immunosorbent assay) is an assay based on the principle of antigen for detection
and quantification of peptides, proteins, antibodies and hormones typically
secreted or released from the cells. An
antigen is immobilized to a surface which is then complexed with an enzyme
linked antibody. Substrate is incubated with conjugated enzyme for detection
signal. A highly specific antibody-antigen interaction is the most critical
element for the detection strategy. They are
sensitive assays because of the binding characteristics of the antibodies and
the amplification or different read-out systems used. ELISA has become a
critical tool in the field of human medicine, veterinary medicine, food
testing, and agriculture. A variety of samples can be used in ELISA for the
detection of specific target including serum, plasma, cell culture supernatant,
cell lysates, saliva, tissue lysates, and urine.
ELISA can measure both
antigens and antibodies and requires 3 necessary reagents:
Solid-phase antigen or antibody
Enzyme-labelled antigen or antibody
Substrate for enzyme action
results achieved by ELISA can be of three different types:
1) Quantitative: ELISA data can be interpreted for measuring the antigen concentration in the sample by using a standard curve.
2) Qualitative: ELISA can also be used to analyse the presence or absence
of the antigen only.
3) Semi-Quantitative: This type of data is used for comparing the relative antigen level in the sample.
ELISAs are of: direct, indirect, sandwich or competitive. The main step is the immobilization of the antigen of interest either by direct adsorption to the assay plate or indirectly with aid of capture antibody already 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).
easy assay set-up
· Each antibody used for detection must be conjugated
· Potential for higher background
· No Signal Amplification
· Less Specificity
· Not amenable to multiplexing
sensitivity than Direct ELISA
of using the conjugated second antibody for multiple assays
antibody will give an amplification of signal
to the number of steps, the protocol typically will take longer and could be
more prone to error
for cross reactivity caused by secondary antibody.
with complex sample matrices
of matched antibody pair can be costly and time consuming.
for quantitative measurement of a sample with low analyte concentration
for small antigens
Advantages of ELISA
1. ELISA technique could be used for both quantitative and qualitative analysis.
2. The ELISA has high sensitivity and specificity achieved by the coating of the plate with a high-affinity antibody.
3. The test is strongly specific due to the selective of antigen or the antibody.
4. Sample volumes can also be adjusted in case of very low abundant protein.
5. ELISA with micro and nano-fabrication strategies and integration of the assay with lab-on-chip (LOC) and lab-on-compact-disk (LOCD) devices provides detection at micro and nano level.
6. ELISA has promising great potential from point-of-care (POC) due to its cost-effectiveness.
7. Multiplexing version of ELISA provides convince of detecting multiple parameters simultaneously in the same sample.
8. Useful in detecting diseases outbreak, can also be used in toxicology as a rapid presumptive screen for certain class of drugs.
9. Useful in the detection of microbes in foodstuff.
Fig: Advantages of Multiplex ELISA.
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Because of its applications advantageous in various fields of biotechnology, clinical diagnosis, plant pathology, diseases conditions, food technology for the detection of disease markers, food allergens and unknown proteins etc. Hence, we being the ELISAkit Distributor welcomes your enquiries at email@example.com .