R-Biopharm AG

Good ELISA Practice

Manual

R-Biopharm Innovative Lsungen fr die Lebensmittel- & Futtermittelanalytik

Good ELISA Practice Manual 2
3

Disclaimer

The Good ELISA Practice (GEP) manual represents a guideline for the establishment of reasonable framework
conditions and conditions of use, which shall be complied with, when using test-kits of R-Biopharm AG and
performing ELISA-analysis.

The GEP manual makes no claim to completeness, but describes only certain minimum standards. 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 the 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 you have the newest version of the manual
prior to performing the ELISA-analysis. The manual can be retrieved, printed and downloaded under the
website www.r-biopharm.com/products/food-feed-analysis.

Introduction

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.

The manual is divided into three chapters, which follow the work flow of an ELISA based analysis:

In the first chapter, basic knowledge about the test principle, ELISA components and required laboratory
equipment is given.

The second chapter describes the implementation of the analysis consisting of sample preparation and
ELISA procedure.

The third chapter explains the process of data evaluation from measurement to interpretation.

Additionally, FAQs are available on request.

Good ELISA Practice Manual 4

Inhalt

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

2 Sample preparation and test implementation 11

2.1 Pipetting techniques 11
2.1.1 General pipetting instructions 11
2.1.2 Forward pipetting 12
2.1.3 Reverse pipetting 12
2.1.4 Pipetting of organic solvents 13
5

2.2 Handling of samples 14

2.2.1 Storage of unprepared samples 14
2.2.2 Sample drawing 14
2.2.3 Sample preparation 14
2.2.4 Use of frozen samples 15
2.2.5 Certified reference material 15
2.3 Preparation and handling of components 16
2.3.1 Storage of kits 16
2.3.2 First in first out 16
2.3.3 Pre-Warming 17
2.3.4 Temperature control 17
2.3.5 Avoiding of contamination and sample mix-up 18
2.3.6 General test handling 18
2.3.7 Time management for pipetting 19
2.3.8 Correct washing 20
2.3.9 Storage of unused components for further experiments 21
2.3.10 Interchange of reagents between tests and batches 21
2.3.11 Security references 21
2.4 Stopping and measuring of the ELISA 22
2.5 Parallel performance of tests 22

3 Data evaluation and interpretation of results 23

3.1 Determination of unknown samples by standard curve 23
3.2 Standard curve fittings 23
3.3 Standard curves of sandwich and competitive ELISAs 24
3.4 Spectrophotometer and software 25
3.5 Compliance of standard curve 26
3.6 Determination of analyte concentration 26
3.7 Measuring range and dilution factor 27
3.8 Units and dimensions 28
3.9 Limit of detection and quantification 28
3.10 Trueness and recovery 29
3.11 Specificity and cross reactivity 30
3.12 Interferences and matrix effects 31
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 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, (4) a group of specific
1.3 ELISA Formats
Currently the following three different systems
exist. In every system, the result is measured on
the basis of the optical density.
1.3.1 Sandwich-ELISA
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 epitope of the antigen (analyte). After
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 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 colour, which can
be measured with a spectrophotometer.
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.
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)
(Figure 1).
7
1.3.2 Competitive ELISA (formats)
There are two possible formats for a competitive
ELISA. The first system consists of a microtiterplate
where the antibody is bound to the surface.
The competition is between the analyte and an
enzyme-analyte conjugate. The second system is
based on a microtiter plate with a fixed amount of
analyte which is bound to the surface. The analyte
from the sample is added and the competition
on antibody binding sites starts after adding the
antibody. After washing, substrate is added and
the measured OD value is inversely proportional
to the amount of analyte in the sample. The more
Fig. 1: Schematic structure of Fig. 2: Schematic structure of
sandwich ELISA competitive ELISA
1.4 ELISA components
As already mentioned above, several components
are needed for an ELISA test system: Microtiter
plates (MTP), enzyme-conjugates, standards
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 though
other materials can be used. The material is
activated by - or -radiation by the manufacturer
analyte that 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 (Figure 2).
For the second system (with an antibody bound
on the surface) a capture antibody is often used
(Figure 3). The advantage of this system is that
the reaction starts at the point of time the capture
antibody is filled in, so there will be nearly the
same incubation time over the whole plate.
Fig. 3: Schematic structure of
indirect competitive ELISA
(calibrators), substrate, stop-solution, and buffers.
Furthermore, it is essential to use controls to be
sure that the test procedure is working correctly.
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.
Good ELISA Practice Manual 8

1.4.2 Conjugate and Substrate

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

the use of standards. Therefore, the samples with 2.2

Std1 8
2.0
0.00 0.058

unknown concentrations and a set of standards 1.8

9.3

Std2 8
5.00 0.294
1.6

with known concentrations are analyzed in parallel A 5.7

b
s 1.4 Std3 8
o 10.00 0.635

on one plate. The result will be a calibration curve

r 4.2
b 1.2
a Std4 8
n 1.0 20.00 1.094
c

(with the associated mathematic formula) built out

2.8
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 4). 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. Fig. 4: Calibration curve

Lot No. Expiry

Microwell plate 15043 2014-12
Standards 11353 2015-04
Conjugate 11353 2015-04
Buffer1 12173 2015-03

1.4.4 Buffers
Substrate/Chromogen 15183 2015-10
Stop solution 15183 2018-04
Washing buffer 11243 2015-11

Please note:

All ELISA systems contain components of biological
origin. For long term storage of these components
and for proper function during the testing
also has an influence on test kit performance. For
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 convenience
sign.: Edda Rohm
of test kit users, these buffer areDate: 2013-09-19
included ready-to-use or as concentrates. They
Quality Assurance Representative

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

procedure, pH-value and ionic strength needs to are also used for sample preparation and washing
be constant. Often the kind of buffer component procedures of the microtiter plates.
The R-Biopharm group is DIN EN ISO 9001 certified.
www.r-biopharm.com

1.4.5 Stop-solution

The stop-solution terminates the enzyme- the reaction the colour will change from blue to
conjugate-reaction. In most cases, sulphuric yellow and will remain stable until measurement
acid at low concentrations is used. By stopping within 10 min.

1.4.6 Additional components

For most systems a positive control is This gives the opportunity to control your test
recommended, for example a spiking solution. system.
9
1.5 Lab equipment and its maintenance
Depending on the requirements of the test
system, various specialized laboratory equipment
is needed for the different steps e.g. pipettes,
equipment for plate washing, incubator for
constant temperature, ELISA reader and a
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, special attention
should be set on maintenance by experts and
proper pipetting technique. Different kinds of
pipettes exist:
Single-channel pipettes with fixed volumes e.g.
50 l
Single-channel pipettes (e.g. with variable
volumes between 10 and 100 l); used normally
for samples and standards
1.5.2 Washer
After every incubation step (except the incubation
with the substrate) the ELISA plate has to be
washed with washing buffer. Washing is a crucial
1.5.3 ELISA reader
The ELISA reader is a spectrophotometer which
allows to measure the optical density (OD) and
to calculate the concentration of your sample.
computer 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 done automatically)
step when performing an ELISA to obtain results
with high precision.
Regular maintenance by experts is essential for
exact results.
Good ELISA Practice Manual
1.5.4 Automation
One possibility for working with an ELISA is the use
of an automated system which allows you to test
your samples without any manual steps. Therefore,
the automated system has to be validated and
calibrated for your test system. Possibilities for that
1.5.5 Additional equipment
In some tests an incubator is required to
guarantee a stable temperature while the test is
running. Sometimes, a seal or a protecting plate
cover are necessary to prevent evaporation or
contamination.
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:
Lab coat Gloves
Eye protection Fume hood
(occasionally)
1.7 Test kit labeling
To ensure the correct handling and storage of your
components please read the Instructions for Use
(IFU) or the test kit insert. On all components the
follow information is stated (if applicable):
Product name Lot number
Article number Storage temperature
Name of the Concentration
component
10
kind of automation are the ChemWell and the
GEMINI. For further information please contact:
sales@r-biopharm.de.
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.
However, it is necessary to study the material
safety data sheets (MSDSs) carefully for all
chemicals substances used. The MSDS contains
information about the dangers when working
with a particular substance, required protective
measures, as well as required actions in case of
emergencies. The MSDS are available on request
for each product. Please contact:
sales@r-biopharm.de.
The expiry date is particularly important, as the
specified behavior can only be guaranteed until
then. After expiration the ingredients can degrade
and the results can decrease in accuracy.
11
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
your 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.
2.1.1 General pipetting instructions
If you use an adjustable pipette please adjust it
to the desired volume before you start pipetting.
Forward
Ready position
First stop
Second stop
Fig. 5: 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.
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. It requires more
liquid volume (dead volume) and is more error-
prone at high volume transfers. Familiarize yourself
with pipetting techniques before you start the
experiments.
Always check the adjusted volume directly before
starting to pipette (Figure 5).
Reverse
Good ELISA Practice Manual 12

2.1.2 Forward pipetting 2.1.3 Reverse pipetting

1
1 Put a new tip on your pipette and check for a 1 Put a new tip on your pipette and check for a
1
firm fit. firm fit.

2
1 Press the operation button to the first stop. 2
1 Press the operation button to the second stop.

3
1 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.
1 Put the pipette tip approx. 1 cm deep into the
3
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.

4
1 Put the pipette tip approx. 1 cm deep into the 1 Remove excessive liquid from the outside of the
4
liquid. Slowly release the operation button to tip.
the ready position and wait until the desired
liquid volume has been aspirated. Ensure that 1 Dispense the liquid into the desired well by
5
no bubbles or foam occurs in the pipette. pressing the operation button to the first stop.
Ensure that no liquid remains on the outside of
5
1 Remove excessive liquid from the outside of the the tip.
tip by touching the test tube with the tip.
1 For repetitive liquid pipetting, press the
6
1 Dispense the liquid into the desired well by
6 operation button to the first stop and repeat
pressing the operation button to the second steps 3 - 5.
stop.
1 Remove the tip to waste.
7
1 Remove the tip to waste.
7
13

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
Fig. 6: 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 6). For the

pipetting of organic solvents we recommend:

1
1 multistep pipettes which use the positive
displacement technique

1
2 serological pipettes for larger sample volumes,
since the graduation allows for the pipetting of
exact volumes

1
3 bottle top dispensers (A) (B)
1
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.
Good ELISA Practice Manual
2.2 Handling of samples
2.2.1 Storage of unprepared 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.
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.
Changes or variations may lead to incorrect
test results. Make sure to use only suitable and
maintained equipment for sample preparation. For
any related questions please contact R-Biopharm AG.
14
Generally, samples should be prepared and tested
immediately whenever possible. If storage cant
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.
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.
15

2.2.4 Use of frozen samples

Before further use, samples must be completely
thawed. Thawing of frozen samples should
be performed at 4C 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
20C to avoid freeze-thaw cycles.

2.2.5 Certified reference material

Certified reference materials (CRM) are naturally Fig. 7: Certified reference materials and standard solutions are
essential quality control tools.
contaminated, homogeneous matrices whose
analyte content has been exactly and reliably
determined (Figure 7). The regular use of CRMs
is recommended to perform internal quality
controls. This allows checking for the trueness and
precision of experimental procedures and for the
testing of handling skills. If no reference material
is available, the use of matrices spiked with
defined analyte concentrations is recommended.
RBiopharm can help you to find suitable certified
reference materials and spiking solutions.
Good ELISA Practice Manual 16

2.3 Preparation and handling of components

2.3.1 Storage of kits

The expiry date printed on the outer label applies Fig. 8: Storage conditions and expiry date are printed on
the outer label of each ELISA test kit.
to all reagents contained in the kit and is also valid
after first use. To maintain the shelf life, store the
kit at conditions noted on the outside label of the
package (Figure 8).

Most kits have to be stored in a dry place and at

4C. Freezing of components needs to be avoided.
If you use 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 you have more than one kit to 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 to avoid a mix-up of this
principle.
17

2.3.3 Pre-Warming

All reagents need to be at room temperature Fig. 9: 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 higher 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 9).

After use it is recommended that all components

are put back into the kit package 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 18

2.3.5 Avoiding of contamination and sample mix-up

A clean and reproducible way of working is crucial Fig. 10: 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 recommend 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 10). Exchange your single use lab ware
as often as needed.

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 shall 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 onto the plate needs to be performed
quickly and without interruptions at every step 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.
19
2.3.7 Time management for pipetting
Pipetting of standards and samples is general the
most time consuming and laborious step when
performing an ELISA. In general it is advised
to keep the pipetting technique as constant as
possible and the absolute pipetting time to a
minimum. This is extremely important when
the last component is added and the analytical
reaction starts (e.g. addition of antibody in a
competitive ELISA when sample and conjugate
is already added). To avoid an optical density
(OD) shift from the first to the last well, it is
recommended to use multichannel or stepper
pipettes for components which are added to all
wells such as antibody, conjugate, substrate/
chromogen or stop solution. However, it is not
recommended to pipette in a rush as mistakes
such as to 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 you have
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 you pipette 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. 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.
Good ELISA Practice Manual 20

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 your 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 l of
washing buffer per washing step and well. Add
the washing buffer firmly and remove the liquid by
pouring out and tapping. Repeat the washing step
3 - 5 times (see instructions for use). For washings
steps we recommend to use a bottle-top dispenser
with manifold (Figure 11).
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 your 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.
Fig. 11: Bottle-top dispenser plate washers with manifolds are
an accurate and affordable option for washing steps.

If you use such an automatic washer, service,

maintain and use the device according to the
manufacturers instructions. Ensure that the device
does not show salt residues that may prevent
liquid flow. Buffers, tubes, manifolds and washing
needles need to be kept free from contamination
of microorganisms. Keep your 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.
21
2.3.9 Storage of unused components for further experiments
Microtiter plates are delivered in a re-selable bag
with a pouch containing a desiccant (Figure 12).
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
Fig. 12: 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
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
material safety data sheets (MSDS). Generally
handle all components with care and take all usual
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 13).
Fig. 13: 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.
laboratory security precautions. While performing
the test procedure use laboratory gloves, wear
a lab coat, do not eat, drink, 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.
Good ELISA Practice Manual 22

2.4 Stopping and measuring of the ELISA

Most ELISAs are measured at a wavelenght of Fig. 14: Addition of stop solution causes a color change from
blue to yellow.
450 nm. The correct wavelength for reading can
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 14).

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.

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 (Figure15). 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. Schedule your
pipetting, washing and OD reading activities before
starting multiplex analysis.
Automation of parallel analysis is possible by
using biochemistry analyzers such as ChemWell
(Awareness Technology Inc.) or Gemini analyzer
(Stratec Biomedical AG). Please contact us for
a list with tests, which are already validated on
biochemistry analyzers.

Fig. 15: Parallel performance of several ELISA tests must be carefully scheduled and organized.
 23

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

QUALITY ASSURANCE CERTIFICATE
Depending
Q U A L Ion
T Y Athe
S S U assay,
RANCE Cthe
E R Tstandard E L I Tcurve
I F I CQAUT A Y A S S Uis
R A N C E C E R T I linear
F I C A T E regression, logit-log, cubic spline and
QUALITY ASSURANCE CERTIFICATE
RIDASCREEN Gliadin
RIDASCREEN Histamin (enzymatic) RIDASCREEN FAST DON
calculated by different curve fittings: 4 parameter 2 order polynomial
nd
(Figure 16)FAST DON
RIDASCREEN
QUALITY ASSURANCE CERTIFICATE
Art. No.: R5901 Lot: 13504 Expiry: 2015-10
Art. No.: R1605 Lot: 12184 Expiry: 2014-11 Art. No.: R7001 Lot: 14383 Expiry: 2014-12
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
1.55.00 0.294
o 1.00 0.106 1.6 0.222 1.328 o 2.2 Std2 4
g 5.7
1.0 0.0 A 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
n
b 1.2 2.0 44.5
standard curve 0.0
0.666
Std2 model
0.862
8 (4PL)
a 3.0
3.000 Std4 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 -0.5
2.5 2.8
b 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
2.0
o 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
a 1.500
0.4 -2.0 0.9 4.7 10.5
33 0,169 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
c 1.000
-2.5 Std6
20.00
2
1.844
56 0,370 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 2015-04
MTP M 15464 2017-04
Negative control12173
11353 2015-04
Buffer1 13084 2015-01 Conjugate 11484 2015-10 Standards 12494 2016-05
OD Enzyme Solution
Spike Solution
13084
12124
Logit of concentration
2015-01
2015-02
Antibody
Red Chromogen Pro
11484
14413
B/B0 in %
2016-04
2016-03
Buffer1
Substrate/Chromogen
15183 OD
2015-03
2015-10
Conjugate
Antibody
11484
11484
2015-10
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
bythe3shape
standards
general polynomial
may decrease
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
product slope
leaflet might
8. change slightly. Furthermore
The absorbance refer
for thetostandards
product leaflet 8.
may decrease during the shelf life of the kit. The general shape o
simple linear regression is regression models. functions with high degree note: bottom of the curve
Indication of instability or deterioration of reagents. Indication of instability or deterioration of reagents. curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet 8
sign.: Edda Rohm Date: 2014-04-29 Please Indication of instability or deterioration of reagents.
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 curve will
Quality
Indication
remain
Assurance
of
EC50
similar, while
Representative
instability or
the slope
deterioration of
might change
reagents.
slightly. Furthermore refer to product leafle
The rangeand(target value without 3 SD) should be within: OD 0,651 1
polynomial pieces (knots). Remark: This document
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.
slope atcreated
has been theelectronically
inflectionand is therefore valid without a signature.
sign.: Edda Rohm Date: 201
Quality Assurance Representative
point of the curve =EC50

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:
Fig. 16: Overview about standard curve fittings 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.
The target value of the Low positive control Low control + has
54,4 mg/kg
been determined to be:
The range (target value 3 SD) should be within: 41,5 67,3 m
Good ELISA Practice Manual Q U A L I T Y A S S U R A N C E C E R T I F I C24
ATE

RIDASCREEN Gliadin
Art. No.: R7001 Lot: 14383 Expiry: 2014-12

3.3 Standard curves of sandwich and competitive ELISAs R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications

Standards
The correct algorithm for the respective ELISA 2.4
Standard curve
Std. n
Conc.(ppb) mean

is depicted on the certificate of analysis and is 2.2

CV(%)

Std1 8
preset in the RIDASOFT Win.net-Software after 2.0
0.00
9.3
0.058

1.8
choosing the method. Depending on assay format 1.6
Std2
5.00
8
0.294
A 5.7

(sandwich or competitive) the standard curve is b

s
o
1.4 Std3
10.00
8
0.635
r 4.2
calculated differently. b
a
1.2
Std4 8
n 1.0 20.00 1.094
c 2.8
e
0.8 Std5 8

In sandwich ELISAs the concentration of the

40.00 1.683
0.6 2.2

Std6 8
standards is plotted on the horizontal xaxis, while 0.4
80.00
2.1
2.366

0.2
QUALITY ASSURANCE CERTIFICATE
OD is plotted on the vertical y-axis (figure 2). 0.0
5.00 10.00 20.00 40.00 80.00

Sometimes B/Bmax, a percentage value is plotted RIDASCREEN

Concentration (ppb)
Chloramphenicol
on the vertical y-axis. For B/Bmax calculation, the Fig. 17: Standard Art.
curveNo.:
of anR1505
ELISA inLot:
sandwich
11084format,
Expiry: 2015-04
standard with the highest analyte concentration is where absorbance is plotted over concentration.
R-Biopharm AG, Darmstadt, Germany certifies thatLotthis
No. batch has been
Expiryapproved by the Qualit
set as 100 % and B/Bmax of the further standards Assurance Department and conforms
Microwell plate
Standards
15043 with specifications
2014-12
11353 2015-04
Conjugate 11353 2015-04
is calculated by dividing the absorbance of the Buffer1
Substrate/Chromogen
12173
15183
2015-03
2015-10 Standards
Standard
Stop solution curve 15183 2018-04
respective standard by the absorbance of the 100 Washing buffer 11243
Std.
2015-11
Conc.(ppt)
CV(%)
n
mean
B/B0

highest standard. The result is multiplied by 100 to Please note:

90
Std1 8
0.00 2.302
obtain percentage units (Figure 17). 80
The absorbance for the standards may decrease during the shelf life of the kit. The general shape of t
2.3 100.0

curve will remain70similar, while the slope might change slightly. Furthermore refer
Std2 to product
8 leaflet 8.
25.00 2.068
Indication of Ainstability
b
or deterioration of reagents. 2.8 89.8
s 60
For competitive ELISAs, the concentration of the o Std3 8
sign.: Edda Rohm
r 50.00 Date:
1.751 2013-09-
Quality Assuranceb Representative 3.1 76.1
a 50
standards is plotted on the horizontal x-axis, n
Remark: This document
c has been created electronically and is therefore valid without a signature.
Std4
100.00
8
1.348
e 40 3.0 58.6
while B/B0, a percentage value is plotted on the (%) Std5 8
30
250.00 0.719
vertical y-axis. For B/B0 calculation standard 1 20
3.4 31.2

Std6 8

which contains no analyte is set as 100 % and B/B0 The R-Biopharm group10is DIN EN ISO 9001 certified.
750.00
4.2
0.306
13.3
www.r-biopharm.com
of the further standards is calculated by dividing 0
25.00 50.00 100.00 250.00 750.00

the absorbance of the respective standard by the Concentration (ppt)

absorbance of standard 1. The result is multiplied Fig. 18: Standard curve of an competitive ELISA, where B/B0
50% inhibition = 132.2

with 100 to obtain percentage units (Figure 18). is plotted over concentration
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
the curve will remain similar, while the slope might change slightly. Furthermore refer to product lea
8. Indication of instability or deterioration of reagents.

sign.: Edda Rohm 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.co
25

3.4 Spectrophotometer and Software

The optical density is read by a microtiter plate
spectrophotometer at a certain wavelength.
There are many different spectrophotometer 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 19, 20). 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.

Fig. 19: Biotek ELx800 microtiter plate spectrophotometer.

Fig. 20: Screenshots of RIDASOFT Win.net:

plate layout, standard curve, results
Good ELISA Practice Manual 26

3.5 Compliance of standard curve

To check compliance of your standard curve
and its parameters (e.g. OD of standard
1, 50 %-dose, B/B0-values, Coefficent of Variation
(CV) of standard duplicates and shape of curve)
please refer to the specific document Acceptance
criteria for RIDASCREEN standard curves, which
is available on request for each product group.
QUALITY ASSURANCE CERTIFICATE
RIDASCREEN Chloramphenicol
Art. No.: R1505 Lot: 11084 Expiry: 2015-04

3.6 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 21. Outside 80 2.3 100.0

Std2 8
70
the measurement range the software will calculate A
b
s
Measured
25.00
2.8
2.068
89.8
60 Std3 8
o
no values. r
b
50
50.00
3.1
1.751
76.1
a
n Std4 8
c 100.00 1.348
e 40 3.0 58.6
(%) Std5 8
30
250.00 0.719
3.4 31.2
20 Std6 8
contentration 750.00 0.306
10 obtained 4.2 13.3

through
0
25.00 50.00 100.00 250.00 750.00

Concentration (ppt)

Fig. 21: Figure 21:50%

Determination
inhibition = 132.2
of analyte concentration
from 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
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

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
27

3.7 Measuring range and dilution factor

If an absorbance or a B/B0 value is obtained The measuring range of the ELISA is determined
which is below or above the standard curve, by the concentration of the standards, and by the
the RIDASOFT Win.net software gives a results dilution factor of the sample preparation method.
< (below) or > (above) standard range. The For example, if milk is diluted 1:4 (1+3) before
software provides the option to extrapolate the applying to the microtiter well, the dilution factor
concentration value. In general, these extrapolated is 4. This means, that the results read from the
values are only estimates and are not reliable. The standard curve have to be multiplied by 4 to obtain
further the sample is below the lowest or above the correct concentration of the analyte in the
the highest standard, the bigger the uncertainty sample. If the analyte in a sample is concentrated
of the calculated concentration is. It is advised, by a factor of 2 during sample preparation, e.g.
to dilute samples which are above the largest by evaporation or column clean-up, the dilution
QUALITY ASSURANCE CERTIFICATE QUALITY ASSURANCE CERTIFICATE
standard and to repeat the analysis until the result factor is 0.5. This means, that the results read from
RIDASCREEN
is within the concentration rangeChloramphenicol
of the standard the standard curve have RIDASCREEN
to be multipliedChloramphenicol by 0.5 to
Art. No.: R1505 Lot: 11084 Expiry: 2015-04 Art. No.: R1505 Lot: 11084 Expiry: 2015-04
curve. obtain the correct concentration of the analyte in
the sample
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications (FigureAssurance
22, 23).
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
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
s
Measured 2.8 89.8 b
s
Measured 2.8 89.8
60 Std3 8 60 Std3 8
o o
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 20
contentration Std6 8 contentration Std6 8
750.00 0.306 750.00 0.306
10
obtained 4.2 13.3
10
obtained 4.2 13.3
through through
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)

Fig. 22: A milk sample was= 132.2

50% inhibition diluted 1:4 before applying to the Fig. 23: A meat sample was
50% inhibition concentrated by factor 2 during
= 132.2

microtiter well. The results have to multiplied by 4 to obtain sample preparation 1. The result has to be multiplied with 0.5
the correct concentration: The concentrationLot15313
Microwell plate
of
No.
the analyte
Expiry
2015-04
in to obtain the correct concentration:
Microwell plate
The concentration
Lot No.
15313
of the
Expiry
2015-04
the sample is 100 ng/kgStandards
x 4 = 400 ng/kg. 12034
Conjugate 14064
2015-09
2015-07
analyte in the sample isStandards
100 ng/kg x 0.5 = 5012034
Conjugate
ng/kg. 2015-09
14064 2015-07
Buffer1 12044 2015-12 Buffer1 12044 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 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 the curve will remain similar, while the slope might change slightly. Furthermore refer to product leaflet
8. Indication of instability or deterioration of reagents. 8. Indication of instability or deterioration of reagents.

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

sign.: Edda Rohm Date: 2014-02-17
Quality Assurance Representative Quality Assurance Representative

Remark: This document has been created electronically and is therefore valid without a signature. Remark: 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.
www.r-biopharm.com www.r-biopharm.com
Good ELISA Practice Manual 28

3.8 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 24). We recommend
using SI units. mg/kg 10-6 parts per million ppm

g/kg 10-9 parts per billion ppb

ng/kg 10-12 parts per trillion ppt

pg/kg 10-15 parts per quadrillion ppq

Fig. 24: Overview about SI-unit and the miscellaneous

dimensionless quantities part per annotation

3.9 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 cant be quantified, as value
is below the LOQ.
80
70 LOQ; 69

experimentally by measuring the concentration 60

concentration

of at least 20 blank matrix samples and then 50

calculated by the formula: Mean concentration of 40

LOD; 53
30
blank samples + (most often but not necessarily 20 MeanBlank; 18

9-fold standard deviation of the concentrations of 10

blank samples (Figure 25). 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
samples

It is recommended to verify these values by Fig. 25: 20 blank samples were analyzed. Mean of blank
samples = 157 ng/kg with a standard deviation of 92,4 ng/kg
spiking experiment with your sample matrix and LOD = 157 ng/kg + 3 x 92,4 ng/kg = 434 ng/kg
laboratory equipment. LOQ =157 ng/kg + 9 x 92,4 ng/kg = 711 ng/kg
29

3.10 Trueness and recovery

Trueness means the closeness of agreement If samples were measured in areas where the
between the average value obtained from a large standard curve is relatively flat, the accuracy
series of test results and an accepted reference of the results mayQ U A be
L I Tlow
Y A especially
S S U R A N Cat E high
CERTIFICATE

value. Trueness can only be established by means concentrations, because

small differences in Fast
OD or
RIDASCREEN Clenbuterol
of certified reference material (CRM). Trueness is B/B0 may cause Art.large differences
No.: R1701C Lot: in concentration
16213 Expiry: 2015-04
calculated by dividing the measured concentration (Figure 26).
R-Biopharm AG, Darmstadt, Germany certifies that this batch has been approved by the Quality
Assurance Department and conforms with specifications
of the CRM by the assigned concentration of the
CRM. The result is multiplied by 100 to obtain a Standards
Standard curve
percentage unit. The absolute difference between 100 Std.
Conc.(ppt)
n
mean
CV(%) B/B0

both values is called bias. 90

Std1 8
A 80 0.00 1.642
b 6.5 100.0

If no CRM is available, e.g. for antibiotics in food

s 70 Std2 8
o 100.00 1.327
2.0 80.8
r
of animal origin, instead of trueness, the recovery b
60
Std3 8
a 300.00 0.964
can be determined. Recovery is determined n
50 1.7 58.7

c Std4 8
experimentally by measuring samples which are e
40
900.00
3.2
0.414
25.2

spiked prior to sample preparation. Recovery is

30
Std5 8
(%)
2700.00 0.173
20 5.3 10.5
calculated by dividing the measured concentration Std6 8
10 8100.00 0.098
of the spiked sample by the spiking concentration. 5.1 6.0

0
The result is multiplied by 100 to obtain a 100.00 300.00 900.00 2700.00 8100.00

Concentration (ppt)
percentage unit. Please note, that spiking on
Fig. 26: In flat areas of the standard curve small differences
the surface of a matrix may be not identical to in B/B0 produces large differences in concentration: 5 % B/B0
50% inhibition = 399.1

naturally contaminated samples, which can lead to gives an 3 times higher results as 10 % B/B0.
Lot No. Expiry
deviations in results between spiked and naturally MTP K
Standards
14113
11193
2015-08
2015-04
Conjugate 14193 2015-04
incurred samples. Antibody 16193 2015-04
Buffer1 12173 2015-04
Red Chromogen Pro 11113 2015-05
Stop solution 11163 2018-03

Recovery or trueness is given in the instructions

for use and represents the mean of the measured
samples The recovery of single samples can vary in
accordance to the precision of the assay.
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
Good ELISA Practice Manual 30

3.11 Specificity and cross reactivity

Antibodies are highly specific to a single analyte,
but sometimes they can also bind other molecules
with different affinities.
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 27). 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 analyte or cross reactive substance

Specificity or cross reactivity = x 100 %
50 %dose of standard substance

If the binding of the antibody affects a substance

100 standard
that is included in the scope of the method, this is 90
substance

called specificity of the method. If this binding is 80

cross
related to unwanted substances that are not within 70 reactive
substance
60
the scope of the method it is called cross reactivity.
B/B0 [%]

50

40

1st Example: The scope of an ELISA describes that 30

20
the system was developed to quantify aflatoxin
10
M1 in milk. Therefore, the specificity for aflatoxin 0
20 200
M1 is 100 %. Furthermore it is stated that a cross- concentration of standards [g/kg]

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
Fig. 27: The 50%-dose of the standard curve of the standard
substance (blue) is more sensitive (170 g/kg) as the 50%-dose
of the standard curve of the cross reactive substance (210 g/kg).
The cross reactivity of the cross reactive substance is therefore:
(170 g/kg / 210 g/kg) x 100 % = 81 %
quantification is only possible if only one analyte
or one cross-reacting substance is present in the
sample.
In this case, the concentration can be calculated as
follows:

measured concentration
Concentration of analyte or cross reactive substance = specificity or cross reactivity of substance in %
100 %
31
2nd example: The scope of a Tetracyclin ELISA
describes that the assay is specific for the
determination of tetracycline, chlortetracycline,
rolitetracycline and demeclocycline in different
matrices. The calibrator material is tetracycline
with a specificity of 100 %. The analysis of
a chlortetracycline containing milk sample
resulted in a concentration of 10 g/L. Since the
specificity of chlortetracycline is 70 % in this
system, the concentration is calculated to 14 g
chlortetracycline per L of milk.
Nevertheless, ELISA test are used as screening
methods. Any positive results or concentration
3.12 Interferences and matrix effects
In general, food and feed are considered as
highly difficult sample matrices, due to their
complex composition. For example, besides its
natural ingredients, (carbohydrates, proteins and
water) honey may contain lots of environmental
contaminants such as heavy metals, radioactive
isotopes, organic pollutants, pesticides,
pathogenic bacteria and their toxins and
genetically modified organisms.
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
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 reactivitys 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.
Consequently, it cannot be excluded that natural
components or any contaminant of a sample
interferes with the reagents used during sample
preparation or in the ELISA test and cause so called
matrix-effects.
R-Biopharm AG

R-Biopharm AG
An der neuen Bergstrae 17
64297 Darmstadt, Germany
Phone: +49 (0) 61 51 - 81 02-0
Fax: +49 (0) 61 51 - 81 02-40
E-mail: info@r-biopharm.de
www.r-biopharm.com
07/2015