Immunoassays

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Immunoassays 1
 Detect Small and Large Analytes
Accurately & Efficiently
Enzo offers hundreds of ELISA kits in both immunometric and
competitive assay formats. As scientists and manufacturers of
kits, we understand the critical nature of your research. Each en-
zyme-linked immunosorbent assay is put through rigorous testing
to ensure high precision, accuracy, sensitivity, and specificity. You
can be confident that you will obtain reproducible results, day-af-
ter day, and lot-after-lot.

What are the Differences Between ELISA Assay Types?

10 Tips for Successful ELISA Assays

In-depth Insights on ELISA:

• %CV in ELISA: How to reduce them and why

they’re important

• Dilutional linearity, parallelism, spike-and-recovery

in ELISA: How to QC your results?

• How to validate sample dilutions and achieve

linearity in new sample types

Immunoassays 2
 What are the Differences Between ELISA
Assay Types?

Morgan Mathieu
Applications Scientist Manager

Overview of ELISA Technology

Enzyme-linked immunosorbent assay (ELISA) is a method allowing the quantification of

a desired marker in a biological sample. The marker can be an antibody, a hormone, a
peptide, or a protein. The quantification of a specific marker using an ELISA-based meth-
od can be very advantageous when compared to a more qualitative or semi-quantitative
method like Western blotting. ELISA is faster, highly sensitive, high-throughput, reproduc-
ible, and flexible with the ability to analyze a variety of different sample types of diverse
origins.

Western Blot ELISA

Quantitative Semi Yes

Sensitivity µg to ng ng to pg

Time 1 day (minimum) 2-3 hours

Throughput Low High

Sample Types Lysates or purified sample Various (lysates, tissues, plasma,serum,urine etc.)

Consistency Low High

Immunoassays 3
 ELISA can also serve as an orthog-
onal assay format to other methods
such as activity assays or Western
blotting to further support and en-
hance previously reported research
results. Below is an example of
how a p62 ELISA kit can be used
to monitor the induction of macro-
autophagy. MDA-MB-231 human
breast cancer cells were treated
with 2µM of witharefin A (WA), an
autophagy-inducing drug or vehi-
cle. Cells were harvested 6 and 12
hours post-treatment, lysed, and
analyzed with our p62 ELISA kit,
and for p62 and LC3-II by Western
blot. Drug treatment correlated with the induction of autophagy as
indicated by the decrease in p62 levels (A and B) and by elevation
of LC3-II levels (C).

The essential components of ELISA are threefold: an antigen to

detect and perhaps quantitate, a specific antibody to this antigen,
and a system to measure the amount of antigen in a given sample.
The crucial component of ELISA-based detection and quantifi-
cation is the specificity of the interaction between the marker of
interest and the antibody. Enzo Life Sciences’ catalog of nearly
300 ELISA kits includes sensitive, specific, and reliable assays for
relevant markers of bioprocess, heat shock response, inflamma-
tion and immune response, oxidative stress, signaling pathways,
steroid and peptide hormones, and much more. Below, we have
highlighted commonly used ELISA assays and their use to deter-
mine which is suitable for your research needs.

Immunoassays 4
 Direct ELISA
This was the ELISA originally developed by Perlmann and Engvall. The surface of the plate is coated
directly with the sample. An enzyme-tagged antibody enables its measurement. Incubation is followed
by washing, which removes the unbound antibodies from the medium. The appropriate substrate is then
added to the medium, producing a signal directly proportional to the amount of antigen in the sample. This
correlation can be used to extrapolate the concentration of antigen in an unknown sample from a standard
curve. Direct ELISA is suitable for determining the amount of high molecular weight antigens. Direct ELISA
is considered to be the simplest type of ELISA. Fewer steps are required making it considerably faster than
other types of ELISA. Cross-reactivity of the secondary antibody is also eliminated. It is, however, relatively
rare as the direct labeling of primary antibodies is time-consuming, expensive, and may adversely affect
the immunoreactivity of the antibody with the targeted antigen.
AP- or HRP-antibody
conjugate 1. Wash
2. Add substrate
3. Incubate
Read plate

Well coated with antigen

Indirect ELISA
Indirect ELISA is a two-step binding process involving the use of a primary antibody and a labeled sec-
ondary antibody. In this method, the primary antibody is incubated with the antigen-coated wells. Next,
a labeled secondary antibody that recognizes the primary antibody is added. This secondary antibody is
often a polyclonal anti-species antibody. A wide variety of labeled secondary antibodies are readily avail-
able. A substrate is then added to the well to produce a signal amplification. This method is commonly
utilized to diagnose infection by bacteria, virus, or a parasite and quantify antibodies against this foreign
antigen. Indirect ELISA detection is versatile as different visualization markers can be used with the same
primary antibody. Since more than one labeled antibody can be bound per antibody target, indirect ELISA
is deemed to be highly sensitive and more flexible than direct ELISA. However, cross-reactivity and a
non-specific signal may occur with the secondary antibody.

AP- or HRP-antibody
conjugate
Sample 1. Wash
antibody 2. Add substrate
3. Incubate
Read plate
1. Wash

Well coated with antigen

Immunoassays 5
 Immunometric/Sandwich ELISA
Immunometric assays, also known as sandwich ELISAs, use two antibodies specific to the antigen to
capture or “sandwich” antigens in the well for detection. Immunometric assays exhibit a direct correlation
between antigen concentration and substrate response. Immunometric assays typically employ a capture
antibody coated on the plate to bind the antigen of interest. During a second incubation, the antigen is
bound by a second detection antibody that is also specific to the antigen. The detection antibody can either
be bound by a secondary antibody-enzyme conjugate, or the detection antibody itself is enzyme-conju-
gated. When chromogenic substrate is added to the assay to develop color, samples with high antigen
concentration generate more signal than those with low antigen concentration, producing a signal directly
proportional to the amount of antigen in the sample. This correlation can then be used to extrapolate the
concentration of antigen in an unknown sample from a standard curve. This type of assay is generally used
for low-to-high molecular weight proteins, such as Endothelin-1 (e.g. Endothelin-1 ELISA kit), HSP70 (e.g.
AMP’D HSP70 high sensitivity ELISA kit), or Survival Motor Neuron (e.g. SMN ELISA kit). Immunometric
ELISAs are highly specific as it relies on a pair of antibodies for capture and detection. They are also con-
sidered to be compatible with a variety of complex samples without the pre-requisite of sample extraction
prior to the analysis.

Immunoassays 6
 Competitive ELISA
In competitive enzyme immunoassays, the antigen in a sample competes for limited antibody binding sites
with antigen conjugated to a reporter enzyme. This produces an inverse relationship between antigen con-
centration and substrate turnover. Competitive ELISAs typically use a single antibody to a low molecular
weight antigen, generally less than 10,000 Daltons. During incubation, samples with high antigen content
result in unlabeled antigen being bound in greater amounts than conjugated antigen. When chromogenic
substrate is added to the assay to develop color, samples with a high antigen concentration generate a
lower signal than those containing low antigen concentration, yielding the inverse correlation between an-
tigen concentration in the sample and color development in the assay. This relationship can then be used
to extrapolate antigen concentration in an unknown sample from a standard curve. This type of reaction is
one of the few methods possible for low molecular weight antigens with a limited number of epitopes or
antibody-binding sites, such as small molecules (e.g. Direct cAMP ELISA kit), peptides (e.g. Oxytocin ELISA
kit), and steroids (e.g. Corticosterone ELISA kit).

“ For decades, Enzo’s ELISAs have been trusted by researchers.

We have long been a manufacturer of consistent, reliable,
functional immunoassays that deliver relevant results at a
cost-effective price per sample. ”

Jonathan Weinreich
Product Manager

Immunoassays 7
 Successful Research Tips for ELISA

Erica Brooks
Scientist

Enzo provides over four decades of experience in the development of kits, biochemicals,
and probes. As Scientists Enabling Scientists™, we realize the value in providing relevant
information to our customers—from discovery to development. We are happy to provide
simple but useful tips for improving daily tasks as well as the overall quality of your re-
search. With this in mind, here is a list of tips for achieving high quality ELISA data.

Immunoassays 8
 1. Consistency is Key to Any Assay
While consistency may seem like a given-in and an obvious goal, there are small assump-
tions that can innocently affect consistency between plates run in an assay. Often, one
of those small assumptions is that lot mixing, in order to conserve reagent, is safe to do.
Actually, you should not ever mix components between kit lots. Each assay is lot-specific
and designed to give you a defined level of performance when used before the assigned
expiry date. If you mix lots, you may inadvertently introduce a reagent that may be de-
graded resulting in a less than optimal assay or ineffectual results. Lot-to-lot consistency
between assay runs may also be ensured by consistently handling the assay reagents,
following the protocol precautions each time, and making sure to adhere to the incubation
times provided. Essentially, consistency should be introduced anywhere and everywhere
possible for the best and most correlative data. A break in consistency could mean less
accurate results for one plate versus another and when you are using samples of the same
source/study, this ultimately can lead to conflicting, hard-to-explain data.

2. Strive for Reproducibility

For ELISA users, reproducibility will lend itself to reliability. Reproducibility can be affect-
ed by mishandling or inconsistent preparation of the kit reagents or even samples. It is
important to make sure that all kit reagents are at room temperature for approximately 30
minutes before every assay. Many of the reagents contain temperature-dependent com-
ponents that may come out of solution when cold. Using the reagents at room tempera-
ture ensures that you have consistent binding kinetics and consistent color development
from one assay to the other. Temperature changes can affect assay behavior, which in turn
can lead to a lack of reproducibility in your data. Striving for reproducibility is also vital
when it comes to handling samples. Samples should always be managed and handled
by researchers in a very consistent manner. This can be done by introducing consistent
sample collections protocols, minimizing freeze-thaw cycles, and optimizing dilution for
your sample group in the assay ahead of time, so that you can be confident that a majority
of your samples should be falling in the most linear portion of the standard curve.

Immunoassays 9
 3. Ensure Assay Accuracy
Increase accuracy by making sure you have duplicates for all points in the assay including
background, standard, and sample wells. It is simply good lab practice to run replicates
for all points; this will allow you to immediately spot any obvious outliers that might affect
your average. For example, if you had an inadvertent pipetting issue with your n=1 of a
sample well in your assay, it would definitely affect the OD, which in turn will affect the
average calculated leading to an inaccurate interpolation from the curve for that particular
sample. More accurate data is, of course, vital to all researchers so that they may have
absolute confidence in their data and can more assertively align their data with other sim-
ilar, published findings.

4. Avoid Contamination
Having low coefficient of variability (%CV) between your sample replicates is key to a suc-
cessful and well run assay. Keep %CV between duplicates low by using fresh pipet tips
for each addition to the assay. New tips can prevent cross-contamination between wells,
which in turn can keep the background and %CVs low and increase data reliability. Also,
it is not recommended to pour reagent from a reservoir back in the original bottle as this
can inadvertently cause contamination as well. Not everyone takes the time to calculate
or review their %CVs for their assay but it can be quite helpful in gauging consistency and
reliability.

5. Produce Reliable Standard Curves

For the most reproducible standard curves, be sure to serially dilute the curve using the
range indicated in the assay manual. Adding points to either end of the curve will not
increase sensitivity and will not allow the kit to detect higher concentrations as well. The
upper and lower asymptotes of the curve are limited by the unique characteristics of the
antibodies used to yield binding to the plate in the assay and expanding the curve will not
change such limitations. Also, keep in mind that it is important to make fresh standard
curves for every assay/plate. Most assays indicate a suggested time limit in which freshly
prepared standard curves should be used. It is vital to follow all recommendations and
precautions surrounding standard curve preparation.

Immunoassays 10
 6. Guarantee Accurate Sample Analysis
For the most accurate sample analysis, it is very important for each end user to optimize
dilution for their unique samples for the levels of the biological marker being detected can
vary as can potential matrix interference effects. It is, therefore, important to not only make
sure that you are falling into a safe place on the standard curve with your sample popula-
tion, but it is also important to make sure you have dilutional linearity using your sample
dilution. Even if there is a recommended dilution ratio for a sample matrix that a researcher
may be using, it is still crucial for each researcher to optimize their dilution ratio for their
samples using the kit protocol recommendations only as a guide. Sometimes extraction,
additional dilution, etc. could be required for some samples and only by optimization of
the dilution can this potential requirement be pre-determined. Some researchers under-
estimate the importance of this optimization process and in some instances, end up with
sample data that is not linear, is falling off the curve, or is too variable rendering the data
essentially unusable.

7. Be Sure to Include All Background

and Control Wells
To make sure your assay worked well and to allow for appropriate troubleshooting should
an issue arise, it is important to include all of the suggested background wells in your
assay. Some assays may call for multiple control wells or background wells and they all
serve a slightly different purpose. If you are unclear about what certain background wells
and control wells are used for in an assay, be sure to reach out to technical support for
clarification.

Did You Know?

All ELISA protocols are available
for download from our website.
Find them directly on the given
product page.

Immunoassays 11
 8. Use the Provided/Recommended Diluents
and Buffers
For the best results, you will want to use the recommended diluents only for your standard
curve and sample preparation. During assay development, manufacturers identify optimal
conditions for not only curve performance but samples detection as well. Detergents, ion-
ic strength, pH etc. can all play a crucial part in finding the most conducive environment
for a happy assay. Following the diluent recommendations is, therefore, always highly
recommended for the best results possible.

9. Follow the Recommended Incubation Times,

Temperatures and Conditions
In order to achieve complete color development, be sure to adhere to the incubation
guidelines indicated in the assay manual. Temperature, time, and shaking (if required or
discouraged) can all greatly affect the status of the assay if they are not followed. Cutting
incubation times short can prevent everything from getting to equilibrium before moving
to the next step in the assay and large temperature changes can also affect binding kinet-
ics. If shaking is suggested, it is always best to follow that guideline as well for in some
assays it does make a bid difference in the resulting OD’s. Accuracy and consistency of
handling during plate incubation really can help enhance assay performance.

Immunoassays 12
 10. Obtain Optimal Detection and Data Analysis
For optimal detection, read the plate at the
wavelength indicated in the assay manual
and use the suggested data analysis soft-
Do You Need Help?
ware. We suggest using a 4Parameter (4P)
Do not hesitate to get in touch
Logistic Curve Fit for data analysis in order
with our Technical Support
to obtain the most accurate sample data.
experts. They are here to help!
Most plate readers do have this 4P soft-
Visit our website:
ware available already. If you are unsure of
www.enzolifesciences.com/support,
whether or not your plate reader has a 4P
or email: techserv-usa@
software, we encourage you to reach out to
enzolifesciences.com
your plate reader manufacturer to confirm.
If you have this software, they can walk you
through accessing it and setting up your
plate template for plate read and data anal-
ysis that will happen immediately following
the read.

In-Depth Insights on ELISA

• %CV in ELISA: How to Reduce Them and Why They’re Important

• Dilutional Linearity, Parallelism, Spike-and-Recovery in ELISA: How to QC Your Results?
• How to Validate Sample Dilutions and Achieve Linearity in New Sample Types

Immunoassays 13
 %CV in ELISA: How to Reduce Them and
Why They are Important

Brian Conrad
Sales Support Specialist

Enzyme-linked immunosorbent assay (ELISA) is a method allowing the quantification

of a desired marker in a biological sample. Achieving high quality quantification data
by ELISA can be very advantageous when compared to more qualitative methods like
IHC and Western blotting. For ELISA users, having a low coefficient of variability (CV
or %CV) between sample replicates is crucial in demonstrating an assay was well-run
and the resultant data is precise and accurate. Reliable assay results are assessed by
standardized measures such as coefficient of variability.

σ
The coefficient of variability is a dimensionless numerical ra-
tio used to describe the level of variability within a population
independently of the absolute values of the observations. In
Cv = –
μ
statistical analysis of numerical data, if your absolute values
are similar, sample populations can be assessed by using
standard deviations; when absolute values vary, you must
consider using a more standardized approach such as %CV, Figure 1. General mathematical
formula for Coefficient of Variability
to assess the precision of a laboratory technique. CV is cal- (%CV).
culated by dividing the standard deviation (σ) of a set of mea-
surements by the mean (µ) of the set which is then expressed
as a percentage of variation to the mean (Figure 1).

Immunoassays 14
 In ELISA data interpretation, %CV can highlight inconsistencies among sample repli-
cates which is demonstrated in the data as variation among Optical Density (OD) read-
outs post-assay. These directly reflect the performance of the assay in the hands of
the end-user. There are two types of %CVs that are used to express the precision of
immunoassay results: intra-assay CV and inter-assay CV. Intra-assay CV is a measure of
the variance between data points within an assay, meaning sample replicates ran within
the same plate. Inter-assay CV is a measure of the variance between runs of sample
replicates on different plates that can be used to assess plate-to-plate consistency. As
a general guideline, to gauge the overall reliability of your immunoassay results, inter-as-
say %CV should be less than 15% while intra-assay %CV should be less than 10%.

It is important to identify the causes of high %CV in ELISAs. Human technical error can
play a role such as inaccurate pipetting technique, splashing of reagents between wells,
drying out of the wells, inconsistent sample handling (variability due to freeze-thaw cy-
cles among samples), and use of differing filter settings to start. Additionally, high %CV
can be the result of machine error such as usage of uncalibrated automated machine
pipettes, uncalibrated plate readers, and inappropriate plate reader software settings
to analyze samples in wells. Lastly, plate, sample, and reagent contamination can lead
to a high %CV. Cross-contamination between reagents can occur from handling er-
rors leading to bacterial or fungal contamination of samples and reagents derived from
compromised sterility. To reduce %CV between your sample replicate ODs would mean
to reduce the incidence of these common sources of error. As a general guideline, to
gauge the overall reliability of your immunoassay results, inter-assay %CV should be
less than 15% while intra-assay %CV should be less than 10%.”

In regards to both inaccurate pipetting and contamination, using fresh pipette tips for
each addition is one common practice. Discarding old tips for new tips between each
addition can prevent cross-contamination between wells which can keep the back-
ground and %CV low amongst your sample replicates. Also, it is not recommended to
pour excess reagent from a reservoir back into the original bottle as this can uninten-

Immunoassays 15
 tionally contaminate your stock solution which will be reflected in your readouts and
may produce a high %CV. In regards to technique, pre-wetting pipette tips 2-3 times
in the solution that is to be pipetted also can help to improve %CV. If using mechanical
air-displacement pipettes, practicing proper technique is important to minimize %CV.
Some general practices include holding the pipette vertically and not at an angle, aspi-
rating slowly and smoothly, having the tip touching the vessel when withdrawn to avoid
extra liquid outside the tip, and making sure the tip is placed just under the surface of
the liquid in the reservoir when dispensing. Consistency with your aspiration point in the
reservoir across duplicates may help to reduce %CV. Regular performance checking by
the end-user or a service technician should be prioritized as well to ensure both me-
chanical and machine pipettes are calibrated. This can go a long way in reducing %CV.
Regular re-calibration should also be a good practice that extends to other instruments
including the plate washer and reader to help reduce variation from machine error that
will impact CVs.

Group: Unknown Run 1

Sample Wells Values Net OD %Bound Result Mean Result Std. Dev. CV%

100 G4 0.541 0.543 58.68 18.449 17.172 1.806 10.5

H4 0.565 0.567 61.276 15.894
102 E4 0.531 0.533 57.599 19.626 21.196 2.22 10.5
F4 0.507 0.509 55.003 22.766
103 C4 0.399 0.401 43.321 45.333 41.39 5.576 13.5
D4 0.428 0.43 46.458 37.447
141 A4 0.451 0.453 48.945 32.312 31.709 0.852 2.7
B4 0.457 0.459 49.594 31.106
150 G3 0.471 51.109 28.482 26.733 2.473 9.3
H3 0.492 0.494 53.38 24.985
151 E3 0.45 0.452 48.837 32.518 34.155 2.315 6.8
F3 0.435 0.437 47.215 35.792
152 C3 0.357 0.359 38.778 60.662 57.035 5.129 9
D3 0.375 0.377 40.725 53.408
157 A3 0.362 0.364 39.319 58.529 60.036 2.132 3.6
B3 0.355 0.357 38.561 61.543
159 G2 0.425 0.427 46.133 38.184 35.664 3.563 10
H2 0.447 0.449 48.513 33.144
Table 1. An example of high and low intra-assay %CV values from data using Aldosterone EIA Kit. Bolded in red are high% CVs (over 10) seen
amongst sample replicates. Bolded in green are low %CV (underneath or at 10) seen between sample replicates.

Immunoassays 16
 Dilutional Linearity, Parallelism, Spike-and-
Recovery in ELISA: How to QC Your Results?

Michael Yan
Sales Support Specialist

Why is ELISA Validation Important?

The enzyme-linked immunosorbent assay (ELISA) provides a methodical quantification

of specific analytes through antibody-analyte binding affinity and colorimetric develop-
ment. For samples validated by ELISAs, high accuracy and reliability in analyte quan-
tification is the expectation. However, samples that have not been validated may not
display similar degrees of reliability. Differing antibody-analyte binding characteristics
and/or interfering effects can be inherent to the sample matrix. This exemplifies the crit-
ical importance of ELISA validation for accurate analyte measurements in new samples.
Validation results may provide verification of sample compatibility and possible sources
of interference with future implications on assay optimization.

Dilutional Linearity
Dilutional linearity determines whether sample matrices spiked with detection analyte
above the upper limit of detection can still provide reliable quantification after dilution
within standard curve ranges. Samples displaying little to no deviation in concentration
after factoring dilutions are known to display linearity. This confirms a high degree of ac-
curacy and shows flexibility of the assay at varying dilutions. This also clarifies whether
low concentration samples with no dilutions exhibit similar concentrations against an
equivalent sample concentration generated from a high concentration spike dilution.
While different considerations can be made for conducting dilutional linearity experi-
ments, the general principles remain the same.

Immunoassays 17
 An example protocol:

• Spike or introduce a known quantity of standard analyte into sample matrices above
the upper detection limit of the standard curve. Commonly used matrices are substi-
tute matrices that attempt to replicate actual test sample matrix or sample matrices
at free or low concentrations of detectable analyte.
• Conduct 1:2 serial dilutions of spiked sample matrix until the lower limit of quantifi-
cation of the standard curve is reached.
• Obtain absorbance and calculate mean concentrations only for sample ranges with-
in the lower and upper limit of the standard curve.

Observed concentration at dilution

Percent Recovery = ×100
Predicted spike concentration after dilution

Observed spiked sample value – unspiked sample value

Samples Percent Recovery
displaying = linearity should detect no changes in observed×100
ideal analyte com-
Actual amount spiked in sample
pared to final analyte concentration after factoring dilutions. However, linearity is also
achievable for sample recoveries within 80% - 120% of the expected values. The high-
est dilution of an analyte that displays linearity within this range is often known as the
limit of dilution. Ranges outside of this 20% threshold display poor linearity and indi-
cates possible interferences from the sample matrix and/or standard diluent. Common
contributors to this discrepancy may come from salts, pH, detergents, protein interac-
tions, or other elements that interfere with antibody binding within matrices. This may
necessitate working within optimal dilutions or possible optimization of sample or sam-
ple diluent for better assay compatibility.

Dilution Expected (pg/ml) Observed (pg/ml) Recovery (%)

Neat --- 390.8 ---

1:2 195.4 194.6 100%

1:4 97.7 105.1 108%

1:8 48.8 67.0 137%

1:16 24.4 27.9 114%

1:32 12.2 12.1 99%

Table 2. A buffer sample containing vasopressin was serially diluted 5 times 1:2 in the kit Assay Buffer and measured in the assay (Catalog
No. ADI-900-017A). The data can be plotted graphically as expected vasopressin concentration versus observed vasopressin concentration.
The line obtained has a slope of 1.0162 with a correlation coefficient of 0.9895.

Immunoassays 18
 Parallelism
Parallelism determines whether actual samples containing high endogenous analyte
concentrations provide the same degree of detection in the standard curve after dilu-
tions. This signifies differences in antibody binding affinity to endogenous analyte and
standard/calibration analyte. Parallelism should generally be performed during valida-
tion of test samples. The key difference between parallelism and dilutional linearity is
that parallelism utilizes samples containing high levels of endogenous levels of analyte
and high concentrations of spiked standard analyte. It is important to use highly con-
centrated samples as true parallelism cannot be detected using sample concentrations
near the lower level of detection. Similar to dilutional linearity, there is not one-set way
of conducting and interpreting tests parallelism. However, the general principles below
hold true for most parallelism tests. An example protocol:

• Identify at least 3 samples that display high concentration of endogenous analyte,

but not exceeding the upper limit of quantification in the standard curve.
• Perform 1:2 serial dilutions using sample diluent until the predicted concentration
falls below the lower limit of quantification of the standard curve.
• Analyze neat and diluted sample OD while factoring in dilution factors. Only use
samples within the standard curve limits.
• Determine mean concentrations of samples with dilutions factored in and calculate
% CV.

%CV within 20-30% of expectations generally display successful parallelism, although

the exact percentage should be decided by end users. Matrices displaying parallel-
ism indicate a comparable selectivity between analyte and antibody from endogenous
sample and standard/calibration analyte. Higher %CV than the usual accepted values
indicates a loss of parallelism and suggests a significant difference in immunoreactivity
between these two analytes. This may be possible due to the presence of post-transla-
tional modifications or unspecified matrix effects affecting analyte.

Immunoassays 19
 Corticosterone (pg/mL) Intra-assay %CV Inter-assay %CV

Low 171 8.0

Medium 403 8.4

High 780 6.6

Low 174 13.1

Medium 415 8.2

High 780 7.8

Table 3: Intra-assay precision was determined by taking samples containing low, medium, and high
concentrations of corticosterone and running these samples multiple times (n=16) in the same assay.
Inter-assay precision was determined by measuring three samples with low, medium and high concen-
trations of corticosterone in multiple assays (n=8). The precision numbers listed represent the percent
coefficient of variation for the concentrations of corticosterone determined in these assays as calculated
by a 4 parameter logistic curve fitting program (Catalog No. ADI-900-097).

Spike/Recovery
Spike/Recovery is often utilized to determine the differences in percent recovery be-
tween sample matrices and standard diluent. The postulated question asks whether the
percent recovery obtained from the standard diluent is identical to the percent recovery
obtained from the natural sample matrix will provide identical percent recovery. Spike/
Recoveries are conducted by spiking a known quantity of standard analyte within the
standard curve to the intended sample testconcentration
Observed matrix and atstandard
dilution diluent. After running
Percent Recovery = ×100
both samples and obtaining thePredicted spike concentration
concentrations, calculateafter dilution
percent recovery.

Observed spiked sample value – unspiked sample value

Percent Recovery = ×100
Actual amount spiked in sample

Immunoassays 20
 Sample Spike Concentration, ng/mL % Recovery Minimum Recommended
Dilution
2 102
Human Serum 1 83 Neat
Extracted
0.5 124
2 101
Human EDTA 1 90 Neat
Plasma Extracted
0.5 112.2
1 89.7
Rat Serum Extracted 0.5 99.6 Neat
0.25 112.4
1 90.9
Mouse Serum Extracted 0.5 105.8 1:2
0.25 115.6
3 102.8
Porcine Serum Extracted 0.6 107.8 1:2
0.12 112.5
5 83.3
Human Saliva Extracted 2.5 98.7 1:2
1.25 108.4
5 100.9
Banana Extracted 2.5 115.7 1:2
1.25 87.6
5 100.1
Plum Extracted 2.5 108.0 1:2
1.25 96.4

Table 4: Purified melatonin was spiked at three concentrations into the minimum recommended dilution
of human, rat, mouse, porcine, and fruit matrices. Matrix background was subtracted from the spiked
values and the average percent recovery for each matrix at the minimum required dilution is presented
below. These results show the tested matrices at the minimum recommended dilution have no obvious
interference with the Melatonin ELISA assay (Catalog No. ENZ-KIT150).

Ideal sample matrices should obtain 100% recovery. However, deviations of 20% or
less are also acceptable. Percent recovery within these ranges suggests a greater con-
fidence of ELISA compatibility with the proposed sample. Recoveries exceeding this
threshold imply potentially significant differences between the natural sample matrix
and the standard diluent. A possible adjustment for improving recovery is to find al-
ternate diluents that align more closely with the proposed sample matrix. Alternatively,
introducing different ratios of sample diluent into the sample matrix may also display
improvements to recovery.

Immunoassays 21
 How to Validate Sample Dilutions and Achieve
Linearity in New Sample Types

Brian Conrad
Sales Support Specialist

Immunoassay Sample Validation Experiments—What is Matrix

Interference?
Enzyme-linked immunosorbent assay (ELISA) is a microplate-based technique frequent-
ly used for detecting and quantifying low-abundance analytes of interest in a variety of
biological samples. The technique depends on two elements: (1) the highly specific anti-
body-antigen interactions measured via development of a colorimetric product generated
by the interaction of an enzyme conjugated to a secondary detection antibody and a com-
plementary colorimetric substrate and (2) accurate comparison of this same interaction
relative to a recombinant protein that is used as a reference standard to generate a stan-
dard curve and interpolate the concentration of analyte present in a biological sample.

In commercially available ELISA kits, a key component needed for accurate measure-
ment of an analyte is performing sample validation for your unique samples and/or sam-
ple types not validated by the kit manufacturer. Samples need to demonstrate robust
detection and quantification by a kit’s antibodies, which can initially be challenging due
to differing antibody-analyte binding characteristics and differences in the endogenous
substances present in the background amongst the analyte of interest that are inherent to
sample matrices. This is otherwise known as sample matrix interference.

These interfering substances are compounds with chemical differences but structural
similarities that cross-react with the antibody targeting the analyte of interest. In this an-
alytical interference, interfering endogenous substances or exogenous contaminants in
your sample matrices can result in false increases or decreases to true analyte concen-
tration levels reflected in optical density (OD). Some examples of protein-rich samples or
samples with potential matrix interference are urine, cell lysates, and blood components
such as serum and plasma.

Immunoassays 22
 Aside from the endogenous substances inherent to the sample and exogenous contam-
inants produced through human error, an end-user may need to assess the compati-
bility of the assay diluent buffering solution in contributing to potential analytical inter-
ference. Interfering components can also be pH-dependent, detergent, organic solvent,
and buffering salt composition and concentration which may dictate buffer compatibility.
Exchanging the buffer may be necessary to resolve the interference in order to detect true
analyte concentrations in your sample. On the other hand, when analyte levels are elevat-
ed significantly above the limit of quantification (LOQ) of the assay, optimizing the dilu-
tion of the sample becomes a strategy to overcome this interference. To ensure accurate
detection and quantification in a sample and mitigate sample matrix effects, an end-user
may need to consider sample validation via spike-and-recovery and linearity-of-dilution
experiments.

Spike-And-Recovery Experiments
In spike-and-recovery experiments, the primary objective is to determine whether ana-
lyte detection is affected by a difference between the assay diluent/buffer used to pre-
pare an end-user’s standard curve and the sample matrix. The sample matrix can be a
neat (undiluted) biological sample or biological sample diluted in assay diluent. This is
accomplished by adding a known amount of the recombinant protein standard (spike)
into the natural test sample matrix and observing its response (recovery) relative to add-
ing the same known amount to the assay diluent. The response (the resulting concen-
tration) is interpolated from the standard curve and OD that resulted due to the addition
of a known quantity of recombinant protein standard. After running the two sets of re-
sponses and calculating concentration from the standard curve, an end-user can cal-
culate percent recovery to identify the percent deviation of the observed spiked sample
value from the observed unspiked sample value relative to the actual known quantity to
determine the compatibility of the assay diluent and sample matrix. An end-user wants
to achieve an identical response for a given amount of analyte of interest in both sample
matrix and assay diluent/buffer. In other words, fully compatible sample matrices should
obtain a 100% recovery because the recovery observed for the spike is identical to the
recovery obtained in both assay diluent and sample matrix. The % recovery may to de-
viate up to 20%. If the recovered value differs significantly from the amount expected
(due to spiking with a known quantity) then this deviation from 100% recovery may sug-
gest the degree of incompatibility/discrepancy of the assay diluent to the sample matrix
in which case adjustments must be made to minimize the deviation. In cases of very
poor sample recoveries, this adjustment may suggest using a different assay diluent
whose composition more closely matches the final sample matrix.

Immunoassays 23
 Linearity-of-Dilution Experiments
Samples that do not exhibit linear dilution at a range of dilutions indicates a matrix compo-
nent is interfering with the accurate detection of the analyte of interest at a given dilution. As
a result, correcting the dilution factor in a dilutional linearity experiment becomes necessary
where the end-user will dilute the sample until the interference is no longer observed. Dilu-
tional linearity experiments are performed to demonstrate that a sample with a spiked con-
centration above the upper limit of quantification can be diluted to a concentration within the
working, standard curve range and still produce an accurate and reliable result. Poor linearity
shown by the hook effect is mitigated by achieving dilutional linearity. The hook effect is seen
as the concentration of analyte begins to exceed the amount of antibody. In this situation,
the dose response curve plateaus and with further increase may start to become negatively
sloped, demonstrating potential matrix interference and reducing the accuracy of quantifying
the true concentration of analyte in a sample matrix as the concentration of analyte begins to
exceed the amount of antibody available. The samples are tested by performing a series of
factored dilutions within the analytical range of the assay using an approved, chosen assay
diluent. When dilutional linearity is seen, the serial dilution scheme will be reflected roughly
in OD change between dilution points in a linear trend or the sample will have roughly the
same calculated concentration after applying the dilution factor to each dilution point. As an
example, if the end-user conducts a two-fold serial dilution, there should roughly be a two-
fold difference in OD between each point. With validated samples where the minimal required
dilution (MRD) is known, this value can ideally be used to as a starting point and a range of
dilutions can be determined to decide if the starting point is appropriate. If the sample has
not been validated then a MRD may need to be identified through titrating a wider range of
dilutions to achieve linearity. Once dilutional linearity is achieved, this confirms a high degree
of accuracy and demonstrates the flexibility of the assay at varying dilutions. Performing
a linearity of dilution experiment can also be done via preparation of several dilutions of a
sample with low level concentration of analyte and then spiking the same known amount
of analyte into each one before testing and assessing assay recovery by comparing the
observed and expected values of non-spiked and/or undiluted samples. Samples detecting
an ideal linearity should not demonstrate changes in
observed analyte concentration relative to final ana-
lyte concentrations after factoring dilutions. Linearity Did You Know?
is achievable for sample recoveries within a range of All ELISA protocols are available
80% to 120% of the expected values. Values that fall for download from our website.
below two times the limit of quantification for the as- Find them directly on the given
say should be avoided in consideration of dilutional product page.
data due to statistical limits at the low end of the as-
say range.
Immunoassays 24
 Highly Cited. Highly Trusted.
Enzo’s ELISA Kits

Accurate and Reliable Analyte Detection You Can Trust, Year After Year
It takes more than just an antibody pair to make an ELISA assay. Building an Reliable Manufacturing Ensures
immunoassay requires screening multiple antibodies, selection of appropriate Consistent Results
standards and conjugates, establishing proper sample prep protocols, and validation
of the assay in relevant matrices. Our expertise delivers reproducible assays that
continue to be cited in peer-reviewed publications by scientists around the world.

Higher Sensitivity for detecting low endogenous levels

Highly Specific with low reactivity with structurally related molecules

Validated with multiple sample matrices from serum and saliva to urine and feces

Trusted with thousands of citations

The graph above demonstrates the robust and

® reproducible nature of the competitive cAMP ELISA
Kit, showing standard curves from 10 kit lots manu-
factured over 10 years.

Life Sciences
scientists enabling scientists™ For Research Use Only.
Not for Use in Diagnostic Procedures
www.enzolifesciences.com/elisa
Immunoassays © 2018 Enzo Life Sciences.
25
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Immunoassays 26