R-Biopharm – dedicated to food safety

Good ELISA Practice

Manual
Good ELISA Practice – Manual 2
3

Disclaimer

The Good ELISA Practice (GEP) manual provides
a comprehensive overview for both - beginners
and advanced analysts - in order to improve the
quality of performed ELISA analysis. It represents
a guideline helping to establish reasonable
framework conditions and conditions of use, which
have to be complied with to achieve the best
possible performance when using R-Biopharm
ELISA test kits.
The GEP manual makes no claim to completeness,
but describes only certain requirements.
The compliance with such minimum standards
does not guarantee the achievement of correct
analytical results, but serves to increase the quality
of assessment at ELISA-analysis. The manual
applies in addition to the respective detailed
instructions of each test-kits. For the proper use
of test-kits, the respective instruction manual
is decisive and shall take priority over this GEP
manual.
The GEP manual is continuously up-dated. Please
make sure that this is the newest version of the manual
prior to performing the ELISA-analysis. The manual
can be retrieved, printed and downloaded from
https://food.r-biopharm.com/technologies/elisa/

Introduction
The GEP manual provides a comprehensive
overview for both beginners and advanced
analysts in order to improve the quality of
performed ELISA analysis.
The enzyme-linked immunosorbent assay
(ELISA) is an antibody-based test method. This
widespread technology is easy to use, sensitive,
fast and reliable. Additionally, ELISA is robust
and mostly used for quantitative analysis.
on a well-established analytical method that
meets the requirements of modern laboratories.
RIDASCREEN® tests show a high sensitivity and are
characterized by robustness. These test systems
are used by food manufacturers to test raw
materials or finished goods.
Furthermore, ELISA requires only basic lab
equipment and is easy to use compared to HPLC
and/or LC-MS/MS.

With more than 30 years of experience in This manual is divided into three chapters, which
developing ELISA test kits, R-Biopharm relies follow the workflow of an ELISA based analysis:

Chapter 1: Chapter 2: Chapter 3:

basic knowledge about the test describing the implementation explaining the process of data
principle, ELISA components of the analysis consisting of evaluation from measurement
and required laboratory sample preparation and ELISA to interpretation.
equipment is given. procedure.

For any comments or feedback please contact your local distributor

trainer@r-biopharm.de
Good ELISA Practice – Manual 4

Content

1 ELISA Basics 6
1.1 Antibody – Antigen Detection 6
1.2 Analyte 6
1.3 ELISA Formats 6
1.3.1 Sandwich-ELISA 7
1.3.2 Competitive ELISA (formats) 8
1.4 ELISA components 8
1.4.1 Microtiter plate (MTP) 9
1.4.2 Conjugate and Substrate 9
1.4.3 Standards (Calibrator) 9
1.4.4 Buffers 9
1.4.5 Stop-solution 10
1.4.6 Additional components 10
1.5 Lab equipment and its maintenance 10
1.5.1 Pipettes 10
1.5.2 ELISA plate washer - automated systems 11
1.5.3 Microtiter plate (MTP) reader for ELISA 11
1.5.4 Automation 12
1.5.5 Additional equipment 12
1.5.6 General words of advice 12
1.6 Good laboratory practice (GLP) 13
1.7 Test kit labeling 13

2 Sample preparation and test implementation 14

2.1 Pipetting techniques 14
2.1.1 General pipetting instructions 14
2.1.2 Forward pipetting using lab pipettes 15
2.1.3 Reverse pipetting using lab pipettes 15
2.1.4 Pipetting of organic solvents 16
2.1.5 Good pipetting techniques to improve the pipetting performance 17
5

2.2 Handling of samples 18

2.2.1 Storage of samples 18
2.2.2 Sample drawing 18
2.2.3 Sample preparation 18
2.2.4 Use of frozen samples 19
2.2.5 Certified reference material 19
2.3 Preparation and handling of components 20
2.3.1 Storage of kits 20
2.3.2 First in – first out 20
2.3.3 Pre-Warming 21
2.3.4 Temperature control 21
2.3.5 Avoiding of contamination and sample mix-up 22
2.3.6 General test handling 23
2.3.7 Time management for pipetting 24
2.3.8 Correct washing 25
2.3.9 Storage of unused components for further experiments 26
2.3.10 Interchange of reagents between tests and batches 26
2.3.11 Security references 27
2.4 Stopping and measuring of the ELISA 27
2.5 Parallel performance of tests 28

3 Data evaluation and interpretation of results 29

3.1 Determination of unknown samples by standard curve 29
3.2 Standard curve fittings 29
3.3 Standard curves of sandwich and competitive ELISAs 30
3.4 Spectrophotometer and Software 31
3.5 Determination of analyte concentration 32
3.6 Measuring range and dilution factor 32
3.7 Units and dimensions 33
3.8 Limit of detection and quantification 33
3.9 Trueness and recovery 34
3.10 Specificity and cross reactivity 35
3.11 Interferences and matrix effects 36
Good ELISA Practice – Manual
1 ELISA Basics
1.1 Antibody – Antigen Detection
Antibodies are proteins produced in plasma cells of
vertebrates as part of the adaptive immune system
against structures (antigens) which are recognized
as foreign to the body.
Antibodies bind to their antigens by a distinct
pattern of ionic and hydrophobic interactions,
hydrogen bridge linkages and Van-der Waals
forces. The interaction between antibody and its
antigen is selective and highly specific, similar to a
lock and key. The Enzyme Linked Immunosorbent
Assay (ELISA) is based on this selective and specific
antibody-antigen recognition. Many formats of
qualitative or quantitative ELISA tests have been
established, a selection of them are illustrated in
the following chapters.
1.2 Analyte
The claimed (antigen) analyte of an ELISA could be:
1 A defined chemical substance e.g.
aflatoxin B1
2 A group of defined chemical substances e.g.
aflatoxins B1, B2, G1 and G2
3 A specific protein e.g. staphyloenterotoxin A
1.3 ELISA Formats
Currently the following three different systems exist
(see page 8 Figure 2-4). In each system, the result
is measured on the basis of the optical density.
6
Performing an ELISA involves at least one specific
antibody for a particular antigen. A main principle
is that one of these immunological components
is immobilized to a solid phase, the cavities of
a microtiter plate. The analyte (antigen) from
the sample interacts with the antibody-antigen
system. This interaction can be visualized by
enzymes, linked to secondary antibodies or
antigens, and indicate if an antibody-antigen
binding has occurred. An added substrate is
converted by the coupled enzyme resulting in a
change of color, which can be measured with a
spectrophotometer.
4 A group of specific proteins e.g.
staphyloenterotoxins A, B, C, D, and E
5 A more or less defined group of proteins
from a food commodity e.g. caseins (as a
part of milk proteins)
6 A food commodity e.g. peanut protein
7

1.3.1 Sandwich-ELISA

Sandwich ELISA are often used in protein analysis
such as food allergens. The analyte (antigen)
needs to be large enough to have two binding
sites (epitopes). In this method a specific antigen
(analyte) in the sample binds to antibodies attached
to the solid phase of a microtiter plate. After
washing, conjugated antibody is added which binds
to a second binding site (epitope) of the antigen
(analyte). After washing, substrate is added and
the results can be measured via the optical density
(OD). The signal is proportional to the amount of
antigen (analyte) (see Figure 1, step 6, Figure 2).

Test Procedure Test Principle

Place the required number of Microwells are coated with antibodies to

1 microwell strips in the frame the target protein

Add 100 µl of standard or sample Standard and samples are added to

2 their respective wells

10 min incubation at room temperature

Wash 3 times with washing buffer Conjugate is added which binds to

3 3x Add 100 µl of antibody - enzyme already bound target protein
conjugate

10 min incubation at room temperature

Wash 3 times with washing buffer Red Chromogen Pro (Substrate/

4 3x Add 100 µl of Red Chromogen Chromogen) is added to produce a
Pro (Substrate/Chromogen) colour change from red to blue

10 min incubation at room temperature in the dark

Add 100 µl of stop reagent H2SO4 stops the substrate/chromogen

5 reaction

Results are read in a MTP The more yellow colour the more target
6 A reader at 450 nm allergen is present in the sample

conc.

Figure 1: Example of test procedure and principle – RIDASCREEN® Allergen Sandwich ELISA
Good ELISA Practice – Manual 8

1.3.2 Competitive ELISA (formats)

There are different formats for a competitive ELISA.
The competitive ELISA (Figure 3) consists of a
microtiterplate where the antibody is bound to the
surface of the well. The analyte from the sample
and the enzyme-analyte conjugate are added to
the well. The competition on antibody binding
sites starts. After washing, substrate is added and
the measured OD value is inversely proportional
to the amount of analyte in the sample. The more
analyte is present, the smaller the OD value. This
method is suitable to measure samples with just
one epitope as well as small analytes such as
mycotoxins or antibiotics.
The difference of the direct to the indirect
competitive ELISA (Figure 4) is that an additional
catcher antibody is bound to the microtiter plate.
There are also competitive ELISA in which the
antigen is bound to the MTP well. This is the case
for RIDASCREEN® Gliadin competitive (R7021).

Substrate
E E

E Substrate

Antigen
Substrate

Figure 2: Schematic structure of Figure 3: Schematic structure of Figure 4: Schematic structure of

sandwich ELISA competitive ELISA indirect competitive ELISA

1.4 ELISA components

Several components are needed for an ELISA test
system: Microtiter plates (MTP), conjugate and
substrate, standards (calibrators), buffers and
stop-solution. Furthermore, it is essential to use
controls to be sure that the test procedure is
working correctly.
9

1.4.1 Microtiter plate (MTP)

The MTP (96 or 48 wells), is the basis for the
analysis. In every well the antibody or antigen
(depends on the format) is bound to the surface.
A common plate material is polystyrene although
other materials can be used. The material is
activated by β- or γ-radiation by the manufacturer
of these plates. Without this activation no or only
minor binding of antibodies or antigens occurs.
In every well the antigen-antibody reaction and
the conjugate-substrate reaction takes place.
Lastly, the reaction is stopped with a specific stop-
solution and the optical density is measured.

1.4.2 Conjugate and Substrate

Antibodies or analytes linked to an enzyme are chromogenic mix which reacts with the conjugate
called conjugates. The linked enzyme converts its enzyme. The result is a colored solution of which
specific substrate into a colored bluish product. the optical density can be measured.
The substrate is usually a hydrogen peroxide/

QUALITY ASSURANCE CERTIFICATE

RIDASCREEN Gliadin
Art. No.: R7001 Lot: 14383 Expiry: 2014-12
1.4.3 Standards (Calibrator) R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications

Standards

All quantitative ELISA systems are calibrated by 2.4

Standard curve
Std.
Conc.(ppb)
CV(%)
n
mean

2.2

the use of standards. Therefore, the samples with 2.0

Std1
0.00
8
0.058
9.3

unknown concentrations and a set of standards 1.8

Std2
5.00
8
0.294
1.6 5.7
A

with known concentrations are analyzed in parallel b

s
o
1.4 Std3
10.00
8
0.635
r 4.2

on one plate. The result will be a calibration curve b 1.2

a Std4 8
n 1.0 20.00 1.094
c 2.8

(with the associated mathematic formula) built out

e
0.8 Std5 8
40.00 1.683
0.6 2.2

of the measured OD values and the concentrations 0.4

Std6
80.00
8
2.366
2.1

of the standard (Figure 5). Based on this, the

0.2

0.0
5.00 10.00 20.00 40.00 80.00

analyte concentration in the sample can be Concentration (ppb)

calculated. Figure 5: Calibration curve of a sandwich ELISA

Lot No. Expiry

Microwell plate 15043 2014-12
Standards 11353 2015-04
Conjugate 11353 2015-04
Buffer1 12173 2015-03
Substrate/Chromogen 15183 2015-10
Stop solution 15183 2018-04
Washing buffer 11243 2015-11

Please note:

1.4.4 Buffers The absorbance for the standards may decrease during the shelf life of the kit. The general shape of the
curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet 8.
Indication of instability or deterioration of reagents.

sign.: Edda Rohm Date: 2013-09-19

Quality Assurance Representative

All ELISA systems contain components of biological has also an influence on test kit performance. For
Remark: This document has been created electronically and is therefore valid without a signature.

origin. For long term storage of these components the convenience of test kit users, these buffer are
and for proper function during the testing included ready-to-use or as concentrates. They
The R-Biopharm group is DIN EN ISO 9001 certified.
procedure, pH-value and ionic strength needs to are also used for sample preparation and washing www.r-biopharm.com

be constant. Often the kind of buffer component procedures of the microtiter plates.
Good ELISA Practice – Manual
1.4.5 Stop-solution
The stop-solution terminates the enzyme-
conjugate-reaction. In most cases, sulphuric acid
at low concentrations is used. By stopping the
1.4.6 Additional components
For most systems a positive and negative control
is recommended, for example a spiking solution.
1.5 Lab equipment and its maintenance
Depending on the requirements of the test
system, various specialized laboratory equipment
are needed for the different steps e.g. pipettes,
equipment for plate washing, incubator for
constant temperature, ELISA reader and a software
1.5.1 Pipettes
A pipette is used to transfer a precisely defined
volume of a solution to e.g. the wells of a microtiter
plate. Pipettes are crucial for results with a high
precision. Therefore, a proper pipetting technique
as well as regular calibrated pipettes are important.
Different kinds of pipettes exist:
• Single-channel pipettes with fixed volumes
e.g. 50 µl
• Single-channel pipettes (e.g. with variable
volumes between 10 and 100 µl); used normally
for samples and standards
10
reaction, the color will change from blue to yellow
and will remain stable until measurement within
10 min.
This gives the opportunity to control the test
system.
to calculate concentrations. However, not all of
the equipment mentioned here is needed for all
test systems. In every case, regular maintenance
and calibration is required for machine and lab
equipment which are used.
• Multistepper pipettes (with the possibility to
pipette multiple times of a specific volume); used
normally for addition of antibody or conjugate
solutions
• Multi-channel pipettes (with the possibility to
pipette the solution in 8 or 12 cavities at the
same time); used normally for washing steps or
addition of antibody or conjugate solutions
• Bottle top dispenser (used normally for washing
steps)
• Fully automatic machines (all pipetting and
incubation steps are performed automatically)
11

1.5.2 ELISA plate washer - automated systems

After every incubation step (except the incubation
with the substrate) the ELISA plate has to be
washed with washing buffer. Washing is a crucial
step when performing an ELISA to obtain results
with high precision.
Washing can be done manually with an 8-channel
pipette or an 8 channel manifold (see Figure 15).
Sometimes customers use automated ELISA plate
washers. These washers are laboratory instruments
where users load a plate and select a program. The
washers then dispense, soak and aspirate wash
buffer from the plate in seconds. When using such
instruments thorough cleaning of the washer is
important to avoid cross contamination. Refer to
the manufacturer’s instructions referring cleaning
between different runs.
The washing procedure is an important step and
has to be validated if deviated from the described
product information.

1.5.3 Microtiter plate (MTP) reader for ELISA

The ELISA reader is a spectrophotometer, which

allows to measure the optical density (OD – The
‘OD’ is the amount of attenuation, or intensity lost,
when light passes through an optical component.
The term ‘absorption’ is also used). To calculate
the concentration of a sample a calibration curve is
used. Regular maintenance by experts is essential
for exact results. The RIDA®ABSORBANCE 96 (Art.
Figure 6: RIDA®ABSORBANCE 96, Art. No. ZRA96FF
No. ZRA96FF) is ideal for the R-Biopharm test kits.
Good ELISA Practice – Manual 12

1.5.4 Automation

One possibility for working with an ELISA is be validated and calibrated for your test system.
the use of an automated system which allows Examples for automation are the ThunderBolt® and
you to test your samples without any manual Bolt™. For further information please contact:
steps. Therefore, the automated system has to sales@r-biopharm.de.

Figure 7: Bolt™, Art. No. ZBOLT Figure 8: ThunderBolt®, Art. No. ZTB

1.5.5 Additional equipment

In some tests, an incubator is required to

guarantee a stable temperature during the test
run. Sometimes, a seal or a protecting plate
cover are necessary to prevent evaporation or
contamination.

1.5.6 General words of advice

To ensure a high precision of the results, the

equipment should be calibrated regularly. Please
ask the manufacturer for the calibration interval
and include this in quality control plans.
13

1.6 Good laboratory practice (GLP)

Dependent on the toxicities and contagiousness
of the used materials, different levels of protective
actions are necessary to guarantee health and
safety of the user. However, basic protective
clothing is already required to avoid contamination
of the samples, which would lead to incorrect
results. The following equipment is a minimum
requirement for every lab:
However, it is necessary to study the Safety Data
Sheets (SDSs) carefully for all chemicals substances
used. The SDS contains information about the
dangers when working with a particular
substance, required protective measures, as well
as required actions in case of emergencies.
The SDS is available on request for each product.
Please contact: sales@r-biopharm.de.

• Lab coat • Gloves

• Eye protection • Fume hood
(occasionally)

1.7 Test kit labeling

To ensure the correct handling and storage of your The expiry date is particularly important, as the
components please read the Instructions for Use specified behavior can only be guaranteed until
(IFU) or the test kit insert. On all components the then. After expiration the ingredients can degrade
follow information is stated (if applicable): and the results can decrease in accuracy.

• Product name • Lot number

• Article number • Storage temperature
• Name of the • Concentration
component
Good ELISA Practice – Manual 14

2 Sample preparation and test

implementation

2.1 Pipetting techniques

Accurate and precise pipetting is crucial in ELISA
analytics, particularly at high sensitivity levels
where a small mistake in pipetting can induce
large differences in the final test results. Be
consistent during pipetting and do not change the
technique while pipetting an assay. Be prepared
before starting pipetting e.g. put all standards in
a row and arrange all samples in a consistent way
to allow uniform pipetting. Ensure that enough
pipette tips are prepared and a waste container is
in place.
The two pipetting techniques used for ELISA are
forward pipetting (standard pipetting) and reverse
pipetting. The forward pipetting technique is
recommended for aqueous solutions. Using this
technique, some liquids may induce bubbles or
foam during pipetting. As an alternative, reverse
pipetting lowers this risk and is recommended for
liquids with higher viscosity. However, it requires
more liquid volume (dead volume) and is more
error- prone at high volume transfers.

2.1.1 General pipetting instructions

Forward pipetting is a technique to dispense Before starting pipetting with an adjustable pipette:
a measured quantity of liquid by means of air 1. Adjust to the desired volume
displacement pipette. The technique is mainly 2. Check adjusted volume
recommended for aqueous solutions, such as
buffers, or diluted acids or alkalis. In case of
solutions with a high viscosity or a tendency to
foam, reverse pipetting is more suitable.

Forward Reverse
Ready position

First stop

Second stop

Figure 9: Schematic illustration of forward and reverse pipetting techniques when using a single channel pipette (piston pipette).
Blue blocks indicate the steps where the pipette needs to be placed in liquid to aspirate. Grey blocks indicate the target wells.
15
2.1.2 Forward pipetting using
lab pipettes
11 Put a new tip on your pipette and check for a
firm fit.
21 Press the operation button to the first stop.
31 Flush pipette tips before use. Pipette tips from
some manufacturers need to be flushed before
aspiration and dispensing of the appropriate
liquid. Please check the according manual.
In case of any doubt, flush the tip before
pipetting.
41 Put the pipette tip approx. 1 cm deep into the
liquid. Slowly release the operation button to
the ready position and wait until the desired
liquid volume has been aspirated. Ensure that
no bubbles or foam occurs in the pipette.
51 Remove excessive liquid from the outside of
the tip by touching the test tube with the tip.
61 Dispense the liquid into the desired well by
pressing the operation button to the second
stop.
71 Remove the tip to waste.
2.1.3 Reverse pipetting using
lab pipettes
11 Put a new tip on your pipette and check for a
firm fit.
21 Press the operation button to the second stop.
31 Immerse the pipette into the liquid. Slowly
release the operating button to ready position
and wait until the desired liquid volume has
been aspirated. Ensure that not bubbles or
foam occurs in the pipette.
41 Remove excessive liquid from the outside of
the tip.
51 Dispense the liquid into the desired well by
pressing the operation button to the first stop.
Ensure that no liquid remains on the outside of
the tip.
61 For repetitive liquid pipetting, press the
operation button to the first stop and repeat
steps 3 - 5.
71 Remove the tip to waste.
Good ELISA Practice – Manual 16

2.1.4 Pipetting of organic solvents

Organic solvents show high vapour pressures
which can affect precise pipetting. The use of
pipettes with air displacement technique to transfer
organic solvents may lead to evaporation of the
Figure 10: Pipetting of organic solvents with pipettes using the
air displacement technique. Particular care should be taken to
prevent evaporation into and leaking out of the tip (A).
Flushing the pipette before liquid transfer helps to transfer the
correct volume (B).

solvent or a leaking out of the tip (Figure 10). For

the pipetting of organic solvents we recommend:

1 Multistep pipettes which use the positive

displacement technique

2 Serological pipettes for larger sample

volumes, since the graduation allows for the
pipetting of exact volumes

3 Bottle top dispensers (A) (B)

4 Pipettes designed especially for the handling
of organic solvents

If single channel pipettes are used to transfer
organic solvents, the pipette tip and the air inside
the pipette needs to be saturated with organic
solvent vapour before pipetting the desired
volume. For this aspirate and dispense the organic
solvent at least 3 times before the desired volume
is transferred. Use appropriate quality control
procedures to monitor the correctness of these
kinds of pipetting steps.
17

2.1.5 Good pipetting techniques to improve the pipetting performance

Immersion depth
Only the top of the pipette tip is immersed into
the standard or sample solution (see Figure 11 a).
When immersing too deep, then too much liquid air
is aspirated. When the pipette tip is too close to bubble

the surface, then air can be aspirated.

Rhythm and speed

A consistent pipetting rhythm helps to avoid jerky
air aspiration (see Figure 11 b). Press the operating
button and then slowly release the button. This a b c
avoids jerky air aspiration and liquid rocketing
<20 °
upwards contaminating the interior of the pipette.

Pre-rinsing
Pre-rinsing equalizes the air temperature and
pressure inside the tip with the temperature of the
sample. During pre-rinsing the plunger is pressed
and released 2 to 3 times (Figure 11 c).
Immersion angle
Best is to hold the pipette close to vertical.
Anything greater than 20 ° of vertical reduces
accuracy of pipetting (see Figure 11 d)
Dispensing
To release sample or standard solution hold the
pipette tip close to the well wall (see Figure 11 e).
Do not touch
well wall
d e
a Only the top of the tip is immersed
b A consistent pipetting helps to avoid jerky air aspiration
c During pre-rinsing the plunger is pressed and released
2-3 times
d The pipette is hold close to vertical during pippeting
e During releasing the top of the pipette is close to the
well wall
Figure 11: Best practice pipetting
Good ELISA Practice – Manual
2.2 Handling of samples
2.2.1 Storage of samples
Unwanted contamination of samples can influence
test results significantly. If there are signs of
unwanted contamination or spoilage do not use
the sample and request a new one. Store the
sample according to the test kit manufacturer
or according to the best scientific knowledge.
Generally, samples should be prepared and tested
2.2.2 Sample drawing
The drawing of a representative sample is a crucial
step. For some parameters, legislations apply
on how to take a representative sample (e.g.
mycotoxins). Please follow these rules precisely. If
there are no rules, samples should
2.2.3 Sample preparation
For sample preparation please follow the
instructions for use provided with the test
kit. Sample preparation involves typically
homogenisation and extraction of the
analyte. Sample preparation involves typically
homogenisation and extraction of the analyte.
Changes or variations may lead to incorrect
test results. Make sure to use only suitable and
18
immediately whenever possible. If storage cannot
be avoided, check for optimal storage conditions
and analyze them as soon as possible. All samples
need to be correctly labelled and sealed to avoid
evaporation or dry-out. Inappropriate storage
conditions may influence later analysis and alter
test results.
be homogenized as much as possible. Inform a
customer beforehand about a minimum sample
amount. State in the test report that the result
refers to the sample as it was sent.
maintained equipment for sample preparation. For
any related questions please contact R-Biopharm.
Depending on the parameter to be tested,
instructions for use may contain information on
how prepared samples can be stored for later
analysis. Please follow these instructions carefully
or prepare samples directly before analysis.
19

2.2.4 Use of frozen samples

Before further use, samples must be completely
thawed. Thawing of frozen samples should
be performed at 4°C or at room temperature,
dependent on analyte stability.
In case of unprepared, non-liquid samples
check the instructions for use for further sample
preparation and homogenization.
Liquid samples must be thoroughly mixed
before they can be used for analysis. To achieve a
homogeneous sample carefully vortex or invert
the sample. Foam formation or intensive mixing
should be avoided since it may denature proteins.
A further sample preparation of liquid samples
(e.g. in case of milk) may be necessary before
analysis. Please check the instructions for use for
further information.
Depending on the sample, freezing and thawing
can lead to crystallization or coagulation. Avoid
freeze-thaw cycles wherever possible, since it can
change sample integrity and alter test results. If
possible, aliquot liquid samples before storage at
‑20 °C to avoid freeze-thaw cycles.

2.2.5 Certified reference material

Certified reference materials (CRM) are naturally Figure 12: R-Biopharm offers a wide range of CRMs for
mycotoxin analysis
contaminated, homogeneous matrices whose
analyte content has been exactly and reliably
determined (Figure 12). The regular use of CRMs
is recommended for quality assurance to establish
traceability. This allows checking the trueness and
precision of experimental procedures and for the
testing of handling skills. If no reference material
is available, the use of control samples or matrices
spiked with defined analyte concentrations are
recommended.

Trilogy® Analytical Laboratory is one of the few

producers of certified, naturally contaminated
reference materials and certified mycotoxin
standards. Additionally, naturally contaminated
quality control materials and analytical standards
for daily quality assurance are available.
Good ELISA Practice – Manual 20

2.3 Preparation and handling of components

2.3.1 Storage of kits

The expiry date printed on the outer label applies Figure 13: Storage conditions and expiry date are printed
on the outer label of each ELISA test kit
to all reagents contained in the kit. To maintain the
shelf life, store the kit at conditions noted on the
outside label of the package (Figure 13).

Most kits have to be stored in a dry place and

at 2 - 8 °C. Freezing of components needs to be
avoided. If using a refrigerator for storage, make
sure the kits are not stored close to the back wall
to avoid freezing. Improper storage of kits or
components may lead to damage. Freezing of test
components may decrease test performance and
lead to invalid test results or –as an extreme- non-
functional test kits. Indicators for false storage are
decreased or non-detectable optical density and
the alteration of test results.

2.3.2 First in – first out

The expiry date of the kit is labelled on the outer The exposure of the ELISA kit to cold/warm cycles
label of the kit package. At least until this date the should be kept as low as possible. We recommend
kit will perform within specifications. Additionally, that samples should be collected. Testing of higher
every kit component has its own expiry date which sample numbers at once reduces the expenditure
is identical or even exceeds the expiry date of the of time per sample. Rather test higher sample
test kit. numbers at once than test only a few samples
consecutively. Please check the instructions for use
If there is more than one kit on hand, it is for relevant limitations.
recommended to use the first in – first out
principle. This means the kit with the shortest
expiry date on the outer label should be used first.
We recommend indicating the date of the first use
on the outer label of the kit box to avoid a mix-up
of this principle.
21

2.3.3 Pre-Warming

All reagents need to be at room temperature Figure 14: Bring all test kit components to room temperature
before use and perform the test at 20 - 25 °C (68 - 77 °F)
before they can be used in the test. Take all
components out of the kit package before use
and allow them to reach room temperature
(20 - 25 °C). Larger bottles and greater 20 - 25 °C/
volumes may require more time to reach room 68 - 77 °F
temperature. Check the temperature of the
components in any case of doubt (Figure 14).

After use it is recommended that all components

are put back into the kit box to avoid the
unintended mixture of components with
components from other kits or lots. Return the
kit back to the recommended storage conditions
as soon as possible (see outer kit label for storage
conditions).

2.3.4 Temperature control

ELISA tests are sensitive to temperature
fluctuations. Therefore, try to stabilize and
control laboratory conditions. This includes the
temperature during photometric analysis. Perform
ELISA tests between 20 and 25 °C and avoid
conditions that are able to drastically change the
temperature or increase evaporation. ELISA tests
should be prevented from direct exposure to
sunlight and ventilations. Cold lab ware and cold
benches may also influence the temperature. It
is helpful to isolate the microtiter plate from the
bench surface by performing the test on a suitable
underlay. A cheap and easy solution is the use of
paper towels.
Good ELISA Practice – Manual 22

2.3.5 Avoiding of contamination and sample mix-up

A clean and reproducible way of working is crucial Figure 15: Carefully label all used containers and document
your labelling.
for optimal results in food and feed analysis. A very
common source of contamination is insufficiently
cleaned re-usable lab ware. To avoid this, it is
highly recommended to use solvent-resistant
single- use lab ware. If this is not possible,
re-usable lab ware should be laboratory sterile
and free of contamination. We recommend using
a laboratory dishwasher or equivalent. Use quality
control blank samples to check for contamination.

Reagents should be handled with calibrated

devices, clean pipettes and containers. Only take
the amount of reagent needed and do not put
reagents back into the container once it has been
removed. Use separate containers and pipette tips
for every reagent to prevent cross contamination.

Make sure to label all containers correctly (Figure

15). Exchange the single use lab ware as often as
needed.
23
2.3.6 General test handling
Before you start, read the instructions for use
enclosed in the kit. Prepare all extraction solutions
and buffers according to these instructions and
follow the described procedures to obtain optimal
results. To allow an unobstructed test procedure
it is helpful to prepare a pipetting scheme before
you start with your experiment.
Depending on the ELISA, antibody and conjugate
solution may need to be diluted prior to use.
These dilutions should be prepared directly before
use and should not be stored for further use.
Contaminated or incorrectly stored conjugate
solutions may have a reduced enzyme activity or
may induce a background signal.
To obtain optimal results, test samples and control
samples (standards, reference samples) have to
be available in the same diluent. Strictly following
the sample preparation protocol ensures this. The
pipetting into the plate needs to be performed
quickly and without interruptions at every step of
Figure 16: The ‘pre-plate’ contains at least 150 µl of standard
or sample per well. To the 7 strips of the pre-plate 5 standards
and 23 samples have been added in duplicate (i.e. 56 wells
in total). Using an 8-channel multi pipette, exactly 100 µl of
the test procedure. For the transfer of samples we
recommend single channel pipettes, or, if a pre-
dilution plate is used, a multi-channel pipette. For
the pipetting of antibody and enzyme solutions,
multistep pipettes are the best option.
Between handling steps a dry-out of the wells has
to be prevented.
Use of pre-plate: In the case of the
RIDASCREEN®FAST Allergen test kits do not use
more than three strips (24 wells) at a time. If more
than three strips are needed, a second uncoated
plate (e.g. low binding from Greiner bio-one Cat.-
No. 655101) should be used as a pre-plate to avoid
a time shift over the microtiter plate. All standards
and samples are pipetted into the uncoated
plate (at least 150 μl per well) and then quickly
transferred to the coated microtiter plate with an
8-channel pipette (see Figure 16). Remember: the
reaction starts when the first solution is added to
the coated plate.
standards or samples are transferred quickly to the coated
plate. The pipette tips are flushed with the standard or sample
solution, each tip is only used once.
Good ELISA Practice – Manual
2.3.7 Time management for pipetting
In general, keep the pipetting technique as
consistent as possible and the absolute pipetting
time to a minimum. Pipetting of standards and
samples is general the most time consuming and
laborious step when performing an ELISA.
Let’s consider the following example using the
sandwich ELISA RIDASCREEN® Gliadin (Art. No.
R7001). Standards and samples, altogether 6 strips
(6 x 8 wells) are pipetted, in 6 minutes. Thereafter
the plate is incubated for 30 minutes. Please note:
• Well A1 will be incubated for 6 + 30 minutes
because it was pipetted first
• Well H6 is incubated for only 30 minutes because
it was pipetted last (so results might be lower).
• It is crucial to understand the underlying ELISA
principle to identify ‘time sensitive’ pipetting
steps.
• In the sandwich ELISA RIDASCREEN® Gliadin
(Art. No. R7001) the ‘antigen-antibody-reaction’
starts when the sample is added to the wells. So
the ‘time sensitive’ step is the sample pipetting.
• In the competitive ELISA RIDASCREEN®
Ochratoxin A (R1311) the plate is already coated
with the specific antibody and the reaction
starts when the sample is added. So the ‘time
sensitive’ step is the sample pipetting.
• In the competitive ELISA RIDASCREEN®FAST
Fumonisin (Art. No. R5602) the reaction starts
when the enzyme conjugate is added. To avoid
an ‘optical density (OD) shift’ from the first to
the last well, it is recommended to use a stepper
pipette.
24
However, it is not recommended to pipette in a
rush as mistakes such as pipetting into wrong
wells, etc. may occur.
It is very important that all samples are handled
in a comparable way. Strictly follow the pipetting
order and the incubation times noted in the
instructions for use. To obtain comparable results
in all wells, the incubation time of each single well
needs to be identical.
To achieve this, start the clock after having
pipetted a component starting a reaction
(standards or samples, antibody solution,
conjugate solution) into the last well. In case of
substrate/chromogen solution, start the clock
before pipetting the solution into the first well.
Stop the substrate/chromogen reaction after the
defined time by adding stop solution in the same
order substrate/chromogen solution has been
added. It is very important to meet the incubation
times noted in the instructions for use.
The activity of the chromogen may be influenced
by light. Cover the plate to protect the chromogen
from light and store it in the brown flask in which
it is delivered. To stop the reaction use the stop
solution delivered with the kit.
25
2.3.8 Correct washing
Washing is a crucial step to remove all unbound
components that might influence reactions or
lead to false results. For washing only use washing
buffers recommended in the instruction for use.
Many kits contain washing buffer salts or solutions
that can be used to prepare ready to use washing
buffers. This removes the necessity to weigh
reagents to prepare the own buffers. For stability
and storage information on the individual washing
buffers delivered with the kits, see the instructions
for use. As all other components, washing buffers
need to be at room temperature before use. Follow
the specific recommendation of the instructions
for use regarding the number of washing steps.
At the end of the incubation steps pour the liquid
out of the wells and tap the microtiter plate holder
vigorously upside down on absorbent paper to
ensure the complete removal of the liquid from
the well. All liquid has been successfully removed
when no signs of liquid remains on the paper towel.
Most ELISA tests require 250 - 300 µl of washing
buffer per washing step and well. Add the washing
buffer and remove the liquid by pouring out and
tapping. Repeat the washing step 3 - 5 times
(see instructions for use). For washing steps it is
recommended to use a bottle-top dispenser (e.g.
Brand, 4720420) connected to a 8-channel manifold
(e.g. Brand, 704526) as shown in Figure 17.
Figure 17: Washing of ELISA
plate using a bottle top
dispenser connected to
8-channel manifold
Buffers, tubes, manifolds and washing needles
need to be kept free from contamination of
microorganisms. Keep the device clean and if
necessary remove contaminated parts. In case of
any doubt, use the manual washing procedure or
contact the manufacturer of the washer.
Automated washing systems are not available at
all testing sites. Due to this we highly recommend
the use of the relatively cheap and easy to
handle bottle top dispensers with an 8 - or 12
-fold manifold for the washing procedure. Other
manual washing techniques like washing bottles
may not allow to treat every well exactly the same
and should not be applied.
Washing steps should be performed fast but
efficiently and accurate. Make sure that the time
between addition of washing buffer to the first
and the last well is as short as possible. This
ensures that wells do not dry-out and it minimizes
differences in incubation times. Despite fast
working speed pay attention to accuracy. Spillover
of liquid from one well to another needs to be
avoided. In case of any doubt add an additional
washing step.
Good ELISA Practice – Manual
2.3.9 Storage of unused components for further experiments
Microtiter plates are delivered in a re-sealable bag
with a pouch containing a desiccant (Figure 18).
In case not all wells of the plate are needed, store
the rest of the wells in this bag. Put the wells and
the microtiter plate together with the desiccant
into the bag and close it. Close all flasks and make
sure to screw the lids on firmly. This is especially
Figure 18: Unneeded plate strips should be stored together
with the desiccant in the re-sealable pouch in which they are
delivered
2.3.10 Interchange of reagents between tests and batches
The components of each lot are thoroughly
adjusted to deliver ELISA kits that show the optimal
performance. The exchange of one or more of
these components between different lots will
change the performance of the tests and is not
26
important for components such as standard
solutions that may contain organic solvents with a
high vapour pressure. We recommend putting all
components back into the kit package for storage.
Store all components upright and under the
indicated conditions (Figure 19).
Figure 19: Until further use all components should be stored
in the kit package in an upright position
allowed. An exchange of components of kits
with the same product number is possible if the
lot number of the kit is identical. However, we
recommend using only components delivered
with the particular kit.
27

2.3.11 Security references

ELISA test kits may contain hazardous substances.
For information on hazards contained in the
substances take note of the warnings on the labels
of the components and refer to the appropriate
safety data sheets (SDS). Generally, handle all
components with care and take all usual
laboratory security precautions. While performing
the test procedure use laboratory gloves, wear
a lab coat, do not eat, drink or smoke and keep
all components away from sources of ignition.
Disposal of waste may differ from country to
country. Please refer to local disposal rules.

2.4 Stopping and measuring of the ELISA

Most ELISAs are measured at a wavelenght of Figure 20: Addition of stop solution causes a color change
450 nm. The correct wavelength for reading can from blue to yellow

be found in the test kit manual under point 3. test

principle. At the end of the test implementation,
stop solution which contains sulfuric acid is added
to each microtiter well. The acid denatures all
proteins including the antibodies and thus stops
the reaction (Figure 20).

Nevertheless it is recommended to read the

microtiter plate directly after addition of stop
solution or at least within the time stated in the
instructions for use. A large delay may still cause a
shift of the absolute values measured.
Good ELISA Practice – Manual
2.5 Parallel performance of tests
If several ELISA tests are performed in parallel,
extraordinary care should be taken. Label microtiter
plates and reagents properly to avoid mistakes and
mixing of reagents between assays. Use a separate
lab timer for each microtiter plate (Figure 21). Take
care, that handling steps of different assays do not
overlap, e.g. washing of one microtiter plate when a
second one has to be stopped.
Figure 21: Parallel performance of several ELISA tests must be carefully scheduled and organized
28
Schedule the pipetting, washing and OD reading
activities before starting different ELISA.
Automation of parallel analysis is possible by using
automates like the Thunder Bolt® or BOLT TM.
Please contact us for a list with tests, which are
already validated on biochemistry analyzers.
 29

3 Data evaluation and interpretation

of results

3.1 Determination of unknown samples by standard curve

The concentration of the analyte in an “unknown” used to generate a standard curve covering the
sample can be determined by comparing the concentration range of interest. There are also
measured signal of the sample with the signal of ELISAs which use single calibration technology
standards containing known concentrations of (SC), where a single standard is used to check
the analyte. In ELISAs usually 5 - 7 standards are compliance with a deposited standard curve.

3.2 Standard curve fittings

Depending on the assay, the standard curve is
calculated by different curve fittings such as
linear regression, logit-log, cubic spline,
4 parameter
Q U A L I Tand
Y A S S2U R A
nd
order polynomial
NCE CE E L I T(Figure
R T I F I CQAUT A Y A S S U R 22).
QUALITY
ANCE CERTIFICATE
ASSURANCE CERTIFICATE
QUALITY ASSURANCE CERTIFICATE
RIDASCREEN
®
Histamin (enzymatic) RIDASCREEN FAST DON
® RIDASCREEN Gliadin ®
RIDASCREEN FAST DON
QUALITY ASSURANCE CERTIFICATE
Art. No.: R1605 Lot: 12184 Expiry: 2014-11 Art. No.: R7001 Lot: 14383 Expiry: 2014-12
Art. No.: R5901 Lot: 13504 Expiry: 2015-10
Art. No.: R5901 Lot: 13504 Expiry: 2015-10 Pag
R-Biopharm
R-Biopharm AG, Darmstadt, Germany certifies that this AG, approved
batch has been Darmstadt, Germany
by the Quality certifies that this batch has been approved by theRIDASCREEN
Quality Gliadin
Assurance Department and conforms with specificationsAssurance Department and conforms withR-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Qualit
specifications
Assurance Department and conforms with specifications
Linear regression Logit-log Cubic spline Art. 4 parameter
Datasheet
No.: R7001 Lot: 14383 Expiry: 2014-12
Standards Standards Standards
Standard curve Standard curve Standard curve R-Biopharm AG,
Standards
3.5 Std. n Std.Darmstadt,
n Germany
Standardcertifies
curve that this batch has been approved by the Qua
2.0 2.4 Std. n
Conc.(ppb) mean
CV(%) Assurance Department and conforms with specifications
Conc.(mg/L) mean Conc.(ppm) mean 3.5 Std. n
Conc.(ppm) mean
3.0 CV(%) CV(%) B/B0
1.8 2.2 3.0 CV(%) B/B0

RIDASCREEN® Calprotectin / One-Point

2.5 Std1 8

Standard curve Calibration and 4 Param

Std1 2 2.0 Std1 4 2.5 Standards
A 0.00 0.009 0.00 0.058 Std1 4
1.6 0.00 1.937
2.0 9.3 Std.
0.00 n
1.937
b 7.9 0.2 100.0 2.4
2.0
L 1.8 Conc.(ppb)
0.2 mean
100.0
s 1.5 Std2 L Std2 8 CV(%)
1.4 o 2 Std2 4
1.5
5.00 0.294
o 1.00 0.106 1.6 0.222 1.328 o 2.2 Std2 4
g A 5.7
1.0 0.0
2.4 conc.
68.6 of calprotectin 0.222
Std1calculated
1.328
68.6 values
r b g 1.0 8
1.2 i Calprotectin mean Standard Curve 2.4
b 0.5 Std3 2
s 1.4
Std3 and
4 corresponding OD i
2.0 Std3 8 0.00 0.058
t o 10.00
0.5 0.635 9.34-parameter logisti
a 5.00 0.484 r 0.666 0.862values of mean 3.5
t3.500
1.8 4.2
Std3 4
1.0 0.0 0.6 1.2
n
b 2.0 44.5
standard curve 0.0
0.666
Std2 model
0.862
8 (4PL)
a 3.0Std4
3.000 8 2.0
5.00
44.5
0.294
c -0.5 Std4 2 n 1.0 Std4 4 1.620.00 1.094 5.7
A 0,0
OD [450 nm; Ref. 620 nm]

0.8 A
e
10.00 0.938 c 2.00 mg/kg
0.422 stool OD b
-0.5
2.5 2.8
2.500
Std4 4
-1.0 0.6 e 3.9 21.8 2.00
Std3 0.422
8
0.8 0 0,010 s 1.4
-1.0
Std5 8 3.9 21.8
0.6
-1.5 Std5 2 Std5 4
o 2.0
2.000
40.00 1.683
10.00
B 0.635
1,5
15.00 1.399 0.6 6.00
19,5
0.204
0,091 r
b 1.2
-1.5
2.2
4.2
Std5 4
1.5
1.500
0.4 -2.0 0.9 4.7 10.5
33 0,169 a
n 1.0 Std6
-2.0 8
Std4
6.00
4.7
20.00
C 8
0.204
10.5
1.094
5,4
0.4
1.080.00
1.000
-2.5 Std6
20.00
2
1.844
56 0,370 c
e 2.1
-2.5
2.366 2.8
D 3,6
0.2 0.8
-3.0 0.5 0.2
95 0,733 0.5
0.500
-3.0
Std5
40.00
8
1.683
0.0 -3.5 0.0 160 1,188 0.6
0.0
0.000 2.2
0.00 5.00 10.00 15.00 20.00 0.222 0.666 2.00 6.00 5.00 10.00 20.00 40.00 80.00 -3.5
275 2,015 1 0.222 10
0.666 100
2.00 1000
6.00 Std6 8
Concentration (mg/L) 0.4
Concentration (ppm) Concentration (ppb) [mg Calprotectin/kg stool] 80.00 2.366
470 2,704 Concentration (ppm) 2.1
0.2
800 3,113
X-axis
Corr.Coeff.: 0.9999 Corr.Coeff.: 0.9983
slope = 0.0918 50% inhibition = 0.519 0.0 Corr.Coeff.: 0.9983
5.00
50% inhibition10.00
= 0.519 20.00 40.00 80.00

Concentration Concentration (logarithmic) Concentration (logarithmic)

Lot No.
Concentration
Expiry
(logarithmic) Concentration (ppb)
Lot No. Expiry Lot No. Expiry
Microwell plate 15043 2014-12 Lot No. Expiry
Y-axis Microwell plate
Standards
14463
12084
2014-11
2015-01
MTP M
Standards
15464
12494
2017-04
2016-05
Standards
Conjugate
11353
11353
2015-04
2015-04
MTP M 15464 2017-04

OD
Buffer1
Enzyme Solution
13084
13084
2015-01
Logit of concentration
2015-01
Conjugate
Antibody
11484
11484 OD
2015-10
2016-04
Negative control12173
Buffer1
Substrate/Chromogen
15183 OD
2015-03
2015-10
Standards
Conjugate
12494
11484
2016-05
2015-10
Spike Solution 12124 2015-02 Red Chromogen Pro 14413 2016-03 Antibody 11484 2016-04
Stop solution 15183 2018-04 Lot No. Expiry
Stop solution
Washing buffer salt
14114
051M8211
2019-02
2016-06
Negative control
Washing buffer 11243Diluent 2015-11 3 QC specification:
Red Chromogen Pro
Microwell plate
Stop solution
14413
15043
14114
2016-03
2014-12
2019-02
OD < 0,05
The relationship In Logit-log is used to Cubic spline is a numeric 4 parameter is a nonlinear Standards
Washing buffer salt
Conjugate
11353
051M8211
11353
2015-04
2016-06
2015-04

plots forPlease note: function that is piecewise- Please note:

Buffer1 12173 2015-03
R-Biopharm between
AG, Darmstadt,variable
Germany certifiesy and that this batch Please
has been linearize
note: approved by the curved
Quality regression model with Substrate/Chromogen
Stop solution
15183
15183
2015-10
2018-04
Assurance Department and conforms with specifications
variable x is linear. Thus, The absorbance further analysis
for the standards by linear
may decrease duringThe shelf life ofdefined
the absorbance forkit.
the theThe
by
standards
general 3 polynomial
may decrease
shape of the 4 parameters:
during the shelf life of the kit. the TheWashing
general shape of
buffer 11243 2015-11

curve will remain similar, while the slope might change curve will
slightly. remain similar,
Furthermore refer to Calibrator
while the slope
product leaflet might
8. change slightly. Furthermore refer
The absorbance tostandards
for the product leaflet 8.
may decrease during the shelf life of the kit. The general shape o
sign.: Eddasimple
Rohm linear regression is Indication ofregression models.
instability or deterioration of reagents.
Date: 2014-04-29 functions
Indication of instability withofhigh
or deterioration reagents.degree Please • bottom of the curve
curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet 8
note: of instability or deterioration of reagents.
Indication
The target value of the Calibrator Calibrator has been determined
applied using the formula: sign.: N. Stork-Heininger
Quality Assurance Representative
sign.: Edda Rohm of smoothness
Quality Assurance Representative to be:
at the
Date: 2014-12-10
The absorbance
• fortop
sign.: N. Stork-Heininger
of the Date:curve
2013-09-19
the standards may decrease during the shelf life of the kit. The general
OD 1, 188shap
Date: 20
•
Quality Assurance Representative
y = mx + b
Remark: This document has been created electronically and is therefore valid without a signature.
connections of the curveQuality
will remain
Indication
EC50
similar,
Assurance
of instability or
while the slope might change slightly. Furthermore refer to product leafle
Representative
deterioration of reagents.
The rangeand(target value without± 3 SD) should becreated
within: OD 0,651 – 1
• slope at the inflection
Remark:
Remark: This document has been created electronically and is therefore valid This document
without has been created
a signature. electronically is therefore valid a signature.
polynomial pieces (knots). Remark: This document has been electronically and is therefore valid without a signature.
sign.: Edda Rohm Date: 201
Quality Assurance Representative
point of the curve =EC50
Figure 22: Overview about standard curve fittings
The R-Biopharm group is DIN EN ISO 9001 certified.
Positive controlRemark: This document has been created electronically and is therefore valid without a signature.
The R-Biopharm group is DIN EN ISO 9001 certified. The R-Biopharm group is DIN EN ISO 9001 certified.
The R-Biopharm group is DIN EN ISO 9001 certified.
www.r-biopharm.com www.r-biopharm.com www.r-biopharm.com
The target value of the Positive control Control + has been www.r-bioph

145,1 mg/kg
determined to be:
The R-Biopharm group is DIN EN ISO 9001 certified.
The range (target value ± 3 SD) should be within: 113,3 – 176,9
www.r-biopharm

Low positive control

The Low positive control can optionally be tested.
Good ELISA Practice – Manual 30

3.3 Standard curves of sandwich and competitive ELISAs

The correct algorithm for the respective ELISA level in a competitive ELISA, the zero standard
is shown on the certificate of analysis and is containing no analyte (antigen) is known as a zero
preset in the RIDASOFT®Win.NET-Software after well or B0. (B0 is originally the binding at zero
choosing the method. Depending on assay format level).
(sandwich or competitive) the standard curve is
calculated differently. For competitive ELISAs, the concentration of the
standards is plotted on the horizontal x-axis, while
In sandwich ELISAs the concentration of the B/B0, a percentage value is plotted on the vertical
standards is plotted on the horizontal x-axis, while y-axis. Then standard 1, which contains no analyte,
OD is plotted on the vertical y-axis (Figure 23). is set as 100 %. Next, B/B0 is calculated, B is the
OD of a standard well and B0 is the maximum OD
Competitive ELISA differs from sandwich ELISA in from the zero standard. The result is multiplied
QUALITY ASSURANCE CERTIFICATE
that the higherQthe
U A concentration
L I T Y A S S U R Aof
N Cthe
E Canalyte
ERTIFICATE with 100 to obtain percentage units (Figure 24).
(antigen) in the sample, the lower the OD reading. Finally the B/B0 for RIDASCREEN
the samples areChloramphenicol
compared to
RIDASCREEN Gliadin
The maximum OD is achieved when no antigen the standard curve andR1505
Art. No.: a quantification
Lot: 11084 isExpiry:
made.2015-04
Art. No.: R7001 Lot: 14383 Expiry: 2014-12
is present in the sample. To assess this maximum
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Qualit
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications Assurance Department and conforms with specifications

Standards Standards
Standard curve Standard curve
Std. n Std. n
2.4 100
Conc.(ppb) mean Conc.(ppt) mean
CV(%) CV(%) B/B0
2.2
90
Std1 8 Std1 8
2.0
0.00 0.058 0.00 2.302
9.3 80 2.3 100.0
1.8
Std2 8 Std2 8
5.00 0.294 70
1.6 A 25.00 2.068
A 5.7 2.8 89.8
b
b s
s 1.4 Std3 8 60
o Std3 8
o 10.00 0.635
r 50.00 1.751
r 4.2
b 1.2 b 3.1 76.1
a 50
a Std4 8
n 1.0 20.00 1.094 B/B 0 c
n Std4 8
c 2.8 100.00 1.348
e 40 3.0 58.6
e
0.8 Std5 8
(%) Std5 8
40.00 1.683 30
0.6 2.2 250.00 0.719
3.4 31.2
Std6 8
0.4 20 Std6 8
80.00 2.366
2.1 750.00 0.306
0.2 4.2 13.3
10

0.0
5.00 10.00 20.00 40.00 80.00
0
25.00 50.00 100.00 250.00 750.00
Concentration (ppb)
Concentration (ppt)

Figure 23: Standard curve of an ELISA in sandwich format, Figure 24: Standard curve of an competitive ELISA, where
50% inhibition = 132.2
where absorbance is plotted over concentration
Lot No. Expiry
B/B0 is plotted over concentration
Microwell plate 15043 2014-12 Lot No. Expiry
Standards 11353 2015-04
Conjugate 11353 2015-04 Microwell plate 15313 2015-04
Buffer1 12173 2015-03 Standards 12034 2015-09
Substrate/Chromogen 15183 2015-10 Conjugate 14064 2015-07
Stop solution 15183 2018-04 Buffer1 12044 2015-12
Washing buffer 11243 2015-11 Red Chromogen Pro 11113 2015-05
Stop solution 11373 2018-08
Washing buffer salt 051M8211 2016-06
Please note:
Please note:
The absorbance for the standards may decrease during the shelf life of the kit. The general shape of the
curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet 8.
Indication of instability or deterioration of reagents. The absorbance for the standards may decrease during the shelf life of the kit. The general shape
the curve will remain similar, while the slope might change slightly. Furthermore refer to product lea
sign.: Edda Rohm Date: 8. Indication of instability or deterioration of reagents.
2013-09-19
Quality Assurance Representative
sign.: Edda Rohm Date: 2014-0
Remark: This document has been created electronically and is therefore valid without a signature. Quality Assurance Representative

Remark: This document has been created electronically and is therefore valid without a signature.

The R-Biopharm group is DIN EN ISO 9001 certified.

www.r-biopharm.com

The R-Biopharm group is DIN EN ISO 9001 certified.

www.r-biopharm.co
31

3.4 Spectrophotometer and Software

The optical density is read by a microtiter plate
spectrophotometer at a certain wavelength. There
are many different spectrophotometers from
different manufacturers available. R-Biopharm
offers spectrophotometers and a software,
called RIDASOFT® Win.NET, which is tailor-made
for the analysis of ELISAs from R-Biopharm
(Figure 25, 26). For the use of the software a
manual (Art. No. R9996) is available on request. If
a spectrophotometer is already available, please
do not hesitate to contact us, to check if your
spectrophotometer can be used in conjunction
with the software and our assays.

Figure 25 RIDA®ABSORBANCE 96, Art. No. ZRA96FF

Figure 26: Screenshots of RIDA®SOFT Win.net:

plate layout, standard curve, results
Good ELISA Practice – Manual 32
QUALITY ASSURANCE CERTIFICATE

RIDASCREEN Chloramphenicol
Art. No.: R1505 Lot: 11084 Expiry: 2015-04
3.5 Determination of analyte concentration R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications

Standards

The software creates the standards curve and 100

Standard curve
Std.
Conc.(ppt)
n
mean
CV(%) B/B0

calculates the concentration of an analyte in an 90

Std1 8
0.00 2.302

unknown sample as shown in figure 27. Outside

80 2.3 100.0

Std2 8
70
A 25.00 2.068

the measurement range the software will calculate b

s
o
60
measured 2.8

Std3
89.8

8
r 50.00 1.751

no values. b
a
n
50
3.1

Std4
76.1

8
c 100.00 1.348
e 40 3.0 58.6
(%) Std5 8
30
contentration 250.00
3.4
0.719
31.2
20
obtained
Std6 8
through 750.00 0.306
10 standard 4.2 13.3

curve
0
25.00 50.00 100.00 250.00 750.00

Concentration (ppt)

Figure 27: Determination of analyte concentration from

50% inhibition = 132.2
unknown sample through standard curve
Lot No. Expiry
Microwell plate 15313 2015-04
Standards 12034 2015-09
Conjugate 14064 2015-07
Buffer1 12044 2015-12
Red Chromogen Pro 11113 2015-05
Stop solution 11373 2018-08
Washing buffer salt 051M8211 2016-06

Please note:

The absorbance for the standards may decrease during the shelf life of the kit. The general shape of

3.6 Measuring range and dilution factor the curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet
8. Indication of instability or deterioration of reagents.

sign.: Edda Rohm Date: 2014-02-17

Quality Assurance Representative

If an absorbance or a B/B0 value is obtained by the concentration of the standards, and by the
Remark: This document has been created electronically and is therefore valid without a signature.

which is below or above the standard curve, the dilution factor of the sample preparation method.
RIDASOFT® Win.NET software gives a results For example, if milk is diluted 1:4 (1+3) before
The R-Biopharm group is DIN EN ISO 9001 certified.

‘<’ (below) or ‘>’ (above) standard range. The applying to the microtiter well, the dilution factor
www.r-biopharm.com

software provides the option to extrapolate the is 4. This means, that the results read from the
concentration value. In general, these extrapolated standard curve have to be multiplied by 4 to obtain
values are only estimates and are not reliable. The the correct concentration of the analyte in the
further the sample is below the lowest or above sample. If the analyte in a sample is concentrated
the highest standard, the bigger the uncertainty by a factor of 2 during sample preparation, e.g.
of the calculated concentration is. It is advised, to by evaporation or column clean-up, the dilution
dilute samples that are above the largest standard factor is 0.5. This means, that the results read from
and to repeat the
Q U A analysis
L I T Y A S Suntil
U R A Nthe
C E Cresult
E R T I Fis
I Cwithin
ATE the standard curve Qhave
U A L I to
T Y be
A S Smultiplied
U R A N C E C Eby
R T 0.5
I F I Cto
ATE

the concentration RIDASCREEN

range of theChloramphenicol
standard curve. obtain the correct concentration
RIDASCREENofChloramphenicol
the analyte in
The measuringArt.range of the
No.: R1505 ELISA
Lot: 11084isExpiry:
determined
2015-04 the sample (Figure Art.
28,No.:
29).R1505 Lot: 11084 Expiry: 2015-04
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications Assurance Department and conforms with specifications

Standards Standards
Standard curve Standard curve
Std. n Std. n
100 100
Conc.(ppt) mean Conc.(ppt) mean
CV(%) B/B0 CV(%) B/B0
90 90
Std1 8 Std1 8
0.00 2.302 0.00 2.302
80 2.3 100.0 80 2.3 100.0

Std2 8 Std2 8
70 70
A 25.00 2.068 A 25.00 2.068
b 2.8 89.8 b 2.8 89.8
s 60 s 60
o Std3 8 o Std3 8
r 50.00 1.751 r 50.00 1.751
b 3.1 76.1 b 3.1 76.1
a 50 a 50
n Std4 8 n Std4 8
c 100.00 1.348 c 100.00 1.348
e 40 3.0 58.6 e 40 3.0 58.6
(%) Std5 8 (%) Std5 8
30 30
250.00 0.719 250.00 0.719
3.4 31.2 3.4 31.2
20 Std6 8 20 Std6 8
750.00 0.306 750.00 0.306
4.2 13.3 4.2 13.3
10 10

0 0
25.00 50.00 100.00 250.00 750.00 25.00 50.00 100.00 250.00 750.00

Concentration (ppt) Concentration (ppt)

Figure 28: A milk sample was diluted 1:4 before applying

50% inhibition = 132.2 Figure 29: A meat sample was concentrated by factor 2 during
50% inhibition = 132.2

to the microtiter well. The results have to multiplied by 4 to sample preparation 1. The result has to be multiplied with 0.5
Lot No. Expiry Lot No. Expiry
obtain the correct concentration:
Microwell plate The concentration
15313 of the
2015-04 to obtain the correct concentration:
Microwell plate The concentration
15313 of the
2015-04
Standards 12034 2015-09 Standards 12034 2015-09
analyte in the sampleConjugate
is 100
Buffer1
ng/kg x 4 = 400
14064
12044
ng/kg.
2015-07
2015-12
analyte in the sample is 100Conjugate
ng/kg
Buffer1
x 0.5 = 50 ng/kg.
14064
12044
2015-07
2015-12
Red Chromogen Pro 11113 2015-05 Red Chromogen Pro 11113 2015-05
Stop solution 11373 2018-08 Stop solution 11373 2018-08
Washing buffer salt 051M8211 2016-06 Washing buffer salt 051M8211 2016-06

Please note: Please note:

The absorbance for the standards may decrease during the shelf life of the kit. The general shape of
33

3.7 Units and dimensions

Concentrations are sometimes not expressed in SI SI-units decimal parts per symbol
units, but in miscellaneous dimensionless quantity
g/kg 10-3 parts per mille ‰
‘part per’ annotation (Figure 30). We strongly
recommend to follow SI units. mg/kg 10-6 parts per million ppm

µg/kg 10-9 parts per billion ppb

ng/kg 10-12 parts per trillion ppt

pg/kg 10-15 parts per quadrillion ppq

Figure 30: Overview about SI-unit and the miscellaneous

dimensionless quantities ‘part per’ annotation

3.8 Limit of detection and quantification

The Limit of Detection (LOD) is the lowest
concentration of an analyte, which can be clearly
distinguished from blank sample readings. The
LOD is determined experimentally by measuring
the concentration of at least 20 blank matrix
samples and then calculated by the formula: Mean
concentration of blank samples + 3-fold standard
deviation of the concentrations of blank samples.
The Limit of Quantification (LOQ) is the lowest
concentration of an analyte which can be
detected quantitatively. The LOQ is determined
Results below the LOD indicate that a sample is
negative or that the concentration of the analyte(s)
is below the LOD.
Results, which are above the LOD and below the
LOQ are qualitative (negative/positive) results only.
This means, that the sample contains the analyte,
but the exact amount cannot be quantified, as the
value is below the LOQ.
80
70 LOQ; 69

experimentally by measuring the concentration 60

concentration

50
of at least 20 blank matrix samples and then
40
calculated by the formula: Mean concentration of
LOD; 35
30

blank samples + (most often but not necessarily 20 MeanBlank; 18

10
9-fold standard deviation of the concentrations of 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
blank samples (Figure 31). samples

Figure 31: 20 blank samples were analyzed. Mean of blank

It is recommended to verify these values by spiking samples = 18 ng/kg with a standard deviation of 5,7 ng/kg
experiment with the sample matrix and laboratory LOD = 18 ng/kg + 3 x 5,7 ng/kg = 35 ng/kg
LOQ = 18 ng/kg + 9 x 5,7 ng/kg = 69 ng/kg
equipment.
Good ELISA Practice – Manual 34

3.9 Trueness and recovery

Trueness means the closeness of agreement The recovery of single samples can vary because of
between the average value obtained from a large • Inhomogeneous sample material
series of test results and an accepted reference • Poor pipetting skills of the technician
value. Trueness can only be established by means • Precision of the assay.
of certified reference material (CRM). Trueness is
calculated by dividing the measured concentration If samples were measured in areas where the
of the CRM by the assigned concentration of the standard curve QU isArelatively
L I T Y A S flat,
S U Rthe ANC accuracy
E CERTIFICATE

CRM. The result is multiplied by 100 to obtain a of the results may be low especially ® at high Fast
RIDASCREEN Clenbuterol
percentage unit. The absolute difference between concentrations, Art.because
No.: R1701CsmallLot:differences
16213 Expiry: in OD or
2015-04
both values is called ‘bias’. B/B0 may cause large differences in concentration
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications
(Figure 32).
If no CRM is available, e.g. for antibiotics in Standards
Standard curve
food of animal origin, instead of trueness, 100 Std.
Conc.(ppt)
n
mean
CV(%) B/B0

the recovery can be determined. Recovery is 90

Std1 8

determined experimentally by measuring samples A

b
80 0.00
6.5
1.642
100.0

which are spiked prior to sample preparation.

s 70 Std2 8
o 100.00 1.327
2.0 80.8
r
Recovery is calculated by dividing the measured b
60
Std3 8
a 300.00 0.964
concentration of the spiked sample by the spiking n
50 1.7 58.7

c Std4 8

concentration. The result is multiplied by 100 to

40
900.00 0.414
e 3.2 25.2

obtain a percentage unit. Please note, that spiking

30
Std5 8
(%)
2700.00 0.173
20 5.3 10.5
on the surface of a matrix generally gives higher Std6 8
10 8100.00 0.098
recoveries as naturally incurred samples.Recovery 5.1 6.0

0
or trueness is indicated in the validation report or 100.00 300.00 900.00 2700.00 8100.00

Concentration (ppt)
in the instructions for use.
Figure 32: In flat areas of the standard curve small differences
in OD produces50%large differences in concentration: 5 % OD
inhibition = 399.1

gives an 3 times higher results as 10 % OD.

Lot No. Expiry
MTP K 14113 2015-08
Standards 11193 2015-04
Conjugate 14193 2015-04
Antibody 16193 2015-04
Buffer1 12173 2015-04
Red Chromogen Pro 11113 2015-05
Stop solution 11163 2018-03

Please note:

The absorbance for the standards may decrease during the shelf life of the kit. The general shape of the
curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet 8.
Indication of instability or deterioration of reagents.

sign.: N. Stork-Heininger Date: 2014-0

Quality Assurance Representative

Remark: This document has been created electronically and is therefore valid without a signature.

The R-Biopharm group is DIN EN ISO 9001 certified.

www.r-biopharm.c
35
3.10 Specificity and cross reactivity
Antibodies are highly specific to a single analyte,
but sometimes they can also bind other molecules
with different affinities. In the case of food
allergens, the cross-reactivity in food samples
(like lentils or quinoa) are evaluated as a pure
commodity (100 % level). Samples well below the
LOQ show no cross-reactivity.
The specificity or cross reactivity is determined by
the measurement of a standard curve consisting of
the analyte or cross reactive substance in a suitable
concentration series (Figure 33). After calculation
of the 50 %-dose of the analyte or cross reactive
substance, the specificity or cross reactivity is
calculated as follows:
50 % – dose of standard substance
Specificity or cross reactivity =
50 % – dose of analyte or cross reactive substance
If the binding of the antibody affects a substance
that is included in the scope of the method, this is
called specificity of the method. If this binding is
related to unwanted substances that are not within
the scope of the method it is called cross reactivity.
1st Example: The scope of an ELISA describes that
the system was developed to quantify aflatoxin
M1 in milk. Therefore, the specificity for aflatoxin
M1 is 100 %. Furthermore it is stated that a cross-
reactivity of 10 % to aflatoxin M2 exists.
Due to the principle of an ELISA system, the
antibody is not able to discriminate between the
specificity to aflatoxin M1 and the cross-reactivity
related to aflatoxin M2. If both are present in an
unknown sample, the result is the sum of both
substances. But, due to the low cross-reactivity of
10 %, a 10-fold higher concentration of aflatoxin
M2 (compared to M1) is necessary to resulting
in a signal comparable to aflatoxin M1. An exact
quantification is only possible if only one analyte
100 standard
substance
90
80
cross
reactive
70
substance
60
B/B0 [%]
50
40
30
20
10
0
20 200
concentration of standards [µg/kg]
Figure 33: The 50%-dose of the standard curve of the standard
substance (blue) is more sensitive (170 µg/kg) as the 50%-dose
of the standard curve of the cross reactive substance (210 µg/kg).
The cross reactivity of the cross reactive substance is therefore:
(170 µg/kg / 210 µg/kg) x 100 % = 81 %
x 100 %
or one cross-reacting substance is present in the
sample.
2nd example: The scope of a Tetracycline-ELISA is
the determination of tetracycline and its derivatives
like chlortetracycline, rolitetracycline and
demeclocycline in different matrices to a certain
degree. The calibrator material is tetracycline with
a specificity of 100 %. The specificity of derivatives
differs, e.g. the specificity of chlortetracycline is
70 % in this system. Assuming the testing of a milk
sample, which is spiked with chlortetracycline,
reveals a measured concentration of 10 μg/L.
The real concentration is calculated to be 14
μg chlortetracycline per L of milk, because the
specificity of chlortetracycline is 70 %.
The specificity of the (tetracycline) ELISA was
determined by analyzing the cross-reactivities to
corresponding substances in buffer system.
In samples, the specificity may deviate from those
determined in the buffer system due to matrix
Good ELISA Practice – Manual
effects. Prior to the analysis of cross-reactive
substances, the user has to determine the Limit
of Detection and the Recovery for the substance
in the respective sample matrix. The test cannot
discriminate between analytes and cross-reactive
substances.
Nevertheless, ELISA tests are often used as screening
methods. Any positive results or concentration
higher than specified threshold should be verified by
a confirmatory method e.g. LC-MS/MS.
In case of an unknown matrix and/or another
3.11 Interferences and matrix effects
In general, food and feed are considered as ‘highly
difficult’ sample matrices, due to their complex
composition. The recovery of a single sample
varies depending on the nature of the food matrix.
The composition (e.g. ingredients, preservatives,
colorants), processing and physical properties of
the matrix material surrounding the analyte has
the largest impact on the recovery. Ingredients
such as high-fat or high-sugar-foods can cause
36
specific analyte which are not included in the
scope of the method, the user must determine the
Limit of Detection and the Recovery of the specific
analyte in the particular sample matrix. Please note
that the specificities and cross reactivity’s were
experimentally determined in the buffer system
only, as it is very time consuming and laborious
to determine every specificity or cross reactivity
of any analyte or cross reactive substance in every
matrix.
different types of interferences compared to
low-fat or low-sugar food. Processing like heat
treatment or hydrolysis can modify the analyte in
such a way that it is no longer recognized by the
antibody. Therefore, the complexity of the food
matrix has the largest influence on the recovery,
this is also called ‘matrix-effects’. A trouble
shooting guide for allergens and validation reports
are available on request.
37
R-Biopharm – dedicated to food safety

R-Biopharm
An der neuen Bergstraße 17
64297 Darmstadt, Germany
Phone: +49 61 51 - 81 02-0
06/2020

Fax: +49 61 51 - 81 02-40

E-mail: info@r-biopharm.de
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