This document provides information about ELISA (Enzyme-Linked Immunosorbent Assay) techniques. It discusses how ELISAs are typically performed in multi-well plates that passively bind antibodies and proteins. Detection enzymes or tags can then be used to detect bound analytes. The document compares direct ELISA detection using a single labeled antibody to indirect detection using unlabeled primary and labeled secondary antibodies. It notes advantages of indirect detection include increased sensitivity through signal amplification. Finally, it provides step-by-step instructions for performing a sandwich ELISA to detect antigens using capture and detection antibodies between wells.

1. 1
Amjad Khan Afridi 7t h
August, 2016
ELISA ( Enzyme Linked Immunosorbent Assay)
Introduction
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which
will passively bind antibodies and proteins. It is this binding and immobilization of
reagents that makes ELISAs so easy to design and perform. Having the reactants
of the ELISA immobilized to the microplate surface makes it easy to separate
bound from nonbound material during the assay. This ability to wash away
nonspecifically bound materials makes the ELISA a powerful tool for measuring
specific analytes within a crude preparation.
A detection enzyme or other tag can be linked directly to the primary antibody or
introduced through a secondary antibody that recognizes the primary antibody. It
also can be linked to a protein such as streptavidin if the primary antibody is biotin
labeled. The most commonly used enzyme labels are horseradish peroxidase
(HRP) and alkaline phosphatase (AP).
Other enzymes have been used as well, but they have not gained widespread
acceptance because of limited substrate options. These include β-galactosidase,
acetylcholinesterase and catalase. 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).
ELISA Formats
ELISAs can be performed with a number of modifications to the basic procedure.
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 (labeled
primary antibody) or indirectly (labeled secondary antibody).
The most powerful ELISA assay format is the sandwich assay. This type of
capture assay is called a “sandwich” assay because the analyte to be measured is
bound between two primary antibodies – the capture antibody and the detection
antibody. The sandwich format is used because it is sensitive and robust.

2. 2
Amjad Khan Afridi 7t h
August, 2016
ComparisonofDirect and Indirect ELISADetection Methods
Direct ELISA Detection
Advantages  Quick because only one antibody and fewer steps are used.
 Cross-reactivity of secondary antibody is eliminated.
Disadvantages  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 Detection
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.
 Different visualization markers can be used with the same primary antibody.
Disadvantages  Cross-reactivity might occur with the secondary antibody, resulting in nonspecific
signal.
 An extra incubation step is required in the procedure.
Fluorescent tags and other alternatives to enzyme-based detection can be used for plate-based assays.
Despite not involving reporter-enzymes, these methods are also generally referred to as a type of ELISA.
Likewise, wherever detectable probes and specific protein binding interactions can be used in a plate-
based method, these assays are often called ELISAs despite not involving antibodies.

3. 3
Amjad Khan Afridi 7t h
August, 2016
Direct vs. Indirect Detection ELISA Strategies
Among the standard assay formats discussed and illustrated above, 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.
Diagram of common ELISA formats (direct vs. sandwich assays)
Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by firs t
attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme-
conjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies
(indirect detection)
Sandwich Assays
A. Sandwich Assays having wells, in which each wells having coated An
Antibodies and an Antigen.
B. In this method we use 4 wells for a single test.
i. Positive Control
ii. Sample well
iii. Negative Control
iv. Blank
C. We use specific solutions for each specific Wells.
D. After the end of the process we absorb and measure the wells by Plate
Reader or Strip Reader, that either test is positive or negative.

4. 4
Amjad Khan Afridi 7t h
August, 2016
Requirements:
1. ELISA Testwell
2. ELISA Plates
3. Test tubes
4. Syringe
5. swab
6. centrifuge
7. Specimen dilution
8. Positive control
9. Sample ( Blood Sample )
10.Negative control
11.RHP-Conjugate
12.Buffer solution
13.Distilled water
14.Chromogen “A”
15.Chromogen “B”
16.Stop solution
17.Micro pipettes and Tips
18.Container
19.Aluminum foil ( Cover)
20.Incubator
21.Plate reader or strip reader
22.Dropper
23.Permanent Marker
24. Tissuepaper
25. Gloves
26. Mask
Sandwich Assays Procedure
1. Bring all the reagents to a roomtemperature .
2. Labeled the wells with specific ID number.

5. 5
Amjad Khan Afridi 7t h
August, 2016
3. Pour 20 µL specimen dilution in each wells except Blank well.
4. Add 100 µL sample in sample well ( S ) .
5. Add 100 µL PositiveControl solution in positive well ( P), while add 100 µL
Negative Controlsolution in negative Well ( N).
6. Covered and Labeled each wells and mix it carefully.
7. Next , replaced all the wells in the incubator and incubated the wells at 37 C
for 60 minutes to react each an Antibodies with their specific an Antigen .
8. After incubation apply HRP- Conjugate to each wells except Blank ( B) well.
9. Again covered and mix the wells and incubated for 30 minutes at 37 C.
10. After incubation washed each wells five times with Buffer Solution to
removed unbounded an antigens and other particles. Each washing steps
should be 30 seconds.
11.After washing apply 50 µL Chromogen“A” and 50 µL Chromogen“B” in
each wells, covered the well and incubated for 30 minutes at 37 C.
12.After incubation add stop solution in each wells to stopped the
Chromogen“A” and Chromogen “B” reactions.
13.Next, Measureand absorb the wells results by Plate Reader or Strips
Reader, either resultNegative or positive.
14.Result : Comparethe colors of the wells with Negative Control (N). If the
colors of all the wells are similar to the Negative control well (N) the result
would be known as Hbs Positive, if the colors are not similar to Negative
Control well (N) , the result would be consider as Hbs Negative (N).
15.Note : the color change can not always be detected by naked eyes.
Demonstrated and particle by Hakeem Zada