MBC 308 Continued…

4. ELISA- Principle, Types and Applications

Enzyme-linked immunosorbent assay (ELISA) test is the most widely used type of
immunoassay. ELISA is a rapid test used for detecting or quantifying antibody (Ab) against
viruses, bacteria and other materials or antigen (Ag). ELISA is so named because the test
technique involves the use of an enzyme system and immunosorbent. ELISA test is being
increasingly used in the detection of antigen (infectious agent) or antibody due to its simplicity
and sensitivity. It is as sensitive as radioimmunoassay (RIA) and requires only microlitre
quantities of test reagents. It has now been widely applied in detection of a variety of antibody
and antigens such as hormones, toxins, and viruses.

Materials needed in ELISA Testing

1. Pipettes, washer system, ELISA plate reader: Readers, washers and pipette are available as
manual or automated system. One of the main factors affecting equipment selection is the
number and types of test samples being run.

1. ELISA Readers: Readers need to have appropriate filter (650 nm and 450 nm).

2. Pipette: Are available as fixed as well as adjustable volume as well as single channel
and multi-channel.

3. Washing system: It can be manual system that washes one row or column at a time or
semi automated systems that wash one strip or plate at a time or fully automated systems that can
process multiple plates

2. Reagents needed for the testing– Concluded in the kit (coated plates, sample diluents,
controls, wash concentrate, conjugate, substrate, stop solution)

 Coated plates: The 96-well plates are made of polystyrene and are coated with either
inactivated antigen or antibody. The function of the plate has to hold the immobilized either
antigen or antibody. Antigen or antibody present in the sample will bind to the plate. This coating
acts as the binding site for the antibodies or antigens in the sample.

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 Controls: Negative and positive controls are provided in each kit. The controls help to
normalize or standardize each plate. Controls are also used to validate the assay and to calculate
sample results. Controls might be pre-diluted and ready to use.

 Conjugates: ELISA conjugates are enzyme labeled antibodies that react specifically to plate
bound sample analytes. Unbound conjugates are washed away after incubation and before the
addition of substrate.

 Wash Concentrate: It acts as a buffered solution containing detergent to wash unbound material
from the plate. (Not all test kits have wash concentrate; in that case distilled water can be used
for washing)

 Stop solution: It stops the enzyme substrate reaction and color development.

Principle

ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated in a well,
and each well contains a different serum. A positive control serum and a negative control serum
would be included among the 96 samples being tested. Antibodies or antigens present in serum
are captured by corresponding antigen or antibody coated on to the solid surface. After some
time, the plate is washed to remove serum and unbound antibodies or antigens with a series of
wash buffer. To detect the bound antibodies or antigens, a secondary antibodies that are attached
to an enzyme such as peroxidase or alkaline phosphatase are added to each well. After an
incubation period, the unbound secondary antibodies are washed off. When a suitable substrate is
added, the enzyme reacts with it to produce a color. This color produced is measurable as a
function or quantity of antigens or antibodies present in the given sample. The intensity of color/
optical density is measured at 450nm. The intensity of the color gives an indication of the
amount of antigen or antibody.

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Types of ELISA Frequently there are 3 types of ELISA on the basis of binding structure between
the Antibody and Antigen.

1. Direct ELISA

2. Indirect ELISA

3. Sandwich ELISA

4. Competitive ELISA

1. Direct ELISA

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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

 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.

2. Indirect ELISA

Antibody can be detected or quantitatively determined by indirect ELISA. In this technique,

antigen is coated on the microtiter well. Serum or some other sample containing primary
antibody is added to the microtiter well and allowed to react with the coated antigen. Any free
primary antibody is washed away and the bound antibody to the antigen is detected by adding an

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enzyme conjugated secondary antibody that binds to the primary antibody. Unbound secondary
antibody is then washed away and a specific substrate for the enzyme is added. Enzyme
hydrolyzes the substrate to form colored products. The amount of colored end product is
measured by spectrophotometric plate readers that can measure the absorbance of all the wells of
96-well plate.

Procedure of Indirect ELISA

1. Coat the micro titer plate wells with antigen.

2. Block all unbound sites to prevent false positive results.

3. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate
the plate at 37°c.

4. Wash the plate, so that unbound antibody is removed.

5. Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).

6. Wash the plate, so that unbound enzyme-linked antibodies are removed.

7. Add substrate which is converted by the enzyme to produce a colored product.

8. Reaction of a substrate with the enzyme to produce a colored product.

Advantages

 Increased sensitivity, since more than one labeled antibody is bound per primary antibody.

 A wide variety of labeled secondary antibodies are available commercially.

 Maximum immunoreactivity of the primary antibody is retained because it is not labeled.

 Versatile because many primary antibodies can be made in one species and the same labeled
secondary antibody can be used for detection.

Flexibility, since different primary detection antibodies can be used with a single labeled
secondary antibody.

 Cost savings, since fewer labeled antibodies are required.

 Different visualization markers can be used with the same primary antibody.
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Disadvantages

 Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.

 An extra incubation step is required in the procedure.

3. Sandwich ELISA

Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on the
microtiter well. A sample containing antigen is added to the well and allowed to react with the
antibody attached to the well, forming antigen-antibody complex. After the well is washed, a
second enzyme-linked antibody specific for a different epitope on the antigen is added and
allowed to react with the bound antigen. Then after unbound secondary antibody is removed by
washing. Finally substrate is added to the plate which is hydrolyzed by enzyme to form colored
products.

Procedure of sandwich ELISA

1. Prepare a surface to which a known quantity of antibody is bound.

2. Add the antigen-containing sample to the plate and incubate the plate at 37°c.

3. Wash the plate, so that unbound antigen is removed.

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4. Add the enzyme-linked antibodies which are also specific to the antigen and then incubate at
37°c.

5. Wash the plate, so that unbound enzyme-linked antibodies are removed.

6. Add substrate which is converted by the enzyme to produce a colored product.

7. Reaction of a substrate with the enzyme to produce a colored product.

Advantages

 High specificity, since two antibodies are used the antigen is specifically captured and detected.
 Suitable for complex samples, since the antigen does not require purification prior to
measurement.

 Flexibility and sensitivity, since both direct and indirect detection methods can be used.

4. Competitive ELISA

This test is used to measure the concentration of an antigen in a sample. In this test, antibody is
first incubated in solution with a sample containing antigen. The antigen-antibody mixture is

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then added to the micro titre well which is coated with antigen. The more the antigen present in
the sample, the less free antibody will be available to bind to the antigen-coated well. After the
well is washed, enzyme conjugated secondary antibody specific for isotype of the primary
antibody is added to determine the amount of primary antibody bound to the well. The higher the
concentration of antigen in the sample, the lower the absorbance.

Procedure

1. Antibody is incubated with sample containing antigen.

2. Antigen-antibody complex are added to the microtitre well which are pre-coated with the
antigen.

3. Wash the plate to remove unbound antibody.

4. Enzyme linked secondary antibody which is specific to the primary antibody is added.

5. Wash the plate, so that unbound enzyme-linked antibodies are removed.

6. Add substrate which is converted by the enzyme into a fluorescent signal.

Advantages

 High specificity, since two antibodies are used.

 High sensitivity, since both direct and indirect detection methods can be used.

 Suitable for complex samples, since the antigen does not require purification prior to
measurement.

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Application of ELISA

1. Presence of antigen or the presence of antibody in a sample can be evaluated.

2. Determination of serum antibody concentrations in a virus test.

3. Used in food industry when detecting potential food allergens.

4. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds,
cholera, STD etc.

5. Agarose Gel Electrophoresis: Definition, Principle, Steps and Uses

Introduction

Agarose gel electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins
based on their size and charge. In other words agarose gel electrophoresis is a method of gel
electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to
separate a mixed population of macromolecules such as DNA , RNA or proteins in a matrix
of agarose.

 Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by
hydrogen-bonding when heated in a buffer and allowed to cool.

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  They are the most popular medium for the separation of moderate and large-sized nucleic
acids and have a wide range of separation.

Principle of Agarose Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size in a solid support medium such as an
agarose gel. Sample (DNA) are pipetted into the sample wells, followed by the application of an
electric current which causes the negatively-charged DNA to migrate (electrophorese) towards
the anodal, positive (+ve) end. The rate of migration is proportional to size: smaller fragments
move more quickly and wind up at the bottom of the gel.

DNA is visualized by including in the gel an intercalating dye, ethidium bromide. DNA
fragments take up the dye as they migrate through the gel. Illumination with ultraviolet light
causes the intercalated dye to fluoresce.

The larger fragments fluoresce more intensely. Although each of the fragments of a single class
of molecule is present in equimolar proportions, the smaller fragments include less mass of
DNA, take up less dye, and therefore fluoresce less intensely. A “ladder” set of DNA fragments
of known size can be run simultaneously and used to estimate the sizes of the other unknown
fragments.

Requirements/ Instrumentation of Agarose Gel Electrophoresis

The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively
simple and include:

1. An electrophoresis chamber and power supply

2. Gel casting trays, which are available in a variety of sizes and composed of
UVtransparent plastic. The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis.
3. Sample combs, around which molten medium is poured to form sample wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
5. Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
“fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.

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 6. Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively,
nucleic acids can be stained after electrophoretic separation by soaking the gel in a
solution of ethidium bromide. When intercalated into double stranded DNA, fluorescence
of this molecule increases greatly. It is also possible to detect DNA with the extrinsic
fluor 1-anilino 8-naphthalene sulphonate.
7. Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in
gels.

Steps Involved in Agarose Gel Electrophoresis

1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and heated in a microwave oven to melt it.

The concentration of Agarose Gel

 The percentage of agarose used depends on the size of fragments to be resolved.

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  The concentration of agarose is referred to as a percentage of agarose to volume of buffer
(w/v), and agarose gels are normally in the range of 0.2% to 3%.

 The lower the concentration of agarose, the faster the DNA fragments migrate.

 In general, if the aim is to separate large DNA fragments, a low concentration of agarose
should be used, and if the aim is to separate small DNA fragments, a high concentration
of agarose is recommended.

2. Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to facilitate
visualization of DNA after electrophoresis.

3. After cooling the solution to about 60oC, it is poured into a casting tray containing a
sample comb and allowed to solidify at room temperature.

4. After the gel has solidified, the comb is removed, taking care not to rip the bottom of the
wells.

5. The gel, still in plastic tray, is inserted horizontally into the electrophoresis chamber and
is covered with buffer.

6. Samples containing DNA mixed with loading buffer are then pipetted into the sample
wells, the lid and power leads are placed on the apparatus, and a current is applied.

7. The current flow can be confirmed by observing bubbles coming off the electrodes.

8. DNA will migrate towards the positive electrode, which is usually colored red, in view of
its negative charge.

9. The distance DNA has migrated in the gel can be judged by visually monitoring
migration of the tracking dyes like bromophenol blue and xylene cyanol dyes.

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA.

 Estimation of the size of DNA molecules

 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting

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  Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to
Northern analysis.

 The agarose gel electrophoresis is widely employed to estimate the size of DNA
fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned
DNA.

 Agarose gel electrophoresis is commonly used to resolve circular DNA with different
supercoiling topology, and to resolve fragments that differ due to DNA synthesis.

 In addition to providing an excellent medium for fragment size analyses, agarose gels
allow purification of DNA fragments. Since purification of DNA fragments size separated
in an agarose gel is necessary for a number molecular techniques such as cloning, it is
vital to be able to purify fragments of interest from the gel.

Advantages of Agarose Gel Electrophoresis

 For most applications, only a single-component agarose is needed and no polymerization

catalysts are required. Therefore, agarose gels are simple and rapid to prepare.

 The gel is easily poured, does not denature the samples.

 The samples can also be recovered.

Disadvantages of Agarose Gel Electrophoresis

 Gels can melt during electrophoresis.

 The buffer can become exhausted.

 Different forms of genetic material may run in unpredictable forms.

5. PCR: Polymerase Chain Reaction

PCR or Polymerase Chain Reaction is a technique used in molecular biology to create several
copies of a certain DNA segment. This technique was developed in 1983 by Kary Mullis, an
American biochemist. PCR has made it possible to generate millions of copies of a small
segment of DNA. This tool is commonly used in the molecular biology and biotechnology labs.

Principle of PCR

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The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment
of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesis is new strands
of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the
pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added
to the 3’ prime end of the DNA polymerase.

Components Of PCR

Components Of PCR constitutes the following:

1. DNA Template– The DNA of interest from the sample.

2. DNA Polymerase– Taq Polymerase is used. It is thermostable and does not denature at
very high temperatures.

3. Oligonucleotide Primers- These are the short stretches of single-stranded DNA

complementary to the 3’ ends of sense and anti-sense strands.

4. Deoxyribonucleotide triphosphate– These provide energy for polymerization and are the
building blocks for the synthesis of DNA. These are single units of bases.

5. Buffer System– Magnesium and Potassium provide optimum conditions for DNA
denaturation and renaturation. It is also important for fidelity, polymerase activity, and
stability.

Types of PCR

PCR is of the following types:

Real-time PCR

In this type, the DNA amplification is detected in real-time with the help of a fluorescent
reporter. The signal strength of the fluorescent reporter is directly proportional to the number of
amplified DNA molecules.

Nested PCR

This was designed to improve sensitivity and specificity. They reduce the non-specific binding of
products due to the amplification of unexpected primer binding sites.

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Multiplex PCR

This is used for the amplification of multiple targets in a single PCR experiment. It amplifies
many different DNA sequences simultaneously.

Quantitative PCR

It uses the DNA amplification linearity to detect, characterize and quantify a known sequence in
a sample.

Arbitrary Primed PCR

It is a DNA fingerprinting technique based on PCR. It uses primers the DNA sequence of which
is chosen arbitrarily.

PCR Steps

The PCR involves three major cyclic reactions:

Denaturation

Denaturation occurs when the reaction mixture is heated to 94 ℃ for about 0.5 to 2 minutes. This
breaks the hydrogen bonds between the two strands of DNA and converts it into a single-
stranded DNA.

The single strands now act as a template for the production of new strands of DNA. The
temperature should be provided for a longer time to ensure the separation of the two strands.

Annealing

The reaction temperature is lowered to 54-60℃ for around 20-40 seconds. Here, the primers
bind to their complementary sequences on the template DNA.

Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length.

They serve as the starting point for the synthesis of DNA.

The two separated strands run in the opposite direction and consequently there are two primers- a
forward primer and a reverse primer.

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Elongation

At this step, the temperature is raised to 72-80 ℃. The bases are added to the 3’ end of the primer
by the Taq polymerase enzyme.

This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds about
1000bp/minute under optimum conditions.

Taq Polymerase can tolerate very high temperatures. It attaches to the primer and adds DNA
bases to the single strand. As a result, a double-stranded DNA molecule is obtained.

These three steps are repeated 20-40 times in order to obtain a number of sequences of DNA of
interest in a very short time period.

Applications of PCR

The following are the applications of PCR :

Medicine

 Testing of genetic disease mutations.

 Monitoring the gene in gene therapy.

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  Detecting disease-causing genes in the parents.

Forensic Science

 Used as a tool in genetic fingerprinting.

 Identifying the criminal from millions of people.

 Paternity tests

Research and Genetics

 Compare the genome of two organisms in genomic studies.

 In the phylogenetic analysis of DNA from any source such as fossils.

 Analysis of gene expression.

 Gene Mapping

SDS PAGE

SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique

used for the separation of proteins based on their molecular weight. It is a technique widely used
in forensics, genetics, biotechnology and molecular biology to separate the protein molecules
based on their electrophoretic mobility.

Principle of SDS-PAGE

The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the
opposite sign when placed in an electric field. The separation of the charged molecules depends
upon the relative mobility of charged species.

The smaller molecules migrate faster due to less resistance during electrophoresis. The structure
and the charge of the proteins also influence the rate of migration. Sodium dodecyl sulphate and
polyacrylamide eliminate the influence of structure and charge of the proteins, and the proteins
are separated based on the length of the polypeptide chain.

Role of SDS in SDS-PAGE

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SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing
agents function to break the disulphide bonds of proteins disrupting the tertiary structure of
proteins.

Materials Required

 Power Supplies: It is used to convert the AC current to DC current.

 Gels: These are either prepared in the laboratory or precast gels are purchased from the
market.

 Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be
used.

 Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled for
10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to
reduce the disulfide linkages to prevent any tertiary protein folding.

 Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running
buffer.

 Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution. The
gel is then destained with the destaining solution. Protein bands are then visible under
naked eyes.

 Protein Ladder: A reference protein ladder is used to determine the location of the
protein of interest, based on the molecular size.

Protocol of SDS-PAGE

Preparation of the Gel

 All the reagents are combined, except TEMED, for the preparation of gel.

 When the gel is ready to be poured, add TEMED.

 The separating gel is poured in the casting chamber.

 Add butanol before polymerization to remove the unwanted air bubbles present.

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  The comb is inserted in the spaces between the glass plate.

 The polymerized gel is known as the “gel cassette”.

Also read: Applications of Biotechnology

Sample Preparation

 Boil some water in a beaker.

 Add 2-mercaptoethanol to the sample buffer.

 Place the buffer solution in microcentrifuge tubes and add protein sample to it.

 Take MW markers in separate tubes.

 Boil the samples for less than 5 minutes to completely denature the proteins.

Electrophoresis

 The gel cassette is removed from the casting stand and placed in the electrode assembly.

 The electrode assembly is fixed in the clamp stand.

 1x electrophoresis buffer is poured in the opening of the casting frame to fill the wells of
the gel.

 Pipette 30ml of the denatured sample in the well.

 The tank is then covered with a lid and the unit is connected to a power supply.

 The sample is allowed to run at 30mA for about 1 hour.

 The bands are then seen under UV light.

Applications of SDS-PAGE

The applications of SDS-PAGE are as follows:

1. It is used to measure the molecular weight of the molecules.

2. It is used to estimate the size of the protein.

3. Used in peptide mapping

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4. It is used to compare the polypeptide composition of different structures.

5. It is used to estimate the purity of the proteins.

6. It is used in Western Blotting and protein ubiquitination.

7. It is used in HIV test to separate the HIV proteins.

8. Analyzing the size and number of polypeptide subunits.

9. To analyze post-translational modifications.

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