substrate to produce a measurable product. The most crucial element of the detection
strategy is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will
passively bind antibodies and proteins. The binding and immobilization of reagents makes
ELISAs simple to design and perform. Having the reactants of the ELISA immobilized to the
microplate surface enables easy separation of bound from non-bound material during the assay.
This ability to wash away non-specifically bound materials makes the ELISA a powerful tool for
measuring specific analytes within a crude preparation.
How ELISA Works?
, Enzyme-linked immunosorbent assays (ELISA) principles are very similar to other
immunoassay technologies. ELISAs rely on specific antibodies to bind the target antigen, and a
detection system to indicate the presence and quantity of antigen binding. In order to maximize
the sensitivity and precision of the assay, the plate must be carefully coated with high-affinity
antibodies. Here are the list of our most popular ELISA kit.
Insulin
Cortisol
TNF alpha
IL-1 Beta
IL-6
IL-10
IFN Gamma
Adiponectin
VEGF
4 TYPES OF ELISA
Comparisons on Direct, Indirect, Sandwich, Competitive ELISA
ELISAs can be performed with a number of modifications to the basic procedure: direct,
indirect, sandwich or competitive. The key step, immobilization of the antigen of interest, can be
accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has
been attached to the plate. The antigen is then detected either directly (enzyme-labeled primary
,antibody) or indirectly (enzyme-labeled secondary antibody). The detection antibodies are
usually labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). A large
selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The
choice of substrate depends upon the required assay sensitivity and the instrumentation available
for signal-detection (spectrophotometer, fluorometer or luminometer).
Among the standard assay formats discussed and illustrated below, where differences in
both capture and detection were the concern, it is important to differentiate between the
particular strategies that exist specifically for the detection step. However, an antigen is captured
to the plate (by direct adsorption to the surface or through a pre-coated "capture" antibody, as in
a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely
determines the sensitivity of an ELISA.
DIRECT ELISA
For direct detection, an antigen coated to a multi-well plate is detected by an antibody
that has been directly conjugated to an enzyme. This detection method is a good option if there is
no commercially available ELISA kits for your target protein.
Advantages:
Quick because only one antibody and fewer steps are used.
Cross-reactivity of secondary antibody is eliminated.
Disadvantages:
Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
, Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes
or tags.
Labeling primary antibodies for each specific ELISA system is time-consuming and expensive.
No flexibility in choice of primary antibody label from one experiment to another.
Minimal signal amplification.
INDIRECT ELISA
For indirect detection, the antigen coated to a multi-well plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an
enzyme-labeled secondary antibody is bound to the first antibody
Advantages:
A wide variety of labeled secondary antibodies are available commercially.
Versatile because many primary antibodies can be made in one species and the same labeled
secondary antibody can be used for detection.
Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Sensitivity is increased because each primary antibody contains several epitopes that can be
bound by the labeled secondary antibody, allowing for signal amplification.
Disadvantages:
Cell Smear: Adhere non-adherent cells on coverslip with chemical bond
Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
