ELISA

Enzyme Linked immunosorbent Assay

Table of Contents
s.no PARTICULARS
1. Introduction
2. History
3. ELISA Principle
4. Types of ELISA
a) Direct ELISA
b) Indirect ELISA
c) Sandwich ELISA
d) Competitive ELISA

5. ELISA Result Interpretation

6. ELISA Applications
7. Advantages of ELISA
8. Limitations of ELISA
9. References

INTRODUCTION
An ELISA is an immunological assay commonly used to measure
antibodies or antigens, including proteins or glycoproteins, in
biological samples. Like other immunoassays, they rely on
binding of antibodies to their targets to facilitate detection.

Typically, ELISA assays are performed in 96-well plates, a

format that makes them amenable to screening many samples
at once. Serum, plasma, cell culture supernatants, cell lysates,
saliva, tissue lysates and urine are all common sample types
used for these assays, but most liquid sample types could be
used in theory. It is, however, important to consider that some
sample types may include inhibitory factors, such as buffer
components that share similar antigenic epitopes1 or factors
like proteases2 that may damage the target or detection
components, that may interfere with the assay’s performance.
There are several different assay formats, but all rely on
binding of either the target itself or an antibody/antigen able to
capture the target to the surface of the plate. A detection step
involving a conjugated antigen, or more often antibody, is then
employed to enable successful binding to be detected and
quantified, most often by colorimetric detection.

Antigens
antigens are composed of proteins, peptides, and
polysaccharides. Any portion of bacteria or viruses, such as
surface protein, coat, capsule, toxins, and cell wall, can as serve
antigens.

Antibodies
Antibodies are proteins that protect you when an unwanted
substance enters your body. Produced by your immune system,
antibodies bind to these unwanted substances in order to
eliminate them from your system.
Antibodies are produced by B cells (specialized white blood
cells). When an antigen comes into contact with a B cell, it
causes the B cell to divide and clone. These cloned B cells — or
plasma cells — release millions of antibodies into your
bloodstream and lymph system.

Antigen –Antibody Interactions

 Antigen-antibody interaction, also known as the
antigen-antibody reaction, is a fundamental process in the
immune system where antibodies bind to specific
antigens. This binding initiates a series of immune
responses that can lead to the neutralization or removal of
harmful agents like viruses, bacteria, and toxins. The
interaction is highly specific, with antibodies recognizing
specific regions on antigens called epitopes.

 Antigen- Antibody Interaction

Radioimmunoassay (RIA)

Radioimmunoassay (RIA) is a highly sensitive

laboratory technique used to measure the
concentration of substances in a sample, typically
antigens, by using radioactive isotopes. It relies on
the principle of competitive binding between labeled
and unlabeled antigens for a limited number of
antibody binding sites.
 History of ELISA
The ELISA (Enzyme-Linked Immunosorbent Assay) was developed in 1971
by Eva Engvall and Peter Perlmann as a safer and more practical alternative
to the radioimmunoassay (RIA). This technique leverages enzyme-linked
antibodies to detect and quantify specific molecules, primarily proteins, in a
sample.
Here's a more detailed look at the ELISA's history:

Early Concepts (1940s-1960s):

 Immunofluorescence: In the 1940s, Albert H. Coons and his colleagues
pioneered the use of fluorescently labeled antibodies to identify antigens in
tissue sections, a technique known as immunofluorescence.
 Radioimmunoassay (RIA): In the 1960s, Yalow and Berson described the RIA
method, which used radioactive isotopes to detect antigens and
antibodies. While effective, RIA had concerns about radioactive waste and
potential health risks, prompting the search for safer alternatives

The Birth of ELISA (1971):

 Engvall and Perlmann's Contribution: Eva Engvall and Peter Perlmann
independently developed the ELISA method by modifying the RIA
approach. They conjugated antigens and antibodies with enzymes rather than
radioactive isotopes.
 Early Applications: They first used ELISA to determine IgG levels in rabbit
serum, and another research team successfully quantified human chorionic
gonadotropin in urine using horseradish peroxidase, according to StatPearls.
 Competitive ELISA in 1976: The competitive ELISA was introduced,
enabling the detection of human chorionic gonadotropin hormone by using a
conjugated substrate to compete with a protein of interest.

 Sandwich ELISA in 1977: The sandwich ELISA method, where the detection
antibody is pre-coated on the plate surface, was developed and tested on
various substrates.
 Indirect ELISA in 1978: The indirect ELISA, which uses a secondary
antibody for detection, was developed and utilized to identify human serum
albumin.
 HIV screening 1985: The ELISA test became the first screening test
commonly used for HIV, marking a significant advancement in diagnostics.
 Present: ELISA is widely used in various applications, including testing for
antibodies against SARS-CoV-2 (COVID-19).
 Principle of ELISA
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.
 Microtiter Plate
 Types of ELISA

Frequently there are 4 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
A direct ELISA (enzyme-linked immunosorbent
assay) is a plate-based immunosorbent assay
intended for the detection and quantification of a
specific analyte (e.g. antigens, antibodies, proteins,
hormones, peptides, etc.) from within a complex
biological sample. Of the four different ELISA
formats, direct ELISA is the simplest and quickest
to perform, but there are some disadvantages
associated with this method
In a direct ELISA the antigen is immobilized
directly onto the surface of a multi-well microtiter
plate such as a 96-well polystyrene plate, and then
complexed with an enzyme-labeled primary
antibody specific for the antigen. Once the
enzyme-labeled primary antibody binds to the
antigen, the conjugated primary antibody catalyzes
a reaction with its respective substrate resulting in
a visible colorimetric output that is measured by a
spectrophotometer or absorbance microplate
reader. Direct ELISAs are suitable for qualitative
and quantitative antigen detection in samples of
interest, antibody screening, and epitope mapping.
 Procedure of Direct ELISA
1. Antigen Coating:
1. Dilute the antigen in a coating buffer to the desired
concentration (e.g., 20 µg/mL).
2. Add the diluted antigen to each well of a microtiter plate
(e.g., 100 µL/well).
3. Incubate the plate at a specific temperature (e.g., 4°C
overnight) to allow the antigen to adhere to the well.

2.Blocking:

1. Add the enzyme-linked antibody (e.g., biotinylated

antibody with HRP) that is specific to the antigen to each
well.
2. Incubate the plate at a specific temperature (e.g.,
room temperature for 1 hour).
3. Remove the antibody solution and wash the plate
several times with the wash buffer.

3. Antibody Incubation:
1. Add the enzyme-linked antibody (e.g., biotinylated
antibody with HRP) that is specific to the antigen to each
well.
2. Incubate the plate at a specific temperature (e.g., room
temperature for 1 hour).
3. Remove the antibody solution and wash the plate
several times with the wash buffer.
 4. Detection:
1. Add a substrate solution (e.g., TMB) that reacts with the
enzyme on the antibody to produce a color change.
2. Incubate the plate at a specific temperature until a visible
color change is observed.
3. Add a stop solution to stop the reaction and measure the
absorbance using a plate reader.

Procedure of Direct ELISA

Advantages:
 Simplicity and Speed: Direct ELISA involves a simpler
workflow with fewer steps, making it faster and easier to
perform than indirect ELISA.
 Reduced Error and Cross-reactivity: By using only one
antibody, the risk of cross-reactivity is minimized, and there
are fewer opportunities for errors due to pipetting or reagent
handling.
 Cost-Effectiveness: The fewer reagents and steps required
in direct ELISA translate to lower overall costs compared to
indirect ELISA, which uses two antibodies .
 Suitable for Small Antigens: Direct ELISA is well-suited
for detecting antigens that may not be easily detectable with
the two-antibody approach used in indirect ELISA .
 No Cross-reactivity from Secondary Antibody: Since
direct ELISA uses a single antibody, there is no risk of
cross-reactivity from a secondary antibody.
 Disadvantages:
 Limited Sensitivity: Direct ELISA relies on a single
antibody to detect the target antigen. Unlike indirect ELISA,
where a secondary antibody binds to the primary antibody,
amplifying the signal, direct ELISA lacks this amplification
step, resulting in weaker signals and reduced sensitivity.
 Time-Consuming Assay Development: Each assay
target in direct ELISA requires a specific, conjugated
primary antibody. This can be time-consuming and labor-
intensive, especially when multiple targets need to be
investigated.
 Potential for Reduced Antibody Reactivity: The
enzyme conjugation process, necessary in direct ELISA, can
potentially affect the antibody's ability to bind to the target
antigen. The enzyme molecule may physically interfere with
the antibody's binding sites, reducing its reactivity.
 Lack of Flexibility: Direct ELISA is less flexible compared
to indirect ELISA. In indirect ELISA, the same secondary
antibody can be used with multiple primary antibodies,
offering more versatility. In direct ELISA, each primary
antibody needs to be individually conjugated, limiting
flexibility.
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 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 microtiter plate with antigen: Dispense 50 μl
antigen solution (coating reagent) into the wells of
a microtiter plate.
2.Wrap coated plates in plastic wrap to seal and
incubate for 2 hr at 37 0C in an incubator.
3.After incubation, uncover the microtiter plate and
discard the solution into a container.
4.Washing Step: Remove coating solution and
wash the plate twice by filling the wells with 200
 µl PBS. The solutions or washes are removed by
pipetting. The remaining drops are removed by
patting the plate on a paper towel.
5.Blocking step: Block the remaining protein-
binding sites in the coated wells by adding 200 µl
blocking buffer and incubate 30 min at room
temperature.
6.Discard the solution and perform the washing
step. Gently flick microplate onto paper towel
7.Add 50 μl of antibody solution using
micropipette from the vial to the wells.
8.Wrap plate in plastic wrap, and incubate for 2 hr
at room temperature. Discard liquid and pat
bottom of plate with dry absorbent paper.
9.Repeat washing step
10. Repeat blocking step.
11. Repeat washing step.
12. Add 50 μl secondary antibody reagent to the
wells.
13. Wrap the micro titer plate in plastic wrap, and
incubate 2 hr at room temperature.
14. Repeat washing step.
15. Add 75 µl substrate solution from vial to the
wells on micro titer plates.
16. Wrap the micro titer plates in plastic wrap and
incubate for 1 hr at room temperature.
17. Add 25 µl stop solution (0.5 M NaOH) from vial
to the wells on micro titer plates.
18. Using a micro titer plate reader to measure
NPP hydrolysis, use a 405-nm filter.
 Procedure of Indirect ELISA

Advantages:
 High Sensitivity: The indirect ELISA method uses a
secondary antibody to bind to the primary antibody, which
in turn binds to the antigen. This allows multiple labeled
secondary antibodies to bind to each primary antibody,
 amplifying the signal and increasing the sensitivity of the
assay. This is particularly useful for detecting low levels of
antigens.
 Greater Flexibility: A single labeled secondary antibody
can be used with different primary antibodies, providing
flexibility in the assay design.
 Cost-Effective: Since fewer labeled antibodies are required
in indirect ELISA, it can be more cost-effective than direct
ELISA, where each primary antibody needs to be labeled .
 Maximum Immunoreactivity: No label interferes with the
binding sites on the primary antibody, ensuring maximum
immunoreactivity.
 Simplified Procedures: Indirect ELISA often involves
fewer steps and is generally more efficient than direct
ELISA, reducing the time and effort required.
 Can be used with complex samples: Indirect ELISA is
suitable for analyzing complex samples without requiring
antigen purification.

Disadvantages:

1. Increased Complexity and Longer Procedure:

Indirect ELISAs require an extra incubation step for the
secondary antibody, making the procedure longer and
more complex than direct ELISAs.

2. Background Noise: The secondary antibody, which

binds to the primary antibody, can potentially cross-react
with other molecules in the sample, leading to nonspecific
binding and increased background noise. This can make it
difficult to distinguish true positives from false positives.

3. Need for Careful Optimization: Due to the increased

potential for background noise, indirect ELISAs require
more careful optimization of incubation times, reagent
concentrations, and washing procedures to minimize
nonspecific binding.

4. Potential for False Positives: Cross-reactivity of the

secondary antibody can lead to false positive results,
where a signal is detected in the absence of the target
antigen.

5. Cost and Availability of Reagents: While indirect

ELISAs offer higher sensitivity, they may require more
specialized reagents, including specific secondary
antibodies, which can increase the cost and may not
always be readily available.
3. Sandwich ELISA
Developed in 19776, as its name suggests, the sandwich
ELISA sandwiches the antigen between antibodies. The
technique can employ the direct or indirect ELISA format
(the sandwich ELISA depicted above is based on the
indirect ELISA) except that rather than non-specific
binding of antigens to the assay plate, the capture
antibody makes this a specific process. This combination
further improves assay sensitivity and specificity.
They do, however, require the determination of compatible
capture and detection antibody pairs to function effectively
with which cross-reactivity can be problematic. This assay
typically has the most steps too, offering greater
opportunity for error. Due to the selective nature of the
antigen binding step, sandwich ELISAs are particularly
useful where the antigen is in a complex mix as antigen
purification is not required.
Procedure of sandwich ELISA
1. Prepare a surface to which a known quantity of
capture antibody is bound.
2.Block any nonspecific binding sites on the surface.
3.Add antigen-containing sample to the plate.
4.Wash the plate, so that unbound antigen is
removed.
5.A specific antibody is added, and binds to antigen
(hence the ‘sandwich’: the Ag is stuck between
two antibodies);
6.Add enzyme-linked secondary antibodies as
detection antibodies that also bind specifically to
the antibody’s Fc region (non-specific).
7.Wash the plate, so that the unbound antibody-
enzyme conjugates are removed.
8.Add substrate that is converted by the enzyme
into a color or fluorescent or electrochemical
signal.
9.Measure the absorbeance or fluorescence or
electrochemical signal (e.g., current) of the plate
wells to determine the presence and quantity of
antigen.

Advantages:
1. It has high specificity since two antibodies are
used and antigen/analyte is specifically captured
and detected.
2. It is suitable for complex samples as antigen does
not require purification prior to measurement.
3. Has good flexibility and sensitivity, since both
direct and indirect detection methods can be
used.
 Sandwich ELISA

Disadvantages:
 Complex Workflow: Sandwich ELISA requires more
incubation steps compared to simpler ELISA formats. This
adds to the overall time and effort required for the assay.
 Need for Specific Antibody Pairs: The assay relies on two
antibodies with different binding sites, one to capture the
antigen and another to detect it. Finding and optimizing
these antibody pairs can be challenging.
 Potential for Cross-Reactivity: If the antibodies are not
perfectly specific, they may bind to unintended antigens,
leading to false positive results or inaccurate
quantification. This requires careful optimization and
selection of antibodies.
 Time and Expense: The additional steps and antibody
optimization required can make sandwich ELISA more time-
consuming and expensive than other methods.
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 then added to the microtitre 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 Competitive ELISA

1.Primary antibody (unlabeled) is incubated with sample
antigen.
2.Antibody-antigen complexes are then added to 96-well
plates which are pre-coated with the same antigen.
3.Unbound antibody is removed by washing the plate.
(The more antigen in the sample, the less antibody will be
able to bind to the antigen in the well, hence
"competition.")
4.The secondary antibody that is specific to the primary
antibody and conjugated with an enzyme is added.
 5.A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.
6.For competitive ELISA, the higher the sample antigen
concentration, the weaker the eventual signal.

Advantages of Competitive ELISA

 High Specificity: Competitive ELISA uses two antibodies to
specifically capture and detect the target antigen, ensuring
accurate results.
 High Sensitivity: This type of ELISA is particularly well-
suited for detecting small antigens or antigens present at
low concentrations.
 Flexibility: Competitive ELISA can be adapted to direct,
indirect, or sandwich formats, providing versatility in
application.
 Robustness: It minimizes the effects of sample dilution and
matrix effects, leading to more reliable results.
 Suitable for Complex Samples: Unlike some ELISA
formats, competitive ELISA doesn't require antigen
purification, making it suitable for complex samples.

 Ease of Use: Competitive ELISA is relatively simple to

perform and can be adapted to various detection methods.
 Rapid Results: Competitive ELISA can be faster than other
ELISA techniques due to fewer steps.

ELISA Result Interpretation:

ELISA results may be interpreted quantitatively,
qualitatively or semi-quantitatively. In a quantitative assay, a
serial dilution of a known standard is used to enable the
generation of a standard curve, normally of optical density
(OD) versus concentration. From this, the precise quantities
of target in the unknown samples can be calculated.
In a qualitative assay, data will still normally be collected
numerically from the use of a microplate reader, but results
will be interpreted as positive or negative by comparison to
blank and/or negative wells with no standard curve.
Semi-quantitative assays also collect numerical data
without the use of a serial dilution or standard curve.
However, the inclusion of both negative and positive
standards, often high and low positives, facilitates the
relative comparison of levels between the wells of unknown
samples to those of known status. With the use of these
standards to monitor assay reproducibility, a cut-off value
may be set with results over the threshold determined to be
positive and those below negative. Some assays may also
incorporate an “amber zone”. For samples whose values fall
in this region, retesting or further investigation is
recommended.
While a quantitative assay may be desirable in some
settings, the inclusion of a full standard curve on each plate
takes up valuable wells. Therefore, a trade-off exists
depending on the information the user hopes to obtain from
the assay result.
Like many assays, the results obtained from an ELISA are
rarely likely to be 100% accurate, with both false positive
and false negative results a possibility. Consequently, most
tests will be associated with measures of sensitivity and
specificity; the closer they are to 100%, the better the test.
There are many factors that can impact these values such
as:
High background from non-specific binding or cross-
reactivit, Poor affinity of antibodies for their
targets ,Suboptimal assay conditions, Sample condition and
complexity.
ELISA Result
ELISA Applications:
Since its conception, this flexible and inexpensive test has
found many uses and continues to do so. Some of these
applications are highlighted below.
Infectious disease diagnostics : The ELISA is particularly
popular in diagnostic testing where is can be applied to the
detection of both the infectious agents themselves and the
antibody response they induce in the host, depending on the
assay conformation chosen. Consequently, ELISAs can be
useful in both identifying infected individuals that may pose
an infection threat or require treatment and in
epidemiological monitoring to identify recovered
individual’s that have previously been infected. Some
chronic infections, particularly where infectious agent load
rises and falls, can be hard to detect if relying purely on the
detection of the infectious agent. However, a sustained
antibody response may be detected8 in these individuals
even when the microbe itself cannot, helping to pinpoint
these individuals.
The first universal test for human immunodeficiency virus
(HIV),9 developed in 1985, was an ELISA to detect human
serum cystatin C. Many other diseases can be diagnosed in
both humans and animals using an ELISA, including dengue
fever,10 hepatitis B,11 SARS-CoV-2,12, 13 Streptococcus
equi8 and Escherichia coli.14
Detection of food allergens : Allergens19 can be
potentially fatal if ingested by affected individuals, so it is
vital that any foods declared free from certain allergens,
such as peanuts,20 are exactly that. The ELISA provides
excellent sensitivity for this purpose, down to parts per
million (ppm) in some cases, and is able to cope with some
food types, such as oils and milks, that alternative methods
like PCR can struggle with. However, some food processing,
such as heating which can change the allergen’s
conformation, can negatively impact21 detection by ELISA.
Biosimilars and biopharmaceutical testing : With the
development of biosimilar drugs, there is a need to prove
their similarity to the reference drugs they seek to emulate.
Here too ELISAs have found utility22 in determining factors
such as total drug concentration and detecting impurities.
Cancer biomarker testing : Cancer biomarkers23 are often
detectable in blood samples. Therefore, ELISA testing can be
utilized as a non-invasive method with which to detect and
quantify known biomarkers.
Forensic drug testing : ELISAs can be used by toxicologists
to screen forensic samples for traces of drugs of abuse24
including amphetamines, cocaine, opiates, methadone,
benzodiazepines and cannabinoids. While liquid samples,
such as urine or blood are easier, it is also possible to
process solid samples, such as hair, for ELISA analysis.

Pregnancy testing : Human chorionic gonadotropin (hCG),

a hormone produced in pregnancy,25 can be detected by
ELISA in urine samples.
Autoimmune disease : Markers indicative of autoimmune
diseases such as rheumatoid arthritis26 and lupus27 may be
detected and quantified by ELISA.

Advantages of ELISA
ELISA (Enzyme-Linked ImmunoSorbent Assay) offers several
advantages, including high sensitivity and specificity, ease of use,
and cost-effectiveness. It's widely used for both quantitative and
qualitative analysis, making it a versatile tool in various
fields. ELISA tests are also known for their speed and ability to be
automated, further enhancing efficiency.
Here's a more detailed look at the advantages:
1. High Sensitivity and Specificity:
ELISA can detect even low concentrations of target molecules
with high accuracy, ensuring reliable results due to the strong
binding between antibodies and antigens. This high sensitivity is a
key feature that allows for the detection of small amounts of a
specific substance.
2. Versatile and Wide Range of Applications:
ELISA is used in diverse fields like clinical diagnostics, drug
discovery, environmental monitoring, and food safety testing. Its
versatility makes it applicable for a wide array of analytes and
sample types.
3. Quantitative and Qualitative Analysis:
ELISA enables both quantitative measurements of analyte
concentrations and qualitative analysis for the presence or
absence of specific targets. This allows for precise determination
of analyte levels or simply confirming the presence of a specific
substance.
4. Easy to Perform and Relatively Cost-Effective:
ELISA protocols are straightforward, and the required equipment
is relatively inexpensive, making it accessible to many
researchers and laboratories. The ease of automation also
contributes to efficiency and cost-effectiveness.
5. Safe and Eco-Friendly:
ELISA assays are generally safe and eco-friendly because they
don't require radioactive substances or large amounts of organic
solvents. This is a significant advantage compared to methods
like radioimmunoassay (RIA).
6. High Throughput:
ELISA can be performed in a high-throughput manner, allowing
for simultaneous testing of multiple samples, which improves
detection efficiency and saves time and costs.
7. Miniaturization and Lab-on-Chip Integration:
Miniaturization of ELISA with microdevices and integration with
lab-on-chip devices enable testing of small sample volumes with
high efficiency and reproducibility.
8. Flexibility in Assay Types:
 Different ELISA formats (direct, indirect, competitive) offer
flexibility in measuring the same analyte, allowing for optimized
detection strategies.

Limitations of ELISA
ELISA has limitations despite its widespread use, including
the potential for false positives and negatives, labor-intensive
antibody preparation, and the need for refrigerated storage and
transport. Additionally, batch-to-batch variability and cross-
reactivity can affect accuracy, particularly in complex biological
samples. The technique may also lack sensitivity for detecting
certain biomolecules or protein isoforms.
Here's a more detailed look at the limitations:
 False Positives and Negatives: ELISA can produce inaccurate
results, leading to misinterpretations and hindering research.
 Laborious Antibody Preparation: Antibodies, which are crucial for
ELISA, are proteins that require time and effort to prepare, making
the process labor-intensive and costly.
 Refrigerated Storage and Transport: Antibodies are proteins that
need to be kept cold during storage and transport, adding to the
complexity and cost of the process.
 Batch-to-Batch Variability: Different batches of antibodies can
exhibit varying reactivity, potentially affecting the accuracy and
reproducibility of ELISA results.
 Cross-Reactivity: Antibodies may react with other molecules
besides the target antigen, leading to false-positive results.
 Lack of Sensitivity: ELISA may not be sensitive enough to detect
certain biomolecules, particularly at very low concentrations.
 Inability to Distinguish Isoforms: ELISA may not be able to
differentiate between closely related protein isoforms, limiting its
ability to provide precise information.

 Hook Effect: In some cases, high concentrations of an analyte can

lead to a decrease in signal or inaccurately low results, known as
the hook effect.
 Sample Matrix Interference: Certain substances in the sample
matrix can interfere with the ELISA assay, leading to inaccurate
results.
 Enzyme-Labeled Antibody Degradation: Enzyme-labeled
antibodies can degrade over time, impacting the accuracy of the
results.
 Need for Large Sample Volumes: ELISA tests often require
relatively large sample volumes, which can be a limitation when
dealing with precious or limited samples.

References
Key ReferencesLinscott’s Directory. See above.Highly
recommended publication listing sourcesof immunological
reagents, kits, andcells/organisms, including addresses
andphone numbers of commercial suppliers(updated
quarterly)
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ours?
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uIiwicGFnZSI6InB1YmxpY2F0aW9uIn19
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acetylcholine_receptor_antibodies_using_biotinylated_a-
bungarotoxin?
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