Discover a Clear and Easy Guide to ELISAs
How To Guide
Last Updated: July 1, 2024
(+ more)
Published: April 8, 2024
Credit: iStock
Enzyme Linked Immunosorbent Assays (ELISAs) are the immunoassay of choice when measuring protein levels in biological fluids. They offer unmatched sensitivity, specificity, and accuracy while tolerating harsh buffers or pretreatments.
A wide array of ELISA kits are readily available, each with unique advantages, from direct and indirect to sandwich and competitive ELISAs. To ensure precision and reproducibility it is essential to choose the right kit, master sample preparation and optimize experimental setup.
This guide explores how to get the most from an ELISA and provides the tools to achieve accurate and consistent immunoassay performance.
Download this guide to explore:
- The latest range of ELISA formats
- Optimization strategies to ensure performance and consistency
- Troubleshooting advice when performing ELISAs
ELISA GUIDE
A CLEAR AND EASY GUIDE TO ELISAS
2
Table of Contents
What is an ELISA? ............................................................................3
The Highest Quality ELISAs Available.......................................3
What's at the core of your immunoassay?.............................3
ELISA Types
Direct ELISA.........................................................................................4
Indirect ELISA ......................................................................................4
Sandwich ELISA...................................................................................4
Competitive ELISA...............................................................................5
Why use an ELISA over other techniques?.......................5
ELISA Formats ....................................................................5
Which Immunoassy is Right for You?.................................................5
The Citation Pack Leader: Quantikine® ELISAs.................................6
Quantikine® Colorimetric Sandwich ELISA Kits ...........................6
Quantikine® QuicKit Colorimetric Sandwich ELISA Kits ..............6
Quantikine High Sensitivity Colorimetric Sandwich ELISA Kit ....6
Quantikine IVD Colorimetric ELISA Kits........................................6
QuantiGlo Chemiluminescent Sandwich ELISA Kits....................6
Parameter Colorimetric Competitive ELISAs................................6
The DIY Solution: DuoSet® ELISAs .....................................................7
DuoSet® ELISA Development Systems.........................................7
DuoSet IC (Intracellular) Assay ELISA Development Systems ....7
DuoSet IC (Intracellular) Phospho-specific ELISA Development
Systems ..........................................................................................7
Your Next Generation ELISA: Simple PlexTM Assay
Simple Plex Assays ........................................................................8
New Quantikine QuicKit Colorimetric Sandwich ELISA Kits .............9
Ensuring ELISA Performance and Consistency..................10
Accurate Detection of Natural Proteins ...........................................10
Confirmed Lot-to-Lot Consistency ....................................................10
Precision & Reproducibility:
Providing Confidence in Your Results ..............................................11
Sensitivity: Measuring Proteins at the pg/ml Range ......................11
Linearity Experiments Idenfity False Positive Signals.....................12
What is the Importance of ELISA Controls?.........................13
Positive ELISA Controls .....................................................................13
ELISA Spike Controls.........................................................................13
Negative ELISA Controls ...................................................................13
Sample Preparation.......................................................................14
Sample Collection & Storage............................................................14
ELISA Assay Timelines..................................................................15
Data Analysis: Calculation of Results.....................................16
Calculating concentration of target protein in the sample.............16
Calculating the coefficient of variation............................................16
Best Practices and Techniques ................................................17
Quantikine® ELISA FAQs...............................................................18
Troubleshooting your Quantikine® ELISA..............................20
Duoset® ELISA Assay Optimization..........................................21
Troubleshooting your DuoSet® ELISA .....................................23
ELISA Kits Offerings.......................................................................25
Supplemental ELISA Development Products......................25
3
ELISAs (Enzyme Linked Immunosorbent Assays) are a type of immunoassay that are commonly
used to quantify levels of a specific target within a sample. Samples routinely used in ELISAs
include serum, plasma, cell culture supernates, cell lysates, saliva, tissue lysates, and urine.
ELISAs are usually run in 96-well microplates coated with a capture antibody specific for the
analyte of interest. Upon incubation with experimental samples, standards, or controls, the
target analyte is captured by this antibody. A conjugated detection antibody binds to a different
epitope on the target analyte. A substrate solution is subsequently added to produce a signal
that is proportional to the amount of analyte bound. ELISAs can have different formats.
Descriptions and diagrams of these can be found in the next section.
R&D Systems®, a Bio-Techne brand, has over 30 years of experience designing, testing, and
optimizing immunoassay kits to ensurethehighestlevel ofperformanceinanalytequantification.
We currently offer more than 600 complete, ready-to-use Quantikine ELISA Kits, 1,000 DuoSet
ELISA Development Systems for numerous different analytes and species, including human,
mouse, rat, canine, primate, and porcine, and an automated Simple Plex platform with over
200 assays available. Choosing quality reagents that will lead to results you can trust is one of
the most critical aspects of scientific research.
Your results matter, so what’s inside your immunoassay should too. R&D Systems® antibodies and proteins are the core of every Bio-Techne
immunoassay platform. Our antibodies and proteins are highly specific, manufactured in-house to ensure reproducibility and tested for suitability
on every application we develop. In addition, the proteins used for the immunoassay standard and as antibody immunogens are typically fulllength, recombinant proteins that are confirmed to be biologically active. The ensures that our standard closely mimics the natural protein and
that the antibodies will recognize the native for of the analyte.
What is an ELISA?
The Highest-Quality ELISAs Available
What’s at the core of your immunoassay?
• Most Referenced ELISA Manufacturer
• Flexible Formats available
• Extensive Analyte Selection
• Rigorous Validation Testing
• Extensive Quality Control Testing
• Long-term Consistency
• Bulk Packaging Available
4
ELISA Types
The four main types of ELISAs are indirect, direct, sandwich, and competitive. Each type of ELISA has its own advantages and disadvantages.
Indirect ELISA
An indirect ELISA is similar to a direct ELISA in that an antigen is
immobilized on a plate, but it includes an additional amplification
detection step. First, an unconjugated primary detection antibody is
added and binds to the specific antigen. A conjugated secondary
antibody directed against the host species of the primary antibody is
then added. Substrate then produces a signal proportional to the
amount of antigen bound in the well.
Sandwich ELISA
Sandwich ELISAs are the most common type of ELISA. Two specific
antibodies are used to sandwich the antigen, commonly referred to as
matched antibody pairs. Capture antibody is coated on a microplate,
sample is added, and the protein of interest binds and is immobilized
on the plate. A conjugated-detection antibody is then added and binds
to an additional epitope on the target protein. Substrate is added and
produces a signal that is proportional to the amount of analyte present
in the sample. Sandwich ELISAs are highly specific, since two
antibodies are required to bind to the protein of interest.
Direct ELISA
In a direct ELISA, an antigen or sample is immobilized directly on the
plate and a conjugated detection antibody binds to the target protein.
Substrate is then added, producing a signal that is proportional to the
amount of analyte in the sample. Since only one antibody is used in a
direct ELISA, they are less specific than a sandwich ELISA.
When to Use
Assessing antibody affinity and specificity. Investigating blocking/
inhibitory interactions
Advantages
• Fast and simple protocol
Disadvantages
• Less specific since you are only using 1 antibody.
• Potential for high background if all proteins from
a sample are immobilized in well.
When to Use
Measuring endogenous antibodies
Advantages
• Amplification using a secondary antibody
Disadvantages
• Potential for cross-reactivity caused by secondary antibody
When to Use
Determining analyte concentration in a biological sample
Advantages
• Highest specificity and sensitivity
• Compatible with complex sample matrices
Disadvantages
• Longer protocol
• Challenging to develop
5
ELISA Formats
Why use an ELISA over other techniques?
Which Immunoassay is Right for You?
Competitive ELISA
Competitive ELISAs are commonly used for small molecules, when the
protein of interest is too small to efficiently sandwich with two
antibodies. Similar to a sandwich ELISA, a capture antibody is coated
on a microplate. Instead of using a conjugated detection antibody, a
conjugated antigen is used to complete for binding with the antigen
present in the sample. The more antigen present in the sample, the
less conjugated antigen will bind to the capture antibody. Substrate is
added and the signal produced is inversely proportional to the amount
of protein present in the sample.
KIT Quantikine ELISA Quantikine QuicKit Quantikine HS ELISA DuoSet ELISAs Simple Plex Assays
Format 96-well plate 96-well plate 96-well plate Flexible Cartridge
Benefit • Most Published
• Low CVs
• Fastest Plate-based
ELISA • Highest Sensitivity
• Economical
• Largest Menu
• Flexible
• Hands Free
• Sensitive
Sample Volume 10-200 mL 50 mL 10-200 mL 100 mL 2.5-25 mL
Number of Analytes 1 1 1 1 Up to 8
Assay Time 3-5 Hours 90 minutes 4-4.5 hours 20 hours 90 minutes
Pre-coated Yes Yes Yes No Yes
When to Use
Determining concentrations of small molecules and hormones
Advantages
• Ability to quantitate small molecules
Disadvantages
• Less specific since you are only using 1 antibody
• Requires a conjugated antigen
There are many different immunoassay platforms available to measure protein levels in biological fluids. ELISAs are preferred in many cases due
to their sensitivity, specificity, accuracy, and ability to tolerate harsh buffers or pretreatments. Comparing an ELISA to a Western blot, sandwich
ELISAs use 2 specific antibodies rather than one and allow for completely quantitative results, while a Western blot can see non-specific bands
and are semi-quantitative at best. An advantage of ELISAs over different multiplexing platforms is the ability to customize the assay for the target
analyte and not having to worry about interference caused by many other antibodies and proteins working together. The diluents used in our
Quantikine ELISA kits are fully optimized to achieve the best performance for that analyte in complex sample matrices. The potential of observing
cross-reactivity or interference is minimized and you can push the sensitivity limits with this technique.
Analyte from sample
HRP - conjugated recombinant analyte Analyte from sample
HRP - conjugated recombinant analyte
6
Quantikine® HS (High Sensitivity) Colorimetric Sandwich
ELISA Kit
Quantikine HS ELISA Kits are complete assays generally used when very low levels of the target protein are
expected. These kits utilize distinct protocols to achieve their impressive levels of analyte detection. Two
different amplification systems are available.
NEW
Quantikine® QuicKit Colorimetric Sandwich ELISA Kits
QuicKit ELISAs allow for rapid quantitation of target protein in only 90 minutes. With a shortened protocol
and only one wash step, these ready-to-use kits allow you to accomplish more in your day, while getting
the same quality of a Quantikine ELISA.
Quantikine® IVD Colorimetric ELISA Kits
Quantikine IVD (in vitro diagnostic) ELISA Kits are complete ready-to-run ELISA kits that have been 510(k)
cleared for in vitro diagnostic use.
QuantiGlo® Chemiluminescent Sandwich ELISA Kits
QuantiGlo ELISA Kits use a chemiluminescent substrate for analyte detection and require a luminometer for
output reading. These kits have a broad dynamic range in comparison to standard colorimetric immunoassays.
Parameter Colorimetric Competitive ELISAs
Parameter ELISA kits are complete microplate-based assays designed to accurately measure the levels of
small molecules using the competitive ELISA format.
The Citation Pack Leader: Quantikine® ELISAs
Kit Components
• Pre-coated 96-well Microplate
• Conjugated Detection Antibody
• Calibrated Immunoassay
Standard
• Assay Diluent
• Calibrator Diluent(s)
• Wash Buffer
• Color Reagent A and B
• Stop Solution
• Plate Sealers
Quantikine Colorimetric Sandwich ELISA Kits
Quantikine ELISA Kits are complete, ready-to-use kits that represent the gold standard in single analyte
detection. Kits are available for measuring a wide range of molecules including cytokines, chemokines,
growth factors, proteases, and more.
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Learn more | rndsystems.com/elisas
The DIY Solution: DuoSet® ELISAs
DuoSet Development Systems
DuoSet ELISA Development Systems contain the basic components required to develop a sandwich
immunoassay for accurately measuring analytes in biological fluids. When complete kits are not an option,
DuoSet ELISA Development Systems are an economical alternative to buying separate antibodies and
protein standards.
DuoSet IC (Intracellular) Assay Development Systems
DuoSet IC ELISA Reagents are sensitive and convenient assays used to measure intracellular protein levels
in cell lysates.R&D Systems DuoSet IC assays measure kinases, apoptosis-related and genotoxic stress
proteins, heat shock proteins, and more. These signal transduction assays make an excellent alternative to
Western blot, especially when used in combination with R&D Systems DuoSet IC Phospho-specific ELISAs.
DuoSet IC (Intracellular) Phospho-specific
ELISA Development Systems
The DuoSet IC Phospho-specific ELISA format is a sensitive and convenient phosphorylation assay for use
with cell lysates. Extensive validation work is done in-house to ensure specificity. These signal transduction
assays are an excellent alternative to Western blot, especially when used in combination with DuoSet IC
ELISAs designed to measure the levels of total protein.
Kit Components
• capture antibody
• biotinylated detection antibody
• mass-calibrated standard
• streptavidin-HRP
• detailed protocol
B
B
P
Accelerate your Biomarker discovery and qualification
with the full support of the R&D Systems
ELISA development team.
•Novel Target ELISA Development'
•New Sample Type Qualification
•New Species Qualification
•DuoSet Serum or Plasma Optimization
•Modification of Existing Kit Contents and Sizes
Learn more | www.rndsystems.com/services/assay-production-custom-development-services
8
Your Next Generation ELISA: Simple PlexTM Assays
Simple Plex Assays
Simple Plex Assays on Ella bring your immunoassays to the next level. In just 90 minutes you get highly
reproducible validated assay data with no manual steps. The assay performance behind that data includes
sub-picogram level sensitivity, 4+ logs of dynamic range and reproducibility that rivals the best laboratory
automation. The entire assay happens within the microfluidic cartridge which allow Simple Plex assays to
achieve a high level of sensitivity, precision and throughput.
Testing across 3 sites, 11 users and 9 Ella
instruments. Four different assays were
processed for CCL2, IL-6, TNF-a and VEGF A.
Eight unique serum samples with 2 controls
for a total of 704 answers.
How Simple Plex Assays Work
Highly Standardized and Reproducible
Simple Plex assays give you single digit reproducibility — even across multiple users and labs. Simple Plex
assays remove the manual steps from executing an immunoassay and enable a high level of precision and
standardization even at very low analyte levels. This ensures that no matter where you measure a sample
you get the same result. And because Simple Plex assays are correlated against our gold standard
Quantikine ELISAs, it’s easy to transfer to another platform whenever you need to.
Sandwich immunoassays happen
in the Glass Nano Reactor (GNR)
Sample
Fluorescent Label
dAb Target/Sample
Capture Ab
GNRs
GNRs
Waste dAb-4
dAb-3
dAb-2
dAb-1
Fluor
Running
Buffer
1.5
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
Normalized Concentration
Multi-Site Test (All Results Histogram)
CV= 7.5%
1. Sample is routed through microfluidic channels.
2. Capture antibody captures target analyte
3. Stringent wash removes unbound analyte
4. Detection antibody migrates through
microfluidic channel
5. Stringent wash removes unbound detection
antibody
6. Scan GNRs
9
Accomplish more in your day without compromising quality. The Quantikine QuicKit ELISAs provide quick,
accurate quantitation of proteins in serum, plasma, and cell culture supernates. Unlike traditional ELISAs,
QuicKit ELISAs have a fast, simplified protocol that only takes 90 minutes to results with just one wash step.
You can expect the same high quality results, sensitivity, and lot-to-lot consistenct to our existing goldstandard Quantikine ELISA kits.
New Quantikine QuicKit Colorimetric Sandwich ELISA Kits
How Quantikine QuicKit ELISA Assays Work
Anti-tag Antibody
Anti-tag antibody coated plates
B Add 50 µL sample
Add Antibody Cocktail
HRP-conjugated
detection antibody
Protein of interest Non-specific proteins
Tag-conjugated
capture antibody
Incubate for 1 hour
C Wash Plate
Add Substrate
D Incubate for 20 minutes
Stop and Read plate
Superior Linearity and Recovery
Linearity and recovery are key performance criteria to ensure your assay is accurately detecting samples, identifying false positives, and
demonstrating your assay is ideal for your sample matrix. R&D Systems Quantikine ELISA kits excel in this area and the Quantikine QuicKit
ELISAs are no different.
Add data and legends from linearity and recovery document attached to email.
Get Quantikine quality in a fraction of the time!
Figure 1. The IL-18 Quantikine® QuicKitTM ELISA has
superior linearity to Brand A. At dilute concentrations,
Brand A is susceptible to matrix effects.
Figure 2. The R&D Systems® IP-10 Quantikine® QuicKitTM
has superior linearity to the Brand A counterpart.
Figure 3. The R&D Systems® G-CSF Quantikine® QuicKitTM
has superior linearity to the Brand A counterpart.
IL-18 Spike Linearity
Dilution
Serum - R&D Systems
250%
Percent Recovery
200%
150%
100%
50%
0% 1:2 1:4 1:8 1:16
Serum - Brand A
Plasma - R&D Systems
Plasma - Brand A
IP-10 Spike Linearity
Dilution
IP-10 Spike Linearity
Plasma - R&D Systems
IP-10 Spike Linearity
Plasma - Brand A
Percent Recovery
200%
150%
100%
50%
0% 1:2 1:4 1:8
G-CSF Spike Linearity
Dilution
250%
Percent Recovery
200%
150%
100%
50%
0% 1:2 1:4 1:8 1:16
Plasma - R&D Systems Plasma - Brand A
10
Confirmed Lot-to-Lot Consistency
All lots are tested to ensure low background, a linear standard curve, consistent assay sensitivity, and a broad dynamic standard curve range.
Consistent standard curve O.D.s, control values, and natural sample values ensure that your samples run consistently over time. Corrected O.D.
Human β-NGF (pg/mL)
0.01
0.1
1
10
10
100 1000 10000
2013 Data
2003 Data
Corrected O.D.
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
1
10
100
10000
1000
Low Control
High Control
Quantitation of Human β-NGF in High and Low Controls. Controls are assayed with every
manufactured lot of β-NGF DuoSet ELISA. Controls must read within a set range of ± two
standard deviations from the mean. Controls for the b-NGF DuoSet ELISA Development
System have remained consistent across 13 years.
Comparison of Human β-NGF Standard Curve O.D.s from 2003 and 2013. Using the
Human β-NGF DuoSet ELISA Development System (Catalog # DY256), standard curve
values generated in 2003 and 2013 were compared for lot-to-lot consistency. Standard
curve O.D.s remained consistent over ten years.
Ensuring ELISA Performance and Consistency
Accurate Detection of Natural Proteins
Antibody pairs recognize the supplied recombinant standard and the
natural proteins in biological samples in a parallel manner, confirming
that this kit can be used to measure the relative mass values of the
natural analyte. R&D Systems has determined the ideal standard
curve range for each assay, ensuring peak sensitivity and reproducibility
of results.
Recognition of Recombinant and Natural Human IL-6. Serial dilutions of rhIL-6 standard
(dark green line) or natural IL-6 produced by unstimulated monocytes (light green line)
were quantitated using the Human IL-6 DuoSet ELISA Development System (Catalog #
DY206). DuoSet ELISAs detect both recombinant and natural proteins in a parallel
manner across a range of concentrations. Corrected O.D.
IL-6 (pg/mL)
0.01
0.1
1
1
10
10
rhIL-6 Standard
Natural IL-6
100 1000
11
Immunoassay precision is defined as the reproducibility of results
within and between assays. This characteristic of an immunoassay is
extremely important in order to: 1) provide assurance that the results
obtained throughout a study are accurate and reproducible from one
experiment to the next and 2) determine if two results are the same or
different. Precision is measured as a coefficient of variation (CV) from
the mean value. Two types of precision should be considered, intraassay precision and inter-assay precision. Intra-assay precision is the
reproducibility between wells within an assay. This allows the
researcher to run multiple replicates of the same sample on one plate
and obtain similar results. Inter-assay precision is the reproducibility
between assays. Inter-assay precision guarantees that the results
obtained will be reproducible using multiple kits over a period of time.
R&D Systems Quantikine Immunoassays typically have CV values less
than 10% across the standard curve for both intra- and inter-assay
precision. These low CV values allow the researcher to perform
repeated assays and be confident that the results are consistent
throughout the study.
50
150
Concentration (pg/mL)
0
0
100
200
6 9 12 15
Time (months)
50
150
Lot 1 Lot 2 Lot 3 Lot 4
Concentration (pg/mL)
0
0
100
200
A B
4 6 9 12
Time (months)
Precision & Reproducibility: Providing Confidence in Your Results
Quantikine ELISA Kits Are Tested for Stability and Reproducibility. A. Three samples with different concentrations of IL-6 (colored lines) were assayed using the same lot of the Human
IL-6 Quantikine ELISA Kit (Catalog # D6050) over a 15 month period. B. Three samples with differing IL-6 concentrations (colored lines) were assayed using four different lots of the
Human IL-6 Quantikine ELISA Kit (Catalog # D6050) over a 12 month period.
4
Number of Observations
0
8
12
16
30–39
40–49
60–69
80–89
100–109
120–129
140–149
160–169
50–59
70–79
90–99
110–119
130–139
150–159
170–179
IL-12 p40 Concentration (pg/mL)
A
2
Number of Observations
0
4
6
8
10
<0.199
0.400–0.599
0.600–0.799
1.200–1.399
1.800–1.999
2.400–2.599
4.600–4.799
0.800–0.999
1.000–1.999
1.400–1.599
1.600–1.799
2.200–2.399
2.000–2.199
2.800–2.999
4.800–4.999
10.000–10.199
IL-6 Concentration (pg/mL)
B
The minimum detectable dose is the lowest measurable value that is
statistically different from zero. It is calculated by adding two standard
deviations to the mean optical density value of several zero standard
replicates and determining the corresponding analyte concentration
from the standard curve. The better the sensitivity of an assay, the
lower the useful working range (standard curve range) will be.
Quantikine ELISAs are optimized to ensure high signal, low background,
and the best sensitivity possible.
Sensitivity: Measuring Proteins at the pg/mL Range
The Minimum Detectable Dose for Many Quantikine ELISA Kits Allows Proteins Present at the pg/mL Range to be Accurately Measured. A. Serum from 86 apparently healthy individuals
was assayed using the Human IL-12/IL-23 p40 Quantikine ELISA Kit (Catalog # DP400). B. Serum from 41 apparently healthy individuals was assayed using the Human IL-6 Quantikine
HS ELISA Kit (Catalog # HS600C).
12
Quantikine ELISA Kits Are Developed to Detect Natural and Recombinant Proteins. A
serum sample containing activated human TGF-β1 was serially diluted (blue line) and
compared to the TGF-β1 standard curve (red line). Results show that the Human TGF-β1
Quantikine ELISA Kit (Catalog # DB100B) measures recombinant and natural TGF-β1 with
equal effectiveness.
Interference Testing of the Human TNF-α Quantikine ELISA. TNF-α, at concentrations of
125–1000 pg/mL, was measured in the presence or absence of soluble TNF receptors
(sTNF RI or sTNF RII) using the Human TNF-α Quantikine ELISA Kit (Catalog # DTA00D). The
results demonstrate that the presence of the soluble TNF receptors at concentrations up
to 1000 ng/mL does not affect the TNF-α concentration determined using the Quantikine
ELISA Kit.
40
100
TNFα Measured (% of expected)
0
0
20
60
80
120
1 10 100 1000
sTNF R present (ng/mL)
sTNF RI
sTNF RII
Optical Density
100
0.01
TGF-β1 Standard
Natural TGF-β1
0.1
1.0
10
102 103 104
TGF-β1 Concentration (pg/mL)
Sample Dilution
Quantikine Kit Competitor Kit
Analyte Concentration Detected (ng/mL)*
4.16 20.87
1:2 105% 73%
1:4 108% ND
1:8 106% ND
Linearity claim 85–115% 89–118%
* Samples were diluted prior to the assay as directed in the product data sheet. All
samples and dilutions were within the standard curve range.
Linearity Experiments Identify False Positive Signals
Learn more about identifying and
eliminating false positive results.
Review our application note.
False Positive ELISA Signals Can Be Identified by Assaying the Linearity of Dilution. Serial dilutions of a cell culture supernate were assayed for
natural linearity using two different TIMP-2 ELISA Kits.
Diluted samples measured using the Human TIMP-2 Quantikine Kit
(Catalog # DTM200) gave recovery results between 105–108% of the
neat sample, supporting the linearity claim of the kit. In contrast, the
target analyte was not detectable beyond the first dilution in samples
measured with the second kit, indicating that the assay was producing
a false positive signal. ND=Not detectable.
Figure 1. The importance of blocking reagents when detecting human GDNF. The
absence of our proprietary blocking reagents resulted in false positives in 9 of 11
samples using the Quantikine ELISA (1A). Conversely, there were no false positive
results in Competitor A’s ELISA when our blocking reagents were added to the
diluent (1B). Note that Competitor A lacked the sensitivity needed to detect GDNF
in positive control supernates from U-118MG and U-87MG cell cultures.
TABLE 1 – FALSE POSITIVE HUMAN GDNF READINGS IN 2 COMPETITOR ELISA KITS
We next determined whether removing blockers from the
diluents in the Human GDNF Quantikine ELISA Kit would lead
to false positive detection. The Competitor B ELISA kit was no
longer commercially available, so they were not included in the
additional testing. Eleven serum samples from apparently healthy
individuals were tested on the Human GDNF Quantikine ELISA Kit
according to protocol. Samples diluted in the Quantikine diluent
were compared to identical samples in diluents that lacked
blocking components. Identical samples were also tested using
Competitor A’s Human GDNF ELISA with our blocking reagents
added to the diluent. U-118MG and U-87MG glioblastoma
conditioned media supernate samples were also tested on both
kits with and without our blocking reagents. These were used as a
positive control7 to ensure appropriate binding with and without
blocking reagents
When using the Quantikine diluents as provided in the GDNF kit,
no serum samples are detectable. When the blocking reagents
are removed from the Quantikine diluents, 9 out of 11 serum
samples were detectable, giving false positive sample values
(Figure 1A). The U-118MG and U-87MG conditioned supernate
samples are detectable whether blocking reagents are used or
not, indicating that the blocking reagents are not suppressing the
real GDNF detection in these positive controls.
When testing the ELISA kit from Competitor A, following the
kit protocol and using the diluents provided, 9 out of 11 serum
samples were detectable (Figure 1B). When blocking reagents
were added to Competitor A’s diluents, the serum samples were
now undetectable. The U-118MG and U-87MG conditioned
supernate samples are not detectable whether blockers are
added to the diluents or left as provided in the kit. This indicates
lower assay sensitivity with Competitor A when compared to
the Quantikine ELISA, although the standard curve range is
actually lower on Competitor A (Quantikine standard curve
range: 9.38 – 300 pg/mL, Competitor A standard curve range:
2.74 – 2000 pg/mL).
QUANTIKINE COMPETITOR A COMPETITOR A +
BLOCKERS COMPETITOR B COMPETITOR B +
BLOCKERS
Standard Curve Range: 9.38 - 300 pg/mL 2.74 - 2000 pg/mL
15.6 - 1000 pg/mL
Sample Dilution Neat 1:2 (AT) 1:10
Serum 1 ND 692 ND 44.4 ND
Serum 2 ND 614 ND 53.9 ND
Serum 3 ND ND ND ND ND
Serum 4 ND ND ND 18.2 ND
Serum 5 ND ND ND ND ND
Serum 6 ND ND ND 15.6 ND
ND = non-detectable, sample values << the lowest standard curve point
Quantikine Serum Values
Quantikine Diluent
0
50
100
150
200
250
300
350
400
450
500
Serum 7
Serum 8
Serum 9
Serum 10
Serum 11
Serum 12
Serum 13
Serum 14
Serum 15
Serum 16
Serum 17
U-118 MG
U-87 MG
Human GDNF Concentration (pg/mL)
Quantikine Diluent w/o Blockers
1A
0
100
200
300
400
500
600
700
800
Serum 7
Serum 8
Serum 9
Serum 10
Serum 11
Serum 12
Serum 13
Serum 14
Serum 15
Serum 16
Serum 17
U-118 MG
U-87 MG
Human GDNF Concentration (pg/mL)
Competitor A Serum Values
Competitor A Diluent Competitor A Diluent + Blockers
1B
HUMAN GDNF SERUM VALUES ON R&D SYSTEMS ELISA VS
COMPETITOR A ELISA.
AVOID FALSE POSITIVE ELISA DATA:
IMPROVE PERFORMANCE WITH THE R&D SYSTEMS®
QUANTIKINE® ASSAY
APPLICATION NOTE
INTRODUCTION
Enzyme Linked Immunosorbent Assays (ELISAs) are commonly
used to quantify biomarkers in serum, plasma, and cell culture
supernates. These samples contain a variety of proteins, blood
components, and other factors that can interfere with the
ELISA results, which is commonly referred to as a matrix effect.
Eliminating these factors is essential to obtaining accurate
sample values. Blocking reagents are commonly used in ELISA
kits to reduce interference from proteins in samples which may
produce false positive results. False positives can also occur
from non-specific binding on the microplate. Solid phase
blocking agents are specifically designed to saturate unoccupied
binding sites on the ELISA plate surface and prevent this nonspecific binding. R&D Systems uses specialized diluents that are
specifically designed to alleviate false positives to give the most
accurate results. Here are some kit-specific examples on how
blocking reagents were used improve performance.
THE IMPORTANCE OF BLOCKING REAGENTS WHEN DETECTING
HUMAN GDNF
Glial Cell Line-derived Neurotrophic Factor (GDNF) is a neurotrophic factor that has been shown to promote the survival of various
neuronal subpopulations in both the central and peripheral nervous systems at different stages of their development. Neuronal
subpopulations shown to be affected by GDNF include motor neurons, midbrain dopaminergic neurons, Purkinje cells and sympathetic
neurons. GDNF is produced by Sertoli cells, type 1 astrocytes, Schwann cells, neurons, pinealocytes, and skeletal muscle cells. GDNF
binding to GFR alpha 1 induces the recruitment of Ret, NCAM-1/CD56, various integrins, Syndecan-3, or N-Cadherin1-3.
When developing the Human GDNF Quantikine ELISA (Catalog # DGD00), it was discovered that the serum sample values were
not matching reported literature values using commercially available ELISAs4-6. These competitor assays were achieving values that
ranged from non-detectable to > 600 pg/mL for serum samples from apparently healthy individuals, while the same samples were
undetectable with the Quantikine ELISA.
FALSE POSITIVE DETECTION IN TWO COMMERCIALLY AVAILABLE
ELISA KITS
Knowing that blocking agents alleviate non-specific binding, a further investigation into our competitors’ assays was conducted by
adding blocking agents to their diluents and testing them side by side with the diluent provided in their kit. As instructed in their
respective inserts, serum samples were diluted 1:2 in Competitor A and 1:10 with acid treatment in Competitor B. Samples were
tested neat on the Quantikine® ELISA. As shown in Table 1, none of the samples were detectable in using the Quantikine kit. However,
competitor kits detected false positive GDNF levels ranging from 15.6 pg/ml to 692 pg/ml. Two of the samples were detectable with
Competitor A using their diluent, but when blockers were added, the samples were no longer detectable. Four of the samples were
detectable with Competitor B, but when blockers were added, the samples were no longer detectable.
13
Quantitation of Human IL-6 in High, Medium, and Low Controls. High (blue line), medium (red line), and low (green line)
controls are assayed with every manufactured lot of the Human IL-6 Quantikine® ELISA Kit (Catalog # D6050). Controls
for the Human IL-6 Quantikine® ELISA Kit fall within acceptable ranges (gray bars) and remain consistent from lot to lot.
What is the importance of ELISA controls?
The importance of including ELISA controls, both positive and negative, in your immunoassay
helps to verify that the assay was run properly and everything is performing accurately.
Positive ELISA Controls
A positive ELISA control can be a recombinant or natural sample that you know will be detectable in the assay. Positive controls help to show
that a negative sample is truly negative. The standard curve is one form of positive control and you can compare your results to the standard
curve data that is provided in your product insert. R&D Systems also sells ELISA controls for the Quantikine ELISAs . Most Human Quantikine
kits have a lyophilized tri-level control with expected ranges that are validated by our Quality Control and our Mouse/Rat Quantikine ELISAs
include one control in the kit. These are great when running multiple plates or when you have multiple users running the assay, to verify that
values are all within the expected ranges.
ELISA Spike Controls
When using complex sample matrices, it is also important to make sure that there is nothing present in the matrix that interferes in the assay.
It is recommended to spike in recombinant or natural protein into your matrix and verify that the amount you spike in is what you read out. For
more information, refer to Section 4f. Our Quantikine ELISAs are already validated for many complex sample types, so refer to the kit insert for
details.
Negative ELISA Controls
Negative controls help to verify that you are not obtaining any false positive results or non-specific binding. Use a sample that you know will
does express the protein you are measuring. If you are quantitating a cell culture supernate, a good negative control would be to test your cell
culture media.
14
Plasma
Collect plasma using EDTA, heparin, or citrate as an anticoagulant.
Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
Assay immediately or aliquot and store samples at ≤ -20 °C. Avoid
repeated freeze-thaw cycles.
Cell Culture Supernates
Remove particulates by centrifugation and assay immediately or
aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw
cycles.
Cell Lysates
Solubilize cell in lysis buffer and allow to sit on ice for 30 minutes.
Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble
material. Aliquot the supernatant into a new tube and discard the
remaining whole cell extract. Quantify total protein concentration using
a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Platelet-poor Plasma
Collect plasma using EDTA, heparin, or citrate as an anticoagulant.
Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
An additional centrifugation step of the plasma at 10,000 x g for 10
minutes at 2-8°C is recommended for complete platelet removal.
Assay immediately or aliquot and store samples at ≤ -20°C. Avoid
repeated freeze-thaw cycles.
Serum
Use a serum separator tube (SST) and allow samples to clot for 30
minutes at room temperature before centrifugation for 15 minutes at
1000 x g. Remove serum and assay immediately or aliquot and store
samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Saliva
Collect saliva in a tube and centrifuge for 5 minutes at 10,000 x g.
Collect the aqueous layer, assay immediately or aliquot and store
samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.
Urine
Aseptically collect the first urine of the day (mid-stream), voided directly
into a sterile container. Centrifuge to remove particulate matter. Assay
immediately or aliquot and store at ≤ -20°C. Avoid repeated freezethaw cycles.
Human Milk
Centrifuge for 15 minutes at 1000 x g at 2-8°C. Collect the aqueous
fraction and repeat this process a total of 3 times. Assay immediately.
Tissue Homogenates
The preparation of tissue homogenates will vary depending upon
tissue type. Rinse tissue with 1X PBS to remove excess blood,
homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20°C.
After two freeze-thaw cycles were performed to break the cell
membranes, the homogenates were centrifuged for 5 minutes at
5000 x g. The supernate was removed immediately and assayed.
Alternatively, aliquot and store samples at ≤ -20°C. Avoid repeated
Tissue Lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a
tissue homogenizer in PBS. Add an equal volume of RIPA buffer
containing protease inhibitors and lyse tissues at room temperature for
30 minutes with gentle agitation. Centrifuge to remove debris. Quantify
total protein concentration using a total protein assay. Assay
immediately or aliquot and store at ≤ -20°C.
Sample Preparations
Sample Collection & Storage
The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated.
(Some proteins require the presence of fetal calf serum for stability)
15
ELISA Assay Timelines
SimplePlex QuicKit Quantikine DuoSet
Sample/reagent Prep
KEY
Analyte Capture
Detection Antibody Incubation
Substrate Incubation
Plate Prep
Wash Step
90
minutes
4.5
hours
5.5
hours
Plate Prep
O/N
90
minutes
16
The values of the unknown samples are assigned in relation to the
standard curve. If samples have been diluted, the concentration read
from the standard curve must be multiplied by the dilution factor.
Always run ELISA samples in duplicate or triplicate This will provide
enough data for statistical validation ofthe results.
Average the duplicate or triplicate readings for each standard, control,
and sample and subtract the average zero standard optical density
(O.D.). The coefficient of variation (CV) of duplicates should be ≤ 20%.
Create a standard curve by reducing the data using computer software
capable of plotting the mean absorbance (y axis) against the protein
concentration (x axis). When possible, utilize the recommended data
reduction method specified in the assay protocol.
If the recommended data reduction method is unavailable, it is
recommended that various methods (e.g. linear, semi-log, log/log, 4 or
5 parameter logistic) be tried to see which curve best fits the data. One
way to determine if the curve fit is correct is to backfit the standard
curve O.D. values. To do this, first plot the standard curve.Next, treat
standards as unknowns and interpolate the O.D. values from your
standard curve. They should read close to the expected values
(+/- 10%). Use the data reduction method that gives the best correlation
value and backfit.
If software is unavailable, the data may be linearized by plotting the log
of the concentrations versus the log of the O.D. on a linear scale. The
best fit line can be determined by regression analysis. This procedure
will produce an adequate but less precise fit of the data.
A representative standard curve is shown in the figure below from
Human IL-6 HS Quantikine ELISA (Cat# HS600C).
(pg/mL) O.D. Average Corrected
0
0.051
0.059 - 0.067
0:156
0.101
0.103 0.044
0.105
0.313
0.148
0.149 0.090
0.149
0.625
0.246
0.251 0.192
0.255
1.25
0.431
0.432 0.373
0.433
2.5
0.798
0.804 0.745
0.809
5
1.407
1.418 1.359
1.429
10
2.485
2.498 2.439
2.510
10
1
0.1
0.1 1
Human IL-6 Concentration (pg/mL)
Optical Density
10
0.01
Calculating the coefficient of variation
The coefficient of variation (CV) is the ratio of the standard deviation to the mean, which is usually expressed as a percentage.
CV = standard deviation mean
Calculating CV is important as it can indicate any inconsistencies or inaccuracies in your
ELISA results. The CV of duplicates should be ≤ 20%. A larger CV indicates greater
inconsistency and possible error.
10
1
0.1
0.1 1
Human IL-6 Concentration (pg/mL)
Optical Density
10
0.01
To determine the concentration of each sample, first find the
absorbance value on the y-axis and extend a horizontal line to the
standard curve. At the point of intersection, extend a vertical line to the
x-axis and read the corresponding concentration. If samples have been
diluted, the concentration read from the standard curve must be
multiplied by the dilution factor.
Calculating concentration of target protein in the sample
Data Analysis: Calculation of Results
17
Best Practices and Techniques
While R&D Systems builds Quantikine ELISA kits to be robust in the hands of even inexperienced users, there are several tips and tricks that can
help even the experienced user get the most from their assay.
Make sure all reagents are brought to room temperature before using (unless instructed to keep
them cold).
If you are not going to run the entire plate, ensure that the remaining strips are sealed in the
plate bag with the desiccant to prevent moisture from degrading the plate.
For standards that are not single use, it is best to aliquot the remaining standard into smaller
volumes and freeze. This allows you to avoid repeated freeze-thaws.
1 2 3 4 5 6 7 8 9 10 11 12
✔
Multichannel pipettes speed the ability to plate your standard and samples and lead to more
consistent results.
✔ When pipetting, dispense liquid with the pipette tips held at an angle and not touching the
bottom of the well.
✔
While it is not necessary to change your pipette tips between each replicate, it is recommended
that you change them between different samples or standards to prevent contamination.
✔
1 2 3 4 5 6 7 8 9 10 11 12
Plate
washer
It is highly recommended that a plate washer is used as manual plate washing can lead to
higher backgrounds.
30
sec
When washing plates, either manually or with a plate washer, be sure to give the wash buffer
time to work by adding a 30 second soak time in between washes.
5min
Pay close attention to the incubation times. As a general guide the incubation time should not
vary by more than +/- 5 minutes per hour of incubation time.
✔
If the assay calls for incubation in a cold environment, at 2–8 °C, and you are running multiple
assays, do not stack the plates on top of each other instead placing them individually on the
shelf.
18
What is included in a Quantikine Kit?
Quantikine Kits are a complete kit consisting of a precoated microplate,
Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop
Solution, Wash Buffer, and plate sealers. They are fully validated
ELISAs for the sample types listed in the specific datasheet. They have
been exhaustively tested for superior quality.
How many samples can be assayed in a Quantikine kit?
Most Quantikine Kits will run the standard curve and 40 samples in
duplicate. Please refer to the datasheet for details on each kit.
What samples can be tested in the kit?
Typically the R&D Systems Quantikine kits are validated for sera, two
types of plasma, and cell culture supernate. However, the samples
validated in an ELISA can vary from product to product. The product
datasheet and product-specific web page states all sample types that
have been validated for use with the ELISA kit. These are the only
samples for which we can support the claims. References may exist for
other sample types. See the “Citations” tab on the product-specific
webpage for any published references citing the use of the kit with an
alternate sample type. Unclaimed sample types should be validated by
the customer.
Has this kit ever been tested with my sample type?
Unfortunately, R&D Systems has not routinely tested many sample
types such as tissue homogenates or bronchoalveolar lavage for ELISA
kits. This does not mean that the ELISA kit is not suitable for other
sample types. One will need to perform a spike and recovery study to
determine if an unvalidated sample type will work with a particular kit.
To perform a spike and recovery experiment, one should divide a
sample into two aliquots. In one of the aliquots, the user should spike
in a known amount of the kit standard. A dilution series is performed
comparing the spiked versus the unspiked sample. Generally, samples
with expected recovery and linearity between 80–120% are considered
acceptable. This method may be used to validate any sample type that
has not been evaluated by R&D Systems. For a more detailed spike
and recovery protocol, please contact Technical Service. Note:
Acceptable ranges should be determined individually by each
laboratory. Please see the Citations tab for peer-reviewed papers
utilizing a wide range of sample types.
Why can I not detect any of my samples?
You will be able to quantify samples down to the lowest point on the
standard curve. In some cases, the standard curve does go down low
enough to detect normal samples. You can check the Sample Values
section in your kit booklet to find out what kind of sample values we
obtained from apparently healthy individuals. You may also want to
review the literature to find out if there is an established normal range
for your target. It is important to recognize that assay platforms and
manufacturers differ in their calibrations for their unique assay
products and reported measurements may not directly correlate.
Can I extend the standard curve (in either direction)?
R&D Systems cannot support kit results outside the stated range
under any circumstances. A specific range was chosen because of
confidence in the reproducibility of the assay.
Quantikine® ELISA FAQs
Why doesn’t the assay range extend to the stated sensitivity?
Sensitivity is the lowest measurable value that is statistically not equal
to zero. It is calculated based on the signal of the background and the
inherent variability of the assay. It is commonly determined by taking
the mean O.D. plus two standard deviations from 20 zero replicates.
This value is converted into analyte concentration from the standard
curve. The low standard is the lowest possible point at which
R&D Systems feels confident that the value is in the linear portion of
the standard curve and, therefore, quantifiable. Values which are
greater than the sensitivity can be distinguished as separate from the
background or the noise of the assay, however the confidence level for
reporting these values is lower than if the sample values fall within the
standard curve range.
Why is a sample dilution necessary in some kits?
There are primarily two reasons for dilutions. In some assays most
samples read above the standard curve, thus requiring a dilution for
analyte levels to fall within the range of the assay. A second reason for
dilution is to limit interference due to factors in complex matrices.
Won’t addition of Assay Diluent cause further dilution of the
sample?
Since the assay diluent is added to all wells, standards and specimens
are treated equally. Therefore, sample concentration can be read from
the standard curve without adjusting for this dilution.
Is there enough Calibrator Diluent for all of my sample
preparations?
The kits are designed with enough calibrator diluent to ensure that the
vast majority of samples fall within the indicated range of the assay.
Should you find that there is not enough diluent provided in the kit to
dilute your samples, you have at least two options. Option 1) Samples
can be diluted in two steps. The initial dilution in culture medium and
a final dilution, of at least 1:10, into the Calibrator Diluent provided in
the kit. Option 2) For a nominal charge, you can purchase additional
diluent provided the same lot included in the kit is still available.
Contact Technical Service for more information.
My diluents appear to contain precipitate. Is this ok?
Due to saturating amounts of some buffer components, some of the
RD1 Assay Diluents contain a light to heavy precipitate. In these
instances, it will be noted in the specific protocol booklet. If it is not
noted in the protocol booklet, please contact Technical Service.
The assay protocol specifies to use the shaker at 500 rpm.
This is too fast for my shaker. Is this correct?
This is 500 rpm with a 0.12 orbit. If the plate shaker has a larger orbit,
then 500 rpm will be too fast. R&D Systems recommends the
ThermoFisher Model # 4625 microtiter plater shaker. Assays requiring
shaker incubations have been optimized for performance with these
shaker specifications only.
19
Are controls available for kits?
R&D Systems offers tri-level control sets for the Human Quantikine
ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity),
and QuantiGlo ELISA kits (chemiluminescent). Please inquire for
specific ordering information.
What is the stability of supplemental ELISA controls?
Controls are assigned an expiration date of 6 months from date of
receipt. They are to be used once and discarded. If the lyophilized
controls are stored properly, it is possible that they will remain stable
for an extended period of time, although we have not conducted
extended stability testing. The controls have not been tested for
stability after reconstitution.
I used your recombinant protein as a control in the
corresponding ELISA kit. Why am I seeing discrepancy in
mass values?
First, a large dilution is required to place the recombinant protein on
the standard curve range. Typically this is a dilution from μg/mL to
pg/mL. Any dilution step can introduce inaccuracy and the larger the
dilution step the greater the potential for error. Any pipetting error or
mis-calibrated pipet can result in apparent over- or under-recovery.
Second, R&D Systems immunoassays have been developed to
measure a level of protein captured by one antibody and detected by a
second antibody. This measurement is calibrated to standards
established when the kit was initially developed. The protein
determination of these initial standards became the Master Calibrators
to which all new standards are formulated. This provides R&D Systems
immunoassay kits with consistency between manufacturing lots. In
general, we would expect +/- 25% recovery of the amount stated on
the vial when using the Quantikine ELISA to determine a protein
concentration. There may be slight differences in the immunologically
recognizable mass between lots of protein, so the apparent
concentration provided on the vial may vary from lot-to-lot when
measured in the ELISA. If you are using proteins to make controls, it is
better to value assign the mass based on measurement in ELISA and
not use the mass on the vial when setting control levels.
Why must I use polypropylene tubes for standard curve
dilutions in certain assays?
Certain proteins or analytes will bind to glass and polystyrene, but do
not readily bind to the polypropylene tubes.
Why are my wells green after adding the stop solution?
This happens when the substrate in the well does not completely mix
with the stop solution. After addition of the stop solution, tap the plate
gently or place on a shaker until the mixture in the wells turns yellow.
Why is there brown precipitate in my wells after addition of the
stop solution?
This is due to incomplete washing after the HRP-labeled detection
antibody (or streptavidin-HRP) incubation. When HRP is present during
the substrate and subsequent stop solution additions, an orangebrown or brown precipitate is observed. This may be remedied by the
addition of a 30 second soak on each wash step followed by complete
removal of all liquid in the wells.
What is a competitive ELISA?
In the competitive immunoassay approach, also termed labeled
analyte technique, there exists a competition between the endogenous
unlabeled antigen and an exogenous labeled antigen for a limited
amount of antibody binding sites. Therefore, a decreasing signal
indicates higher concentrations of the analyte being measured.
What is a sandwich ELISA?
A sandwich ELISA uses an immobilized capture antibody specific for
the analyte of interest in a sample. After the analyte is bound to the
immobilized antibody, a labeled secondary antibody specific for the
analyte is used for detection. The analyte is “sandwiched” between the
two antibodies. The sandwich ELISA is extremely sensitive, and the
values obtained are quantitative when compared with a standard
curve.
Can a partial Quantikine ELISA plate be used?
The Quantikine ELISA plates have removable strips of wells. Unused
wells may be removed from the plate, returned to the foil pouch
containing the desiccant pack, and stored at 2–8°C for up to one
month.
Can I stop an assay at any point, extend an incubation time or
change the suggested incubation temperature?
R&D Systems has optimized the assays for both incubation times and
temperatures. Each kit has only been validated for the protocol
described in the kit datasheet. We cannot guarantee the performance
of our kits when the protocol has been altered in any way.
Can reagents from different kits be interchanged?
Assay Diluent(s), Calibrator Diluent(s), and substrate may be
interchanged if they have the same part number AND lot number. R&D
Systems does “whole kit QC” which means that we cannot support the
use of reagents from other lots or sources being substituted into an
assay. Plates and Conjugate cannot be interchanged under any
circumstance.
Why do I need to use a 4-PL curve fit for generating my
standard curve?
R&D Systems develops and QCs most of our Quantikine ELISA Kits
using a 4-parameter logistic (4-PL) curve-fit. As an alternative, construct
a standard curve by plotting the mean absorbance for each standard
on the y-axis against the concentration on the x-axis and draw a best fit
curve through the points on the graph. The data may be linearized by
plotting the log of the concentrations versus the log of the O.D., and
the best fit line can be determined by linear regression. This procedure
will produce an adequate but less precise fit of the data.
Why am I seeing high variability between sample duplicates?
The two main reasons for high variability in an assay is related to
pipetting & washing technique.
20
Problem Possible Cause Solution
No signal or low signal
Reagents added in incorrect order, or incorrectly
prepared
• Repeat assay
• Check calculations, standard reconstitution, etc.
Standard has been damaged (if there is a signal in the
sample wells)
• Check that standard was handled according to directions. Avoid vortexing.
• Use new vial
Incorrect incubation conditions • Check incubation conditions were for the specified length, at the appropriate
temperature, and shaker specifications were met if required.
Incorrect filters • Check specified signal and correction wavelengths in the protocol
Incorrect Storage/Handling • Check that kit was stored properly according to conditions indicated on
the box label
Too much signal – whole plate
turned uniformly blue
Insufficient washing/washing step skipped – unbound
peroxidase remaining • See washing procedure
Substrate Solution mixed too early and turned blue • Substrate Solution should be mixed and used immediately
Plate sealers or reagent reservoirs reused, resulting in
presence of residual HRP. This will turn the TMB blue
non-specifically
• Use fresh plate sealer and reagent reservoir for each step
Work surface cleaned with bleach • Residual bleach fumes can oxidize TMB and cause non-specific high signal
Standard curve achieved but poor
discrimination between points
(low or flat curve)
Plate not developed long enough
• Increase Substrate Solution incubation time
• Use recommended time
Incorrect procedure • Eliminate modifications, if any
Improper calculation of standard curve dilutions • Check calculations, make new standard curve
Insufficient washing
• See washing procedure
• If using an automatic plate washer, check that all ports are clean and free of
obstructions, add a 30 second soak step and rotate plate halfway through
the wash
Plate sealer reused • Use a fresh plate sealer for each step
Poor Duplicates
No plate sealers used • Use plate sealers
Insufficient washing
• See washing procedures
• If using an automatic plate washer, check that all ports are clean and free of
obstructions
Variations in incubation temperature • Avoid incubating plates in areas where environmental conditions vary
Variations in protocol • Adhere to the same validated assay protocol
Variation in pipetting
• Ensure all pipette tips are securely fastened and dispensing consistent volumes
• Establish use of either forward or reverse pipetting for entirety of the assay
Improper shaker
• Check that shaker orbit and speed meet specifications indicated in the kit insert.
Any splashing on the plate sealer or foaming of liquid in the sample can also result
in poor precision.
Saliva contamination • Wear a mask to avoid contamination
Poor assay to assay reproducibility
Plate sealers reused • Use fresh plate sealer for each step
Improper calculation of standard curve dilutions
• Check calculations, make new standard curve
• Use internal controls
No signal when a signal is
expected, but standard curve
looks fine
No cytokine in sample or levels below assay range
• Repeat experiment
• Reconsider experimental parameters
• Obtain fresh samples, minimize freeze-thaw cycles
• Use enzyme inhibitors
Sample matrix is masking detection
• Dilute samples at least 1:2 in appropriate diluent, or preferably do a series of
dilutions to look at recovery
• If specified in the kit protocol, the assay may only recognize the sample after
specific treatment. Follow any sample treatments specified in assay insert.
Samples are reading too high, but
standard curve looks find Samples contain cytokine levels above assay range • Dilute samples further and run again
Very low readings across the plate
Incorrect wavelengths • Check filters/reader
Insufficient development time • Increase development time
Green color develops upon addition
of stop solution when using streptavidin-HRP
Reagents not mixed well enough in wells • Tap plate
Edge Effects Uneven temperatures around work surfaces • Avoid incubating plates in areas where environmental conditions vary
• Use plate sealers
Drift
Interrupted assay set-up • Assay set-up should be continuous – have all standards and samples prepared
appropriately before commencement of the assay
Reagents not at room temperature • Ensure that all reagents are at room temperature before pipetting into the wells
unless otherwise instructed in the antibody inserts
Troubleshooting your Quantikine® ELISA
21
There are many parameters which influence the results obtained in an
ELISA. These include: antibody quality and concentrations, incubation
times, incubation temperatures, detection reagent quality and
concentration, and substrate type and quality. For this section, it is
assumed that all recommended reagents are being used.
Antibody concentration—the best way to determine the optimal capture
and detection antibody concentrations is to perform a grid experiment.
A grid experiment provides a method to test many antibody pair
concentrations using only one plate. Antibody starting concentrations
will vary depending on antibody type (monoclonal versus polyclonal)
used for capture and detection, see Table 1. Refer to the product
inserts for capture and detection antibody types as well as
recommended starting concentrations.
Table 1. Recommended antibody starting concentrations
Table 2. Grid experiment for monoclonal capture-polyclonal detection assay
Monoclonal Capture/ Polyclonal Detection Monoclonal Capture/ Monoclonal Detection Polyclonal Capture/ Polyclonal Detection
Capture Concentration 1, 2, 4 and 8 µg/mL 0.5, 1, 2 and 4 µg/mL 0.2, 0.4 and 0.8 µg/mL
Detection Concentration 50, 100, 200 and 400 ng/mL 0.25, 0.5, 1 and 2 µg/mL 50, 100, 200 and 400 ng/mL
To form the grid, divide a 96-well plate into 4 quadrants. See Figure 2
for an example of a monoclonal capture-polyclonal detection grid
experiment. The 6 columns in each quadrant represent capture
antibody concentrations, the 4 rows in each quadrant represent
standard curve points, and each of the 4 quadrants represents a
different detection antibody concentration. Each quadrant is a “minigrid”, identifying different capture antibody and standard
concentrations at one particular detection antibody concentration. In
the grid experiment in Figure 2, each quadrant contains all the possible
combinations of capture antibody at 1, 2 and 4 µg/mL and standard
curve points of ø (Diluent stated on the product insert), 1000, 2000,
and 4000 pg/mL, at one detection antibody concentration.
From the multiple combinations of antibody pair concentrations
illustrated on the grid, select the concentrations that give the best
signal to noise ratio. The ø standard points give the “noise” or the
background value that can be expected at each of the antibody pair
concentrations. The 1000, 2000 and 4000 pg/mL standard curve
points give the “signal” resulting from each of the many antibody pair
concentrations. Select the highest signal to noise ratio that still gives
an acceptable background. A signal to noise ratio of at least 10 is
excellent, but the ratio should be at least five.
50 ng/mL detection 100 ng/mL detection
1 2 3 4 5 6 7 8 9 10 11 12
1 µg/mL
capture
1 µg/mL
capture
2 µg/mL
capture
2 µg/mL
capture
4 µg/mL
capture
4 µg/mL
capture
1 µg/mL
capture
1 µg/mL
capture
2 µg/mL
capture
2 µg/mL
capture
4 µg/mL
capture
4 µg/mL
capture
A Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø
B 1000 pg/mL
standard 1000 1000 1000 1000 1000 1000 pg/mL
standard 1000 1000 1000 1000 1000
C 2000 pg/mL
standard 2000 2000 2000 2000 2000 2000 pg/mL
standard 2000 2000 2000 2000 2000
D 4000 pg/mL
standard 4000 4000 4000 4000 4000 4000 pg/mL
standard 4000 4000 4000 4000 4000
E Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø Ø
F 1000 pg/mL
standard 1000 1000 1000 1000 1000 1000 pg/mL
standard 1000 1000 1000 1000 1000
G 2000 pg/mL
standard 2000 2000 2000 2000 2000 2000 pg/mL
standard 2000 2000 2000 2000 2000
H 4000 pg/mL
standard 4000 4000 4000 4000 4000 4000 pg/mL
standard 4000 4000 4000 4000 4000
200 ng/mL detection 400 ng/mL detection
DuoSet® ELISA Assay Optimization
22
Background <0.2 O.D. units. Factors that influence background
include: blocking reagent, capture and detection antibody
concentrations, detection system, incubation times, diluents and
washing technique.
Curve height preferably above 1.0, usually between 1.0 and
3.0 O.D. units. Factors that influence curve height include: capture and
detection antibody concentrations (see grid experiment in Figure 2),
incubation times and temperatures, detection system concentration,
avidity of antibodies for antigens, pH, diluents and quality of reader.
Detection system assay sensitivity may increase with increasing
detection reagent concentration or alternate detection system.
However, this may result in higher background readings.
Dilution of serum and plasma samples serum and plasma samples
may require a dilution of at least 2-fold in an appropriate buffer to
overcome matrix effects. Empirically determine the dilution of the
samples required to result in linearity of dilution. When diluting
samples, remember that the diluent used for the standard curve
should be the same as that used for samples. If samples are diluted,
include the appropriate dilution factor when calculating results.
BSA bovine serum albumin, used as a blocking and carrier protein.
Since different grades of BSA exist and may contribute to background,
an ELISA grade BSA should be chosen and validated.
Incubation temperatures the sample and detection antibody
incubations should be performed at room temperature. Sample
incubation overnight at 4°C or 1 hour at 37°C may increase assay
sensitivity, but may also increase the background.
Interfering substances it is important to be aware of the possible
presence of interfering substances such as heterophilic antibodies or
rheumatoid factors. Please refer to The Immunoassay Handbook,
edited by David Wild, Nature Publishing Group, copyright 2001, for
suggestions on how to control for these substances.
Reagent reconstitution and storage conditions reconstitution and
storage instructions provided with each reagent must be followed to
ensure proper reagent perfomance.
Sample preparation and storage while not every analyte has the
same stability within a given matrix, there are general precautions
which should be followed. Samples that are not used immediately after
preparation should be stored in single use aliquots at -70°C. A -20°C
freezer may be acceptable, depending on analyte, if it is a manual
defrost freezer. It is best if the samples contain carrier protein. Multiple
freeze-thaw cycles should be avoided.
Samples/standard volume use of a larger sample/standard size
(200 µL per well vs. 100 µL per well) may increase sensitivity.
Substrate substrates can vary. However, choosing an alternate
substrate will require additional assay condition optimization. Some
substrates require a longer incubation time to get the curve to a
reasonable height. If the substrate is functioning as expected,
sensitivity may be enhanced by increasing incubation time. Monitor
the plate as it is developing to avoid excessively high backgrounds.
Typically, the incubation time ranges from 10 to 30 minutes. Use the
correct filters required to read the appropriate wavelength for the
substrate chosen. This information is available from the substrate
vendor.Incubation times sensitivity may be increased with a longer
incubation time at room temperature. Be aware that the top of the
curve may flatten out and become unusable, limiting the assay range.
Additionally, background may increase.
Use of a shaker at room temperature may increase sensitivity.
Shakers may be used for some or all of the incubation steps. Incubation
times should be determined empirically.
Washing, follow instructions given in your ELISA Protocol. Insufficient
washing can result in high coefficients of variation (CVs), high
background and poor results.
Sensitivity varies for each antibody pair. Sensitivity is defined by
reliable discrimination from the zero standard. Factors which influence
sensitivity include: capture and detection antibody concentrations
(refer to the grid experiment shown in Figure 2), incubation times and
temperatures, avidity of antibodies for antigens, sample/standard
volumes, pH, diluents and wash buffer formulation. However, there is a
limit to the sensitivity that can be achieved with each antibody pair.
23
Problem Possible Cause Solution
High Background
Insufficient washing
• See washing procedure
• Increase number of washes
• Add a 30 second soak step in between washes
Too much streptavidin-HRP or equivalent • Check dilution, titrate if necessary
Insufficient blocking
• Check blocking solution calculations
• Increase blocking time
BSA impurities • Use high-quality BSA and consider evaluating a different preparation of BSA
Incubation times too long • Reduce incubation times
Interfering substances in samples or standards • Run appropriate controls
Buffers contaminated • Make fresh buffers
No signal
Reagents added in incorrect order, or incorrectly prepared
• Repeat assay
• Check calculations and make new buffers, standards, etc.
Contamination of HRP with azide • Use fresh reagents
Not enough antibody used • Increase concentration
Standard has gone bad (if there is a signal in the sample wells)
• Check that standard was handled according to directions
• Use new vial
Buffer containing FCS used to reconstitute antibodies • Requalify your reagents of choice
BSA impurities • Use high-quality BSA and consider evaluating a different preparation of BSA
Capture antibody did not bind to plate
• Use an ELISA plate (not a tissue culture plate)
• Dilute in PBS without additional protein
Buffers contaminated • Make fresh buffers
Too much signal—whole
plate turned uniformly
blue
Insufficient washing/washing step skipped – unbound peroxidase
remaining • See washing procedure
Substrate Solution mixed too early and turned blue • Substrate Solution should be mixed and used immediately
Too much streptavidin-HRP • Check dilution, titrate if necessary
Plate sealers or reagent reservoirs reused, resulting in presence of
residual HRP. This will turn the TMB blue non-specifically • Use fresh plate sealer and reagent reservoir for each step
Buffers contaminated with metals or HRP • Make fresh buffers
Standard curve achieved
but poor discrimination
between points (low or
flat curve)
Not enough streptavidin-HRP • Check dilution, titrate if necessary
Capture antibody did not bind well to plate
• Use an ELISA plate (not a tissue culture plate)
• Dilute in PBS without additional protein
Not enough detection antibody • Check dilution, titrate if necessary
Plate not developed long enough
• Increase Substrate Solution incubation time
• Use recommended time
Incorrect procedure • Go back to General ELISA Protocol; eliminate modifications, if any
Improper calculation of standard curve dilutions • Check calculations, make new standard curve
Poor Duplicates
Insufficient washing
• See washing procedure
• If using an automatic plate washer, check that all ports are clean and free of
obstructions, add a 30 second soak step and rotate plate halfway through
the wash
Uneven plate coating due to procedural error or poor plate quality
(can bind unevenly)
• Dilute in PBS without additional protein
• Check coating and blocking volumes, time and method of reagent addition.
Check plate used
• Use an ELISA plate (not a tissue culture plate)
Plate sealer reused • Use a fresh plate sealer for each step
No plate sealers used • Use plate sealers
Buffers contaminated • Make fresh buffers
Troubleshooting your DuoSet® ELISA
continued on next page
24
Problem Possible Cause Solution
Poor assay to assay
reproducibility
Insufficient washing
• See washing procedures
• If using an automatic plate washer, check that all ports are clean and free of
obstructions
Variations in incubation temperature
• Adhere to recommended incubation temperature
• Avoid incubating plates in areas where environmental conditions vary
Variations in protocol • Adhere to the same protocol from run to run
Plate sealers reused, resulting in presence of residual HRP which will
turn TMB blue • Use fresh plate sealer for each step
Improper calculation of standard curve dilutions
• Check calculations, make new standard curve
• Use internal controls
Buffers contaminated • Make fresh buffers
No signal when a signal
is expected, but standard
curve looks fine
No cytokine in sample or levels below assay range
• Use internal controls
• Repeat experiment, reconsider experimental parameters
Sample matrix is masking detection • Dilute samples at least 1:2 in appropriate diluent, or preferably do a series
of dilutions to look at recovery
Samples are reading too
high, but standard curve
looks fine
Samples contain cytokine levels above assay range • Dilute samples and run again
Very low readings across
the plate
Incorrect wavelengths • Check filters/reader
Insufficient development time • Increase development time
Coated plates are old and have gone bad • Coat new plates
Capture antibody did not bind to the plate
• Use an ELISA plate (not a tissue culture plate)
• Dilute in PBS without additional protein
Buffer containing FCS used to reconstitute antibodies • Requalify your reagents of choice
Green color develops
upon addition of stop
solution when using
streptavidin-HRP
Reagents not mixed well enough in wells • Tap plate
Edge Effects Uneven temperatures around work surfaces
• Avoid incubating plates in areas where environmental conditions vary
• Use plate sealers
Drift
Interrupted assay set-up • Assay set-up should be continuous – have all standards and samples
prepared appropriately before commencement of the assay
Reagents not at room temperature • Ensure that all reagents are at room temperature before pipetting into the
wells unless otherwise instructed in the antibody inserts
Troubleshooting your DuoSet® ELISA continued
25
ELISA Kit Offerings
Quantikine Kits
Species # of Kits
Human 458
Mouse 140
Rat 53
Porcine 15
Canine 15
Multi-Species 7
Cynomologus Monkey 1
Rhesus Macaque 1
Viral 1
DuoSet Kits
Species # of Kits
Human 732
Mouse 297
Rat 61
Canine 17
Porcine 13
Feline 10
Equine 8
Bovine 5
Rabbit 5
Multi-Species 5
Cotton Rat 4
Guinea Pig 3
Primate 2
Viral 1
C. botulinum 1
Viral 1
Supplemental
ELISA Development Products
Product Catalog #
DuoSet ELISA Ancillary Reagent Kit 1 DY007
DuoSet ELISA Ancillary Reagent Kit 2 DY008
DuoSet ELISA Ancillary Reagent Kit 3 DY009
Clear Microplates DY990
Black Microplates DY991
ELISA Plate Sealers DY992
ELISA Plate-Coating Buffer DY006
Glo Substrate Reagent Pack DY993
Stop Solution DY994
Reagent Diluent Concentrate 3 DY004
Reagent Diluent Concentrate 2 DY995
Reagent Diluent Concentrate 1 DY997
Reagent Additive 1 DY005
Streptavidin-HRP DY998
Substrate Reagent Pack DY999
Wash Buffer Concentrate WA126
Sample Diluent Concentrate 1 (5X) DYC001
Sample Diluent Concentrate 1 (2X) DYC002
EvenCoat™ Streptavidin Microplates CP003, CP004
EvenCoat™ Goat Anti-mouse IgG Microplates CP001, CP002
Sample Activation Kit 1 DY010
Cell Lysis Buffer 1 890713
Cell Lysis Buffer 2 895347
Cell Lysis Buffer 3 895366
Cell Lysis Buffer 5 895890
Lysis Buffer 6 895561
Lysis Buffer 16 895935
Lysis Buffer 17 895943
26
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Global info@bio-techne.com bio-techne.com/find-us/distributors TEL +1 612 379 2956 North America TEL 800 343 7475
Europe | Middle East | Africa TEL +44 (0)1235 529449 China info.cn@bio-techne.com TEL +86 (21) 52380373
For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners.
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