APPLICATIONNOTE
Influence of coating buffer
and incubation conditions
on ELISA performancewww.gbo.com /bioscience1/ INTRODUCTION
ELISA (Enzyme-Linked Immunosorbent Assay)
is one of the most widely used techniques inboth basic immunology research and diagnostic analyses. Because ELISA enables peptides,
proteins, antibodies and hormones to be selectively detected in small concentrations among
a multitude of other substances with relatively low cost and high simplicity, the method
provides an important and useful tool for disease monitoring, diagnostics and doping tests,
as well as environmental and food analytics.
ELISA methods yield both sensitive and accurate results, and employment of automatedhandling with a microplate platform allows
rapid conduction of tests in a high-throughput manner.
Although ELISA methodology is used in awide variety of different assays (e.g. direct,
indirect, sandwich, competitive), all variants
are based on the same principle: the bindingof one assay component – antigen or specificantibody – to a solid surface by passive adsorption, and its selective interaction with subsequently added assay components. Moleculesthat do not specifically interact with the component bound to the solid surface are washedaway during the assay process.
The specific binding of antigen and antibody
can then be visualised via an appropriate detection system (e.g. radioactive, colorimetric,
fluorescence, or luminescence) either by direct labelling or indirectly with an enzyme
conjugate. In the latter case the assay is developed by adding an enzymatic substrate toproduce a measurable signal. The strength of
the signal is correlated to the amount of analyte present within the sample, enabling ameans of quantitation.
Two enzyme labels that remain in widespread use today are horseradish peroxidase(HRP) and alkaline phosphatase (AP). When

2/8

Application Noteconjoined with a chemical substrate, a colorimetric reaction is produced and the resultant
color can be measured by means of absorbance or optical density readings taken withina spectrophotometer.
There are a number of key elements to beconsidered when developing an ELISA: thenature and size of the analyte, number andlocalisation of epitopes, binding properties
to the solid surface, and which or how many
antibodies are available for use. Other factors that can have direct bearing on achieved
results are substrate concentration, buffer
composition and incubation conditions.This
application note discusses the influence ofdifferent coating buffers and incubation conditions on ELISA performance using three exemplary assays established in our laboratory:(1) Biotin-labelled insulin (insulin-biotin) wasbound by adsorption to the surface of a highbinding microplate and detected with a streptavidin-horseradish peroxidase conjugate(Fig. 1). This assay is based on the specificnon-covalent binding of biotin and streptavidin. The interaction between the 52.8 kDa
protein streptavidin from Streptomyces avidinii and the vitamin biotin with a dissociation constant in the order of approx. 10 -14 M [1]is one of the strongest non-covalent bindings
known in nature.

Streptavidin-HRP
Biotinylated
proteinFigure 1: Biotin-Insulin assay.
Biotin-labelled insulin is coated to the surface of a highbinding microplate. A streptavidin-HRP conjugate interacts with the bound insulin-biotin and enables detection.

(2) The 10 AA peptide Strep-tag II was coatedto the plate surface of a high-binding microplate and detected with a StrepTactin® HRP
conjugate (Fig. 2). The Strep-tag II peptide
is usually used as affinity tag for the expression and purification of recombinant fusion
proteins. Strep-Tactin® is a variation of streptavidin optimised for the binding of Strep-tag
II fusion proteins [2].

StrepTactin®-HRP
10 AA Strep-tag

Figure 2: Strep-tag II assay.
A high-binding microplate is coated with Strep-tag II. In asecond incubation step a Strep-Tactin® HRP conjugate
binds to the Strep-tag II peptide and enables detection.

(3) Insulin, a peptide hormone of about 5.8 kDa,
was bound to the plate surface and detected using a monoclonal mouse anti-insulin
antibody as primary antibody and an antimouse-IgG-horseradish peroxidase (HRP) conjugate as secondary antibody (Fig. 3).

2/ MATERIALS
2.1/ MATERIAL AND REAGENTS
Item

Supplier

Item No.

Strep-tag II peptide

IBA GmbH

2-1018-002

Strep-Tactin®-HRPIBA GmbH

2-1502-001

Insulin-biotin

Sigma-AldrichI2258-2MG

Streptavidin-HRP

Sigma-Aldrich

S5512-1MG

Insulin (bovine)

Sigma-Aldrich

I5500

Anti-insulin
antibody (mouse)

Sigma-Aldrich

I2018

Anti-mouse-IgG-HRP (goat)

Sigma-Aldrich

A9917

EASYsealsealing tape

Greiner Bio
One GmbH

676001

96 well F8 Strip
high-binding ELISA(MICROLON 600)
microplate

Greiner Bio
One GmbH

762071

Carbonate coating
buffer pH 9.6(0.05 M carbonate,
pH 9.6)

Sigma-Aldrich

C3041

PBS coating buffer
pH 7.4

Biochrom

L1825

Sigma-Aldrich

P3563

Roth

8076.3

TMB

Sigma-Aldrich

T0440

Citric Acid

Sigma-Aldrich

C0759

Sodium Acetate

Sigma-Aldrich

71183

Sulphuric acid
0.5 M

Roth

K027.1

Acetate coating
buffer pH 5.5(0.2 M Na-Acetate,
pH 5.5)
Washing buffer
PBS-T (0.14M NaCl,
0.01 M phosphate,
0.05 % Tween 20)

Anti-Mouse
AB-HRP (goat)

Blocking buffer(3 % BSA in
washing buffer)

Anti-Insulin AB(mouse)

BSA Fraction V

InsulinFigure 3: Insulin ELISA.
In an indirect ELISA, insulin is coated to the surface ofa microplate. A monoclonal mouse anti-insulin antibody
as primary antibody interacts with the bound insulin. In asecond incubation step a labelled secondary antibody(anti-mouse-IgG-HRP) that recognises the primary antibody
is employed to bind the primary antibody-insulin complex
and enables detection.

Substrate solution(1 part of TMB tothree parts ofcitric acid/acetate,each 0.1 M, pH 4.9)

Influence on ELISA performance

3/8

2.2/ INFLUENCE OF COATINGBUFFER ON BINDING
Two assays were established to investigate
the influence of the coating buffer on binding
properties to the high-binding surface. For the
insulin-biotin assay, a 96 well F8 Strip highbinding ELISA microplate (Item no. 762071)
was coated with 100 µl per well biotin-labelled insulin (serial dilution from 1 mg/ml to
0 mg/ml) in carbonate, PBS or acetate coating
buffer, respectively, and incubated overnight.
The plate was washed three times with 350 µl
per well washing buffer (BioTek, Elx405Select),
blocked with 150 µl blocking buffer per welland incubated for one hour. Blocking buffer
was removed and 100 µl per well streptavidinHRP (8 ng/ml) in blocking buffer was added.
After incubation for one hour and subsequentwashing, 100 µl per well substrate solution wasadded. The reaction was stopped by adding
100 µl per well 0.5 M sulphuric acid. The optical density was measured in a Tecan SpectraFluorPlus at 450 nm. All incubation steps were
performed at 23 °C in a humidified chamber.
The Strep-tag II assay was performed according to the insulin-biotin assay procedure.
Coating was done with Strep-tag II peptide(serial dilution from 2 mg/ml to 0 mg/ml) instead of biotin-labelled insulin. For detection
a 1:10,000 dilution of Strep-Tactin®- HRP in
blocking buffer was used.

2.3/ INFLUENCE OF INCUBATIONCONDITIONS ON ASSAYPERFORMANCE
The influence of temperature and incubation
conditions on assay performance was studied using an insulin ELISA established in ourlab [3]. 96 well F8 high-binding ELISA strip
plates (Item no. 762071) were coated with 100 µl
per well insulin (800 ng/ml) in carbonate coating buffer and incubated overnight. The plates

4/8

Application Note

were blocked with 3 % BSA in washing buffer(150 µl per well) and incubated for one hour.
After removal of the blocking buffer 80 ng/ml
anti-insulin antibody in washing buffer (100 µl
per well) was added. After incubation for one
hour and subsequent washing, an anti-mouseIgG-HRP secondary antibody was added(300 ng/ml in washing buffer, 100 µl/well). The
plate was incubated for one hour and washedbefore addition of 100 µl/well substrate solution for a 20 min incubation. The reaction was
stopped by adding 100 µl per well 0.5 M sulphuric
acid, after which optical density was measured in a Tecan SpectraFluorPlus at 450 nm.
For all incubation steps the plates were either
stored stacked (5 plates per stack) on the labbench at room temperature (approx. 20 °C) orin a humidified chamber at 23 °C. The upper
most plates of the stacks were each sealed with
an EASYseal sealing tape. Incubation with thesubstrate solution was done for all plates nonstacked, without a sealing tape and in a humidified chamber at 23 °C.

3/ RESULTS ANDDISCUSSION
3.1/ INFLUENCE OF COATINGBUFFER ON BINDING
During the coating procedure ionic strengthand pH value of the buffer can play an important role. The most widely used coating buffer
for ELISA is 0.05 M Carbonate buffer at pH 9.6.
Nevertheless, for some molecules a different
pH value may result in a more favourable
structure and charge for an adsorptive binding to the microplate surface. Alternative
coating buffers are 10 mM PBS at pH 7.4 andacetate buffer at pH 5.5 [4]. PBS is often
used in sandwich ELISA applications to coatthe capture antibody to the plate surface.
Cuvelier et al. describe the positive effectThe influence of different coating buffers was
first tested with an assay where biotinylated
insulin was bound to the plate surface andthen detected with a streptavidin-HRP conjugate. With this assay no significant impactof the coating buffer was detectable (Fig. 4).
In a second assay the 10 amino acid (AA)peptide Strep-tag II was bound by passive
adsorption to the surface and detected with a
StrepTactin®-HRP conjugate. Adsorptive binding of small molecules like a 10 AA peptide
is generally difficult to achieve [6]. Often directbinding is not possible and instead a sandwich
or a competitive ELISA format, which involves
the initial binding of a capture anti-body tothe plate surface, is applied. Biotin labellingof the molecule and subsequent binding to a
streptavidin coated surface is another alternative. Results from this experiment did notexhibit significant binding of Strep-tag II to
the plate surface when carbonate or acetate
coating buffer were used.
A dramatic increase in binding was apparent
when PBS buffer was employed (Fig. 5), demonstrating that the choice of coating buffer canhave a major influence on binding properties.

OD 450 nm

BINDING OF INSULIN-BIOTIN TO A HIGH-BINDING
SURFACE WITH DIFFERENT COATING BUFFERS
1.2
1.0

Carbonate

0.8

PBS

Acetate

0.6
0.4
0.2
0.0
1000

500

250

125

62,5

31,25

15,6

7,8

0

Insulin-biotin concentration [ng/ml]

Figure 4: Choice of coating buffer
has no influence on the binding of biotinylated insulin
to the surface of a high-binding F8 strip plate

BINDING OF STREP-TAG II PEPTIDE TO A HIGH-BINDING
SURFACE WITH DIFFERENT COATING BUFFERS
OD 450 nm

of substituting carbonate by acetate buffer
for some IgG species for coating in sandwich
ELISA [5].

0.0
0.0

Carbonate

0.0

PBS

Acetate

0.0
0.0
0.0
0.0
0.0
2000

1000

500

250

125

62,5

31,25

15,6

0

Strep-tag II concentration [ng/ml]

Figure 5: Usage of PBS buffer
enhances binding of the Strep-tag II peptide
significantly in comparison to the usage of carbonate
or acetate as coating buffer.

3.2/ INFLUENCE OF INCUBATIONCONDITIONS
Adsorptive binding of molecules to a solid
surface, the specific interaction between antigen and antibody or primary and secondary
antibody, as well as the enzyme-substrate
reaction, are kinetic reactions and therefore
sensitive to incubation time and temperature.
Hence, coating is often done overnight to allow
maximum saturation of the available bindingsites on the plate surface. For the ELISA steps
subsequent to coating, shorter incubation times
are frequently used. In this context consideration should be given that incubation temperature has a major influence on the resulting
signal intensity. In general, a higher incubation temperature with a consistent incubation
time will result in higher OD values. Therefore, an increase of incubation temperature
will allow a shorter incubation time to achieve
the same signal intensity as those obtainedwith longer incubations at lower temperatures.
Incubation temperatures can typically range
from 4 °C to room temperature and even be as
high as 37 °C. When selecting the appropriate incubation temperature, it is imperative
to take into account the temperature stability of the molecules involved. In the depictedinsulin ELISA, an increase of incubation tem-

Influence on ELISA performance

5/8

INFLUENCE OF PLATE SEALING ON INNER PLATEHOMOGENEITY OF SIGNAL INTENSITYOD 450 nm

perature from about 20 °C (RT) to 23 °C for the
incubation steps with the primary and secondary antibody resulted in an increase of OD
values from about 0.87 to 1.08 units (Fig. 6).

1.2
1.0
0.8
0.6

OD 450 nm

INFLUENCE OF INCUBATION TEMPERATURE
ON SIGNAL INTENSITY

0.4
stacked unsealed at RT

0.2

1.2

sealed with EASYseal at RT

0.0

1.0

1

2

3

4

5

6

7

8

0.8

row

0.6

RT

0.2

23 °C

0.0
1

2

3

4

5

6

7

8

row

Figure 6: An increase in incubation temperaturefrom about 20 °C up to 23 °C results in significantly
higher OD values with an identical experimental set-up.
The graph shows mean OD values of microplate
wells per row. Microplates were sealed with EASYseal
for incubation.

Figure 7: Sealing of microplates
during incubation steps with primary and secondary
antibody in an environment without humidity control
prevents the edge effect. The graph displays mean OD
values of microplate wells per row.

INFLUENCE OF STORAGE CONDITIONS OF STACKED
PLATES ON INNER PLATE HOMOGENEITY
OF SIGNAL INTENSITYOD 450 nm

0.4

1.2
1.0
0.8

Not only does temperature play an importantrole for signal intensity, it can also, besides
evaporation, significantly affect consistencyof ELISA results. A temperature and evaporation gradient between the inner and outerwells of a microplate may result in a significant difference in standard readings between
outer and central wells, known as edge effect [6].
The edge effect is based on the fact that atemperature adaption is taking place more
rapidly in outer vs. inner microplate wells.
This effect is often enhanced if non-covered
microplates are stored in stacked formation,
when, in addition to the temperature gradient,a gradient caused by evaporation from theinner to the outer area of a microplate may
become relevant. For this reason, the edge
effect is most pronounced when non-covered microplates are stored stacked in anenvironment lacking temperature and humidity control. To overcome edge effect, useof an appropriate sealing tape like EASYseal(Fig. 7), storage of openly stacked plates
within a humidified chamber (Fig. 8), or imple-

6/8

Application Note

0.6
0.4
bench top

0.2

humidiﬁed chamber

0.0
1

2

3

4

5

6

7

8

row

Figure 8: Storage of openly stacked microplates
during incubation with primary and secondary
antibody in a humidified chamber significantly reduces
differences in OD values due to the edge effect.
The graph depicts mean OD values of microplate
wells per row.

mentation of a storage system with temperature and humidity control can dramaticallyreduce data inconsistency.
When developing and performing EnzymeLinked Immunosorbent Assays, there are a
multitude of parameters that should be considered to achieve optimal accuracy, reliability
and sensitivity of the test. Optimisation of
coating buffer and incubation conditions, suchas temperature, incubation time and environment, are fundamental steps to establish an
ELISA successfully.

4/ LITERATURE
4. Raem A. M, Rauch P (2007) Immunoassays.
Spektrum Akademischer Verlag, Elsevier GmbH,
München.

1. Green,N.M. 1975. Avidin. Adv. Protein Chem.
29 pp 85-133
2. Voss S., Skerra A. (1997) Mutagenesis of a flexible
loop in streptavidin leads to higher affinity for the
Strep-tag II peptide and improved performancein recombinant protein purification. Protein
Engineering vol.10 no.8 pp.975–982.

5. Cuvelier A. et al. (1996) Substitution of carbonate
by acetate buffer for IgG coating in sandwich
ELISA. J Immunoassay. 17(4):371-82.
6. Gosling J.P. (2000) Immunoassays. Practical
approach. Oxford University Press, Oxford.

3. Application Note Greiner Bio-One: „Insulin ELISA
on high-binding MICROLON 600 and CELLSTAR®
microplates“

5/ ORDERING INFORMATION
96 Well ELISA Microplates
U-Bottom / V-Bottom / F-Bottom / Half Area
Well format: 96, Bottom: solid, Raw material: PS, Lid: no
Item no.

Well profile

Bindingcharacterisitc

Binding brand
nameProductcolour

655001

F-bottom/ST

med.-binding

MICROLON 200

655061

F-bottom/ST

high-binding

655080F-bottom / Chimney Well

655081

Plate
designWorking volume(well)

Qty. inner /
outer

clear

25 µl - 340 µl

10 / 40

MICROLON 600

clear

25 µl - 340 µl

10 / 40

med.-binding

MICROLON 200

clear

25 µl - 340 µl

10 / 40

F-bottom / Chimney Well

high-binding

MICROLON 600

clear

25 µl - 340 µl

10 / 40675001

F-bottommed.-binding

MICROLON 200

clear

half area

15 µl - 175 µl

10 / 40675061

F-bottomhigh-binding

MICROLON 600

clear

half area

15 µl - 175 µl

10 / 40

Plate design

Working volume(well)

Qty. inner /outer

96 Well ELISA Strip Plates
Well format: 96, Bottom: solid, Raw material: PS, Lid: no
Item no.

Well profile

Bindingcharacterisitc

Binding brand
name762070F-bottommed.-binding

MICROLON 200

clear

strip plate 12x8

20 µl - 350 µl

10 / 100

762071

F-bottomhigh-binding

MICROLON 600

clear

strip plate 12x8

20 µl - 350 µl

10 / 100

Product colour

96 Well ELISA Single-Break Strip Plates
Well format: 96, Well profile: C-bottom, Bottom: solid, Raw material: PS, Plate design: strip plate 12x8,
Working volume (well): 20 µl - 300 µl, Lid: no
Colour stripe

Qty. inner /outerItem no.

Binding characterisitc

Binding brand name705070medium-binding

MICROLON 200

clear

10 / 100

705071high-binding

MICROLON 600

clear

10 / 100

Influence on ELISA performance

7/8

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F073118 AN Influence on ELISA performance english [Rev.01.2023]

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