Kayode Adeyemi presented on the types and principles of ELISA. ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique developed in 1972 to detect and quantify peptides, hormones, glycoproteins, antigens, and antibodies. There are several types of ELISA techniques including direct, indirect, sandwich, competitive, and multiplex. ELISA offers advantages over previous techniques like RIA by not requiring radioactive materials.

1. JOURNAL CLUB
PRESENTATION ON
TYPES AND PRINCIPLES OF ELISA, REVIEW.
Kayode Adeyemi. G
Training and Clinical Trial Center,
ISTH, Edo State.

2. E L I S A
(ENZYME LINKED IMMUNOSORBENT ASSAY)1972: Peter Perlmann and his Graduate student, Eva Engvall invented the ELISA technique at Stockholm University
WHAT IS ELISA ASSAY?
 Quantitative or Qualitative
 Multi-well Microtiter Plate-based Assay
 Detect and quantify Peptides, Hormones, Glycoprotein, Antigens, and Antibodies.
 Absorbent 96 or 384 well polystyrene, polypropylene or polycarbonate plate
 Biological fluids, culture media or cell lysate.

3. PRE-ELISA ERA: RIA
Advantages
Highly specific: Immune reactions are specific
High sensitivity : Immune reactions are sensitive
Disadvantages
Radiation hazards:
Uses radiolabelled reagents, such as tyrosine conjugated gamma isotopes of iodine; like 125-I
Requires specially trained personnel.
Labs require special license to handle radioactive material.
Storage of radioactive material and radioactive waste disposal.

4. TYPES OF ELISA TECHNIQUES
Direct
Indirect
Sandwich
Competitive
Multiplex

5. DIRECT ELISA TECHNIQUE
known antibody for targeted antigen will be linked with an enzyme and
immobilized on a plate.
Sample with targeted antigen will be added and incubated.
Antibody-antigen complex will be formed leaving behind the enzyme.
Displaced enzyme will bind to a given substrate to form detectable colored
Enzyme-Substrate Complex
Optical density will be measured by a colorimeter at wavelength λ (higher
than 450nm) and 450nm.
λ difference = λ– 450nm will be used for data interpretation for accuracy.

6. final.luckincsolutions, 2020

7. INDIRECT ELISA TECHNIQUE
Suspected antigen in sample is bounded to wells.
Specific antibody that possess two epitopes will be added.
One epitope specific for suspected antigen, and the other for a secondary
antibody and incubated.
Secondary antibody linked to an enzyme, will be added then incubated and
washed.
The presence of the suspected antigen in the sample will lead to a formation of
Antigen-10 Antibody-20 Antibody complex.
Enzyme will be released for substrate to act on for color development.

8. .
MICROBIONOTES.COM, 2019

9. COMPETITIVE/INHIBITION ELISA
Plate is precoated with same antigen of test.
Sample with suspected antigen is incubated with Capture antibody.
Capture antibody-Sample antigen complex will be added to precoated plate.
In the presence of antigen in sample, the Capture antibody will not bind to
precoated antigen.
Secondary antibody linked with enzyme will be added to bind to Precoated
antigen as the capture antibody binds to the sample antigen.
Enzyme on the Secondary antibody will be made free to be detected by
substrate and Optical density measurement.

10. MICROBIONOTES.COM, 2019

11. SANDWICH
Plate is precoated with a monoclonal antibody selective for
targeted antigen.
Sample antigen added to enzyme-linked secondary monoclonal
antibody leading to conjugate formation and incubated with
antibody precoated in the microwells.
Sample antigen will bind to antibody in microwell and
secondary antibody to form a sandwich conjugate complex.
Substrate will bind to displaced enzyme for color development
only if the conjugate complex is formed.

12. MICROBIONOTES.COM

13. MULTIPLEX ELISA TECHNIQUE
Multiplex refers to an assay with the highest number of analyte that can be measured per assay
(up to millions) and "low-plex" or "mid-plex" refers to procedures that process fewer (10s to
1000s).
Multiplex involves the use of magnetic beads to bind capture antibody.
Analytes can be interrogated by large numbers of capture antibodies spotted on microarrays, such
as with semi-quantitative membrane antibody arrays.
Physical isolation of capture antibodies on a shared solid surface, or 1–25 capture antibodies
immobilized in small arrays at the bottom of individual wells of 96- or 384-well plates.
Microspheres of designated colors are coated with a specific antibodies read by flow cytometry as
the beads are distinguishable by fluorescent signature.
The number of analytes measured is determined by the number of different bead colors.
The Multiplex assay requires specialized equipment and a well-validated antibody pair.

15. VALIDATION OF ELISA RESULT
For the result of an ELISA assay to be accepted, there is need for the
inclusion of blank, positive and negative controls for every strip, or plate
done.
Most ELISA technique requires the use of two to three positive and negative
controls.
If the optical density of this controls are not consistent with their expected
value, such run will be tagged invalid or otherwise.

16. RESULT INTERPRETATION
Intensity of the color formed is directly proportion to the concentration of the target
analyte in the sample.
The color intensity is measured at specific wavelength in term of optical density with
the use of ELISA Spectrophotometer also known as Microplate reader.
In interpreting the result generated by the plate reader, a cut-off value is obtained
by the addition of a specific given value with the mean of the negative controls
obtained.
Individual sample OD-Ratio will be obtained by dividing the optical density of
individual well by the calculated cut-off value. The result will be used to ascertain
the positivity of the samples using a given scale.
Samples with ambiguous OD-ratio that lies within the scale of positive and negative
are repeated.