APPLICATIONNOTE
Insulin ELISA on high-binding
MICROLON 600 andCELLSTAR® microplates
www.gbo.com /bioscience1/ INTRODUCTION
ELISA (enzyme-linked immunosorbent assay)
is one of the most widely used laboratory
techniques in analysis and diagnostics. Analytes such as peptides, proteins, antibodies
and hormones can be selectively detected andquantified in small concentrations among a
multitude of other substances with relatively
low cost and high simplicity.
Although ELISA methodology is used in a widevariety of different types of assays (e.g. direct
ELISA, indirect ELISA, sandwich ELISA, competitive ELISA), all ELISA variants are based on
the same principle: the binding of one assay
component – antigen or specific antibody –to a solid surface, and its selective interaction
with a subsequently added assay component.
Molecules that do not specifically interact withthe solid surface bound assay component are
washed away during the assay. ELISA is typically carried out in microplates or in tubes (Fig.1).

surface and detected using a monoclonal
mouse anti-insulin antibody as primary antibody
and an anti-mouse-IgG-horseradish peroxidase (HRP) conjugate as secondary antibody(Fig.2). The clinical application of an ELISA
based on the binding of insulin to a solid surface
is to monitor the presence of insulin-binding
antibodies in the sera of patients with type I
diabetes mellitus and of patients with administered insulin [1, 2]. An additional aspect of
this application note is to compare the performance of high-binding plates with CELLSTAR®
adherent cell culture plates for ELISA methods,
as the suitability of CELLSTAR® plates for thepurpose of ELISA is a frequently asked question.

anti-mouse
antibody-HRP (goat)

Tetramethylbenzidine(TMB)

anti-insulin
antibody (mouse)
InsulinFigure 2: Insulin ELISA:
Insulin is coated to the surface of a microplate.
A monoclonal mouse anti-insulin antibody as primary
antibody interacts with the bound insulin. In a second
incubation step a labelled secondary antibody (antimouse-IgG-HRP) that recognizes the primary antibody isemployed to bind the primary antibody/Insulin complex
and enable detection.

2/ MATERIALS
2.1/ MATERIAL AND REAGENTS
Item

Figure 1: 96 well ELISA strip plate and immuno tubes

This application note describes the development of an indirect ELISA for quality assurance applications in our lab. Insulin, a peptide
hormone of about 5,8 kDa, is bound to the plate

2/8

Application Note

Supplier

Item No.

Insulin (bovine)

Sigma-Aldrich

I5500

Anti-insulin
antibody (mouse)

Sigma-Aldrich

I2018

Anti-mouse-IgGHRP (goat)

Sigma-Aldrich

A9917

Dianova

009-000-003

Human IgG

ItemSupplier

Item No.

Peroxidase
AffiniPure Goat
anti-human-IgG

Dianova

109-035-003

96 well highbinding ELISA(MICROLON 600)
plate

Greiner BioOne GmbH

655061

96 well CELLSTAR®
plate

Greiner BioOne GmbH

655160Coating buffer(0.05 M carbonate,pH 9.6)

Sigma-Aldrich

C3041

Washing buffer
PBS-T (0.14 M NaCl,
0.01 M phosphate,
0.05 % Tween 20)

Sigma-Aldrich

P3563

Roth8076.3

Blocking buffer(3 % BSA inwashing buffer)
BSA Fraction V
Substrate solution(1 part of TMB tothree parts ofcitric acid/acetate,each 0.1 M, pH 4.9)
TMB

Sigma-Aldrich

T0440

Citric Acid

Sigma-Aldrich

C0759

Sodium Acetate

Sigma-Aldrich

71183RothK027.1

Sulphuric acid 0.5 M

2.2/ DETERMINATION OF
OPTIMAL WORKING
CONCENTRATIONS

insulin antibody in blocking buffer (100 µl perwell) was added in a concentration from
19.5 ng/ml to 2.5 µg/ml. After incubation for
1 h and subsequent washing, an anti-mouse
antibody horseradish peroxidase-conjugate(anti-mouse-HRP) was added (5 µg/ml inblocking buffer, 100 µl/well). The plate wasincubated for 1 h and washed before additionof 100 µl/well substrate solution for a 20 minincubation. The reaction was stopped byadding 100 µl per well 0.5 M sulphuric acid,after which optical density was measured ina Tecan SpectraFluorPlus at 450 nm. All incubation steps were performed at 23 °C in ahumidified chamber.

2.2.2/ CHECKERBOARD
TITRATION OF ANTI-INSULIN
ANTIBODY AND ANTIMOUSE-IGG-HRP
A 96 well flat bottom high-binding ELISAMICROLON 600 plate (Item No. 655061) wascoated with 800 ng/ml insulin and processed
as described above (Section 2.2.1). Anti-insulin
antibody was added in concentrations from
19.5 ng/ml to 2.5 µg/ml. Anti-mouse-IgG-HRP
was added in a concentration from 2.4 ng/ml
up to 5 µg/ml.

2.2.1/ CHECKERBOARD
TITRATION OF INSULIN AND
ANTI-INSULIN ANTIBODY

2.3/ COMPARISON OF THE
PERFORMANCE OF A HIGH
BINDING 96 WELL ELISAMICROLON 600 PLATE WITH A
CELLSTAR® PLATE

A 96 well flat bottom high-binding ELISA MICROLON 600 plate (Item No. 655061) was coatedwith 100 µl per well insulin (0 ng/ml to 8 µg/ml)
in coating buffer and incubated overnight.
The plate was washed three times (BioTek,Elx405Select) with 350 µl washing buffer perwell, blocked with 3 % BSA in washing buffer(150 µl per well) and incubated for 60 min. Anti-Two different assays were performed to compare binding of insulin and IgG within a highbinding 96 well flat bottom ELISA MICROLON
600 plate (Item No. 655061) and a corresponding CELLSTAR® plate for adherent cell culture(Item No. 655160). For the insulin assay, bothplate types were coated with 800 ng/ml insulin. For the immunological reactions 80 ng/ml

Insulin ELISA

3/8

anti-insulin and 300 ng/ml anti-mouse IgG-HRP
were used, respectively. For the IgG assay,
both plates were coated with 100 µl/well human
IgG (5 µg/ml) in coating buffer. For detection
goat anti-human IgG-HRP was added in a concentration of 3.3 ng/ml in blocking buffer. Bothassay procedures were conducted according
to the insulin assay protocol as described inSection 2.2.1.

3/ RESULTS ANDDISCUSSION
3.1/ DETERMINATION OF
OPTIMAL WORKING
CONCENTRATIONS
3.1.1/ CHECKERBOARD TITRATIONOF INSULIN AND ANTIINSULIN ANTIBODY
When establishing a new assay optimal working concentrations for all components mustbe determined empirically. This is done by performing a so-called checkerboard titration. In
such an experiment serial dilutions of two
assay components, e.g. coating concentrationand primary antibody, are screened in a matrix(Fig. 3). In a first step the insulin coating concentration was titrated against the concen-

123456789 10 11 12 13
1:1

B

1:2

C

1:4

D

1:8

EFGH
1:1 1:2 1:4 1:8
Component AFigure 3: Checkerboard titration

4/8

Application NoteComponent BAtration of the primary (anti-insulin) antibody todetermine the optimal coating concentration.
Carbonate-buffer was used for coating as described in the literature [1, 2]. In case the ideal
coating buffer is not known it is recommendable to try coating buffers with differing
pH-values, e.g. carbonate buffer at pH 9.6,phosphate buffer at pH 7.4 and acetate-buffer
at pH 5.5 as some proteins or peptides bindbetter to the plastic surface at neutral or acidic
pH [3]. The plates were incubated overnightat room temperature in a humidified chamber.
Incubation in a humidified chamber preventsthe formation of a temperature gradient andwater evaporation within the plate which may
result in a shift of OD values between outerand central wells. Alternatively the plates canbe covered with a sealing tape (EASYseal,
Item No. 676001). Incubation times and temperature were adopted from a protocol alreadyestablished in our lab. Even though incubation
time and temperature can be optimized to
improve assay performance, because existing
protocol incubation times and temperature
worked well to achieve results, these parameters were not modified. After coating, theplates were blocked with 150 µl/well of 3 %BSA in washing buffer. It is essential to block
any remaining unoccupied binding sites.
Blocking with an appropriate blocking reagent
prevents non-specific binding of assay components in a later step of the assay that could
lead to increased background signals. BSA is
the most commonly used reagent for blocking.
Alternative blocking reagents are e.g. gelatine, casein or commercially available solutions.
After blocking, the primary antibody – a monoclonal mouse anti-insulin antibody – was addedin a concentration ranging from 19.5 ng/mlto 2.5 µg/ml. The plates were washed witha washing buffer containing the detergent
Tween 20. The addition of a detergent enhances the removal of non-specifically boundantibody molecules and therefore helps tominimise background. The secondary antibody,an anti-mouse-IgG-HRP conjugate, was added

bination were (1) an optical density of about 0.8
to 1.5 units, (2) a high signal to noise ratio,and (3) the location in the linear part of thecurve to assure assay sensitivity. A combinationof an anti-insulin concentration of 80 ng/ml andan anti-mouse-IgG-HRP concentration of
300 ng/ml was chosen for further experiments(Fig. 5). The resulting protocol is shown in Fig.6.

OD [450 nm]
Anti-insulin-antibody

3 .5
3.0
2.5
2.0
1.5
1.0
0.5

2,4 ng/ml

9,8 ng/ml

4,9 ng/ml

19,5 ng/ml

78 ng/ml

39 ng/ml

156 ng/ml

313 ng/ml

625 ng/ml

1,25 μg/ml

5 μg/ml

0.0
2,5 μg/ml

in excess to eliminate any influence on the test
results. A colorimetric detection was performed using 3, 3’, 5, 5’ tetramethylbenzidine (TMB)
as the substrate. Another frequently usedenzyme label for colorimetric assay is alkaline phosphatase (AP) with p-nitrophenyl
phosphate (pNPP) as the substrate. The enzymatic reaction was stopped after 20 minutes
by inactivating HRP with sulphuric acid. The
optical density at 450 nm was plotted against
the serial dilution of insulin resulting in a saturation curve showing the maximum concentration of insulin bound to the plate surface
under the chosen conditions (Fig. 4). Because
saturation of the high-binding plate surface
occurred at approximately 800 ng/ml, this value
was chosen as coating concentration for further proceedings.

Anti-mouse-lgG-HRP

Anti-insulin-antibody

OD [450 nm]
3 .5

2,5 μg/ml

1,25 μg/ml

625 ng/ml

313 ng/ml

156 ng/ml

78 ng/ml

39 ng/ml

19,5 ng/ml

3.0

Figure 5: Checkerboard titration of the anti-insulin
antibody concentration against anti-mouse-IgG-HRP.

2.5
2.0
1.5
1.0
0.5

Coat plate with 800 ng/ml insulin incoating buffer (100 µl/well)

2,5 μg/ml

1,25 μg/ml

625 ng/ml

313 ng/ml

156 ng/ml

78 ng/ml39 ng/ml

19,5 ng/ml

0 ng/ml

25 ng/ml

50 ng/ml

100 ng/ml

200 ng/ml

600 ng/ml

400 ng/ml

1 μg/ml

800 ng/ml

2 μg/ml

6 μg/ml

4 μg/ml

8 μg/ml

0.0

Insulin Coating

Overnight, 23 °C

Wash 3x with washingbuffer (350 µl/well)

Block with 3 % BSA in
washing buffer (150 µl/well)
1 h, 23 °C

Figure 4: Checkerboard titration of the insulin coating
concentration against the anti-insulin antibody conc.

Primary antibody: 80 ng/ml mouse anti-insulin antibody in blocking buffer (100 µl/well)
1 h, 23 °C

3.1.2/ CHECKERBOARD
TITRATION OF ANTI-INSULIN
ANTIBODY AND ANTIMOUSE-IGG-HRP

Wash 3x with washingbuffer (350 µl/well)

Secondary antibody: 300 ng/ml anti-mouseIgG-HRP in blocking buffer (100 µl/well)
1 h, 23 °C

Wash 3x with washingbuffer (350 µl/well)

Substrate solution (100 µl/well)
20 min, 23 °C

In a second step the high-binding microplate
was coated with 800 ng/ml insulin and theprimary antibody (anti-insulin antibody) concentration was screened against the concentration of the secondary antibody (anti-mouseIgG-HRP) to determine appropriate antibodyconcentrations. The criteria for a suitable com-0.5 M sulphuric acid (100 µl/well)

Measurement of OD at 450 nm

Figure 6: Insulin ELISA protocol

Insulin ELISA

5/8

The attachment of biomolecules to a solid
surface is based upon passive adsorption.
Therefore physicochemical forces such ashydrophobic bonds, hydrophilic interactionsand H-bonding are relevant. Polystyrene, themost widely used material for immunologicalmicroplates, has hydrophobic characteristics.
Because for most ELISA applications thepre-sence of hydrophilic groups is beneficial,high-binding ELISA MICROLON 600 plates are
physicallytreatedtointroduceadefinednumberof hydrophilic groups. CELLSTAR® plates, withintended application for adherent cell culture,undergo a different kind of physical treatment
to improve cell adhesion. This type of treatment
renders the plate surface more hydrophilic,similar to the treatment of high-binding plates,
but the number of polar groups introduced on
the CELLSTAR® surface is much higher than ona high-binding surface intended for ELISA application. As the usability of CELLSTAR® platesfor ELISA is a frequently asked question, highbinding ELISA and CELLSTAR® plates werecompared with the insulin assay describedabove as well as another assay based on theadsorption of human IgG to the plate surface.
Both IgG and insulin bound very well on thehigh-binding ELISA MICROLON 600 surface(Fig. 7). However, insulin did not bind significantly to the CELLSTAR® surface, resulting
in a very low OD value, and the binding of IgG
to the CELLSTAR® surface was much weakerin comparison to the high-binding ELISAMICROLON 600 surface. Coefficients of variation (CV) for both assays were well below
5 % in each plate type. Non-specific binding on the CELLSTAR® surface, as is often
discussed for tissue culture plates, was notdetected under the tested conditions (datanot shown).

6/8

Application Note

OD [450 nm]

1 .6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

CV [%]

3.2/ COMPARISON OF THE
PERFORMANCE OF A HIGH
BINDING ELISA MICROLON
600 PLATE AND
A CELLSTAR® PLATElgG

InsulinlgG

Insulin

5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.51.00.5
0.0

Figure 7: Optical density values and coefficients ofvariations for the insulin and the IgG ELISA on highbinding and CELLSTAR® microplates.

These results demonstrate that the performance of ELISA applications on the CELLSTAR®
surface is highly dependent on the nature of themolecule being bound to the plate. It is likely
the high charge density on the CELLSTAR®
surface is not beneficial for binding of insulin and IgG, but rather prevents binding byrejection. This effect is more pronouncedwith the smaller insulin molecule than with thesignificantly larger IgG molecule. It is important to note that, due to intended application
for use, CELLSTAR® plates are not subject tothe same quality assurance procedure that is
implemented for immunological plates. For
production of Greiner Bio-One immunologicalproducts, raw material batches are checked
as a part of an integrated quality control process to ensure specific criteria are satisfied
prior to the release of the finished goods. To
guarantee consistent binding properties theELISA products are continuously monitored
for inter and intra plate homogeneity. Quality
testing is performed to assure that the coefficient of variation in one plate does notexceed 5 % for colorimetric assays and theratio of OD values of new plates to referenceplates is in the range of 100 +/- 10 %. As
CELLSTAR® plates have a designated intent forcell culture, the quality control procedures forthese products are vastly different and involve
comprehensive monitoring and testing surrounding that intended purpose. In addition to

the superior comparative results achieved
using ELISA vs. CELLSTAR® plates within thisapplication note, for the quality testing reasons outlined above ELISA plates are infinitely
preferable over CELLSTAR® plates for ELISA
applications.

4/ LITERATURE
1. Wilkin T. et al. (1984), A Micro Enzyme-Linked
Immunosorbent Assay for Insulin Antibodies
in Serum, Journal of Immunological Methods,
76: 185-194

Enzyme Immunoassay for Anti-Insulin
Antibody in Diabetes Mellitus Patiens,
Journal of Immunological Methods, 84: 245-250
3. Raem A.M., Rauch P. (2007) Immunoassays,
Spektrum Akademischer Verlag

2. Kobayashi N. et al. (1985), A Solid-Phase

5/ ORDERING INFORMATION
96 Well ELISA Microplates – 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/Standard

med.-binding

MICROLON 200

655061

F-bottom/Standard

high-binding

655080F-bottom / Chimney Well

655081

Plate
designWorking volume(well)

Qty. inner /outerclear

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 600clear

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: noItem no.

Well profile

Bindingcharacterisitc

Binding brand
name701070F-bottommed.-binding

MICROLON 200

clear

strip plate 12x8

20 µl - 350 µl

10 / 100

701071

F-bottomhigh-binding

MICROLON 600clear

strip plate 12x8

20 µl - 350 µl

10 / 100

Product colour

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: noColour stripe

Qty. inner /outer

Item no.

Binding characterisitc

Binding brand name705070med.-binding

MICROLON 200

clear

10 / 100

705071high-binding

MICROLON 600

clear

10 / 100/ Please note this is only an extract of immunological products. You can find more information in our catalogue or on our website: www.gbo.com.

Insulin ELISA

7/8

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E-MAIL

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F073106 AN Insulin ELISA english [Rev.12.2022]

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