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 ensure the highest level of performance in analyte quantification. 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 full- length, 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

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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

https://www.rndsystems.com http://www.rndsystems.com/elisas http://www.rndsystems.com/services/assay-production-custom-development-services

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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

N or

m al

iz ed

C on

ce nt

ra tio

n

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

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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 gold- standard 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%

Pe rc

en t R

ec ov

er y

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

Pe rc

en t R

ec ov

er y 200%

150%

100%

50%

0% 1:2 1:4 1:8

G-CSF Spike Linearity

Dilution

250%

Pe rc

en t R

ec ov

er y

200%

150%

100%

50%

0% 1:2 1:4 1:8 1:16

Plasma - R&D Systems Plasma - Brand A

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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.

C or

re ct

ed O

.D .

Human β-NGF (pg/mL)

0.01

0.1

1

10

10

100 1000 10000

2013 Data 2003 Data

C or

re ct

ed O

.D .

2 0

0 0

2 0

01

2 0

0 2

2 0

0 3

2 0

0 4

2 0

0 5

2 0

0 6

2 0

07

2 0

0 8

2 0

0 9

2 01

0

2 01

1

2 01

2

2 01

3

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 β-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.

C or

re ct

ed O

.D .

IL-6 (pg/mL)

0.01

0.1

1

1

10

10

rhIL-6 Standard Natural IL-6

100 1000

http://www.rndsystems.com/products/human-beta-ngf-duoset-elisa_dy256 http://www.rndsystems.com/products/human-il-6-duoset-elisa_dy206

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, intra- assay 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

C on

ce nt

ra tio

n (p

g/ m

L)

0 0

100

200

6 9 12 15 Time (months)

50

150

Lot 1 Lot 2 Lot 3 Lot 4

C on

ce nt

ra tio

n (p

g/ m

L) 0

0

100

200 BA

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

N um

be r

of O

bs er

va tio

ns

0

8

12

16

3 0

– 3

9 4

0 –

4 9

6 0

– 6

9

8 0

– 8

9

10 0

– 10

9

1 2

0 –

1 2

9

14 0

– 14

9

16 0

– 16

9

5 0

– 5

9

7 0

– 7

9

9 0

– 9

9

11 0

– 11

9

13 0

– 13

9

15 0

– 15

9

17 0

– 17

9

IL-12 p40 Concentration (pg/mL)

A

2

N um

be r

of O

bs er

va tio

ns

0

4

6

8

10

< 0

.1 9

9

0 .4

0 0

– 0

.5 9

9 0

.6 0

0 –

0 .7

9 9

1 .2

0 0

– 1

.3 9

9

1 .8

0 0

– 1

.9 9

9

2 .4

0 0

– 2

.5 9

9

4 .6

0 0

– 4

.7 9

9

0 .8

0 0

– 0

.9 9

9 1

.0 0

0 –

1 .9

9 9

1 .4

0 0

– 1

.5 9

9 1

.6 0

0 –

1 .7

9 9

2 .2

0 0

– 2

.3 9

9 2

.0 0

0 –

2 .1

9 9

2 .8

0 0

– 2

.9 9

9

4 .8

0 0

– 4

.9 9

9

10 .0

0 0

– 10

.1 9

9

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).

http://www.rndsystems.com/products/human-il-6-quantikine-elisa-kit_d6050 http://www.rndsystems.com/products/human-il-6-quantikine-elisa-kit_d6050 http://www.rndsystems.com/products/human-il-12-il-23-p40-quantikine-elisa-kit_dp400 http://www.rndsystems.com/products/human-il-6-quantikine-hs-elisa-kit_hs600c

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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

TN F- α

M ea

su re

d (%

o f e

xp ec

te d)

0 0

20

60

80

120

1 10 100 1000 sTNF R present (ng/mL)

sTNF RI sTNF RII

O pt

ic al

D en

si ty

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 r eagents whe

n detecting h uman GDNF

. The

absence of o ur proprietar

y blocking re agents result

ed in false p ositives in 9

of 11

samples usin g the Quanti

kine ELISA (1 A). Converse

ly, there were no false pos

itive

results in Co mpetitor A’s

ELISA when our blocking

reagents we re added to

the

diluent (1B). Note that Co

mpetitor A la cked the sen

sitivity neede d to detect G

DNF

in positive co ntrol superna

tes from U-11 8MG and U-8

7MG cell cul tures.

TABLE 1 – FA LSE POSITIVE

HUMAN GD NF READING

S IN 2 COMP ETITOR ELISA

KITS

We next de termined wh

ether remov ing blockers

from the

diluents in th e Human GD

NF Quantikin e ELISA Kit w

ould lead

to false posi tive detectio

n. The Comp etitor B ELIS

A kit was no

longer comm ercially availa

ble, so they were not inc

luded in the

additional te sting. Eleven

serum samp les from appa

rently healthy

individuals w ere tested on

the Human G DNF Quantik

ine ELISA Kit

according to protocol. Sa

mples dilute d in the Qua

ntikine diluen t

were compa red to iden

tical sample s in diluents

that lacked

blocking com ponents. Ide

ntical sample s were also t

ested using

Competitor A ’s Human GD

NF ELISA wit h our blockin

g reagents

added to th e diluent. U

-118MG and U-87MG g

lioblastoma

conditioned media super

nate samples were also te

sted on both

kits with and without our b

locking reage nts. These we

re used as a

positive cont rol7 to ensure

appropriate binding with

and without

blocking rea gents

When using the Quantikin

e diluents as provided in

the GDNF kit ,

no serum sa mples are de

tectable. Wh en the block

ing reagents

are removed from the Q

uantikine dil uents, 9 out

of 11 serum

samples wer e detectable

, giving false positive sam

ple values

(Figure 1A). The U-118M

G and U-87M G conditione

d supernate

samples are detectable w

hether block ing reagents

are used or

not, indicatin g that the blo

cking reagen ts are not sup

pressing the

real GDNF d etection in th

ese positive controls.

When testin g the ELISA

kit from Co mpetitor A,

following the

kit protocol a nd using the

diluents pro vided, 9 out

of 11 serum

samples wer e detectable

(Figure 1B). When block

ing reagents

were added to Competito

r A’s diluents , the serum s

amples were

now undete ctable. The

U-118MG an d U-87MG

conditioned

supernate sa mples are n

ot detectabl e whether b

lockers are

added to the diluents or l

eft as provid ed in the kit.

This indicate s

lower assay sensitivity w

ith Competit or A when c

ompared to

the Quantik ine ELISA, a

lthough the standard cu

rve range is

actually low er on Comp

etitor A (Qu antikine stan

dard curve

range: 9.38 – 300 pg/mL

, Competitor A standard

curve range:

2.74 – 2000 p g/mL).

QUANTIKINE COMPETITO

R A COMPETITO

R A +

BLOCKERS COMPETITO

R B COMPETITO

R B +

BLOCKERS

Standard Cu rve Range:

9.38 - 300 pg /mL

2.74 - 2000 p g/mL

15.6 - 1000 p g/mL

Sample Dilut ion

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-de tectable, sam

ple values << the lowest st

andard curve point

Quantikine S erum Values

Quantikine D iluent

0 50

100 150 200 250 300 350 400 450 500

Se ru

m 7

Se ru

m 8

Se ru

m 9

Se ru

m 1

0

Se ru

m 1

1

Se ru

m 1

2

Se ru

m 1

3

Se ru

m 1

4

Se ru

m 1

5

Se ru

m 1

6

Se ru

m 1

7

U -1

18 M

G

U -8

7 M

G

H um

an G

D N

F C

on ce

nt ra

tio n

(p g/

m L)

Quantikine D iluent w/o Bl

ockers

1A

0 100 200 300 400 500 600 700 800

Se ru

m 7

Se ru

m 8

Se ru

m 9

Se ru

m 1

0

Se ru

m 1

1

Se ru

m 1

2

Se ru

m 1

3

Se ru

m 1

4

Se ru

m 1

5

Se ru

m 1

6

Se ru

m 1

7

U -1

18 M

G

U -8

7 M

G

H um

an G

D N

F C

on ce

nt ra

tio n

(p g/

m L)

Competitor A Serum Value

s

Competitor A Diluent

Competitor A Diluent + Blo

ckers

1B

HUMAN GD NF SERUM V

ALUES ON R &D SYSTEM

S ELISA VS

COMPETITO R A ELISA.

AVOID FA LSE POSIT

IVE ELISA DATA:

IMPROVE P ERFORMAN

CE WITH T HE R&D SY

STEMS ®

QUANTIKI NE® ASSAY

APPLICATIO N NOTE

INTRODUCT ION

Enzyme Link ed Immunos

orbent Assay s (ELISAs) ar

e commonly

used to qua ntify biomark

ers in serum , plasma, and

cell culture

supernates. T hese sample

s contain a v ariety of pro

teins, blood

components , and other

factors tha t can interf

ere with th e

ELISA results , which is co

mmonly refe rred to as a

matrix effect .

Eliminating these factor

s is essenti al to obtain

ing accurate

sample value s. Blocking r

eagents are commonly u

sed in ELISA

kits to reduc e interferenc

e from prote ins in sample

s which may

produce fals e positive re

sults. False positives can

also occur

from non-sp ecific bindin

g on the m icroplate. So

lid phase

blocking age nts are speci

fically design ed to saturat

e unoccupied

binding sites on the ELIS

A plate surfa ce and prev

ent this non-

specific bind ing. R&D Sys

tems uses sp ecialized dilu

ents that are

specifically d esigned to a

lleviate false positives to g

ive the most

accurate res ults. Here ar

e some kit-s pecific exam

ples on how

blocking rea gents were u

sed improve performance

.

THE IMPOR TANCE OF B

LOCKING R EAGENTS W

HEN DETEC TING

HUMAN GD NF

Glial Cell Lin e-derived Ne

urotrophic Fa ctor (GDNF)

is a neurotro phic factor th

at has been shown to pro

mote the sur vival of vario

us

neuronal sub populations

in both the central and

peripheral n ervous syste

ms at differe nt stages of

their develop ment. Neuro

nal

subpopulatio ns shown to b

e affected by GDNF includ

e motor neur ons, midbrain

dopaminerg ic neurons, Pu

rkinje cells an d sympatheti

c

neurons. GD NF is produc

ed by Sertoli cells, type 1

astrocytes, Sc hwann cells,

neurons, pin ealocytes, an

d skeletal mu scle cells. GD

NF

binding to G FR alpha 1 in

duces the rec ruitment of R

et, NCAM-1/ CD56, variou

s integrins, S yndecan-3, o

r N-Cadherin 1-3.

When develo ping the Hum

an GDNF Qu antikine ELIS

A (Catalog # DGD00), it

was discover ed that the s

erum sample values were

not matching reported lite

rature values using comm

ercially availa ble ELISAs

4-6. These comp etitor assays

were achievi ng values tha

t

ranged from non-detecta

ble to > 600 pg/mL for s

erum sample s from appar

ently healthy individuals,

while the sam e samples w

ere

undetectable with the Qua

ntikine ELISA .

FALSE POSIT IVE DETECT

ION IN TWO COMMERC

IALLY AVAIL ABLE

ELISA KITS Knowing tha

t blocking ag ents alleviate

non-specific binding, a fu

rther investig ation into ou

r competitor s’ assays was

conducted b y

adding block ing agents to

their diluent s and testing

them side b y side with th

e diluent pro vided in thei

r kit. As instr ucted in thei

r

respective in serts, serum

samples wer e diluted 1:2

in Competit or A and 1:1

0 with acid t reatment in

Competitor B . Samples we

re

tested neat o n the Quantik

ine® ELISA. A s shown in Ta

ble 1, none o f the samples

were detecta ble in using t

he Quantikin e kit. Howeve

r,

competitor k its detected

false positive GDNF levels

ranging from 15.6 pg/ml t

o 692 pg/ml. Two of the s

amples were detectable w

ith

Competitor A using their d

iluent, but wh en blockers w

ere added, th e samples we

re no longer detectable.

Four of the s amples were

detectable w ith Competit

or B, but whe n blockers w

ere added, th e samples we

re no longer detectable.

http://www.rndsystems.com/products/human-tgf-beta-1-quantikine-elisa-kit_db100b http://www.rndsystems.com/products/human-tnf-alpha-quantikine-elisa-kit_dta00d http://www.rndsystems.com/products/human-timp-2-quantikine-elisa-kit_dtm200

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 freeze- thaw 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)

O pt

ic al

D en

si ty

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)

O pt

ic al

D en

si ty

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

http://www.rndsystems.com/products/human-il-6-quantikine-hs-elisa-kit_hs600c

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.

2 3 4 5 6 7 8 9 10 11 121

� 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.

�

2 3 4 5 6 7 8 9 10 11 121

P late

w ash

er

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 orange- brown 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 strepta- vidin-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 “mini- grid”, 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

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26

Notes _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________

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Notes _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________ _____________________________________________________

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