N° 9

FORUM
TECHNICAL NOTES & APPLICATIONS FOR LABORATORY WORKCONTENT
1. Introduction
2. Impact of the solid
surface

MICROPLATES FOR ENZYMELINKED IMMUNOSORBENTASSAYS (ELISA)
1/ INTRODUCTION
ELISA (Enzyme-Linked Immunosorbent Assay) is one of the most widely used biochemical
methods in laboratory analysis and diagnostics. Analytes such as peptides, proteins, antibodies and hormones can be detected selectively and quantified in low concentrations
among a multitude of other substances. Additionally, ELISAs are rapid, sensitive, costeffective and can be performed in a high-throughput manner. An ELISA is used in a vastvariety of different types of assays (e.g. direct ELISA, indirect ELISA, sandwich ELISA,
competitive ELISA, see technical appendix). Nevertheless, all ELISA variants are basedon the same principle, the binding of one assay component – antigen or specific antibody –to a solid surface and the selective interaction between both assay components. Molecules not specifically interacting with the assay component bound to the solid surface
are washed away during the assay. For the detection of the interaction the antibody or
antigen is labelled or linked to an enzyme (direct ELISA). Alternatively, a secondary
antibody conjugate can be used (indirect ELISA). The assay is developed by adding an
enzymatic substrate to produce a measurable signal (colorimetric, fluorescent or luminescent). The strength of the signal indicates the quantity of analytes in the sample.

3. Performance ofGreiner Bio-One highbinding microplates
4. Microplate format andwell profile
5. Technical Appendix
6. Ordering Information

FORUM

N° 9

Oxygen content [atom%]

2/ IMPACT OF THE SOLID SURFACE
A key step in an ELISA is the binding of one assay component – antigen or antibody – to the solid surface. Therefore,
the features of this surface, most often the wells of a 96 well
microplate, are crucial for the performance of the assay.

3.5
2.5
2.5
1.5
1.5
0.5

2.1/ SURFACE PROPERTIESThe attachment of antigens or antibodies to a solid surface,also referred to as coating, is based upon passive adsorption. Therefore, physiochemical forces like hydrophobicbonds and H-bonding are relevant. Hydrophobic bonds are
based on close interactions between hydrophobic groups.
Molecules containing hydrophilic groups can be bound byhydrophilic interactions if hydrophilic groups are present
on the solid surface. Bonds based upon passive adsorption
are generally not that strong and their nature is transient.
Binding depends on many factors like size and propertiesof the molecule to be bound, pH of the coating buffer andtemperature. Nevertheless, once the interactions between
the solid surface and the molecules are established in large
numbers, the binding is very stable.
Polystyrene is the most widely used material for immunological microplates. The resin is highly transparent andideally suited for optical measurements. Due to its chemical nature polystyrene is a hydrophobic compound andnon-treated polystyrene plates feature hydrophobic characteristics. As for most ELISA applications the presenceof hydrophilic groups at the surface is beneficial, ELISA
plates are most often physically treated to introduce adefined number of hydrophilic groups to the plate surface.
Non-treated polystyrene microplate surfaces contain
about 0.5 atom% oxygen, whereas the oxygen content ofGreiner Bio-One high-binding microplates is approximately
2.5 atom% (Fig. 1).

0.0
non-treatedhigh-binding

Figure 1: Oxygen content on polystyrene microplate surfaces
determined by XPS (X-ray photoelectron spectroscopy).

usage of high-quality virgin polystyrene and a well-established and monitored manufacturing process. At Greiner
Bio-One incoming raw material lots are quality tested ina sample production and a subsequent immunoassay.
The new raw material lot is only accepted for production
if the sample plates fulfill the following criteria:/

For intra-plate homogeneity the coefficient of variation(CV) must not exceed 5% for colorimetric or 10% forfluorescence and luminescence assays.

/

For all immunological products, to guarantee constantbinding properties, the CV for five tested plates must notexceed 10%. Additionally, the ratio of new sample plates
to reference plates has to be in the range of 100 +/- 10%.

The CV is a measure of the variation of the optical density
values in different wells. It is a benchmark for the homogeneity of a surface with regard to binding properties.
The lower the CV the more consistent are the binding properties of the tested surfaces.
The CV is calculated according to the following formula:

CV [%] =standard deviationmean• 100%

2.2/ CONSISTENCY OF SURFACE PROPERTIES
Microplates used for immunological assays must feature defined and consistent binding characteristics. Keyfeatures besides the physical surface treatment are the2 / 16www.gbo.com

Fig. 2A-C and 3A-C show the CVs and optical density
values of samples of three different microplate formats
taken randomly from ten production lots over four years.
To verify the consistency of the binding properties, theN° 9microplates were tested with two ELISA protocols: a) a
direct ELISA where human IgG, a 150 kDa glycoprotein, isbound to the plate surface and b) an indirect ELISA where
the plates were coated with the 5.8 kDa peptide hormone
insulin. With both assays CVs for all tested plates were
well below the staked 5% limit and in each experimental
set up the ratio of the measured optical densities waswithin the given 10% tolerance (Table 1).

Item No.

Description

F-bottom/ standard
96 well microplates
C8 Single-break
strip plates

655061705071762071
Table 1:

F8 Strip plates

FORUM

IgG

Insulin

94.3%

98.8%

92.8%

91.9%

92.3%

93.8%

Maximum ratio within the tested plates

CV [%]OD 450 nm

2A: HIGH-BINDING MICROPLATES. STANDARD 96 WELL. F-BOTTOM (ITEM NO. 655061)
2.0
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 102.0

CV [%]OD 450 nm

2B: HIGH-BINDING C8 SINGLE-BREAK STRIP PLATES (ITEM NO. 705071)
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10CV [%]OD 450 nm

2C: HIGH-BINDING F8 STRIP PLATES (ITEM NO. 762071)
2.0
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10Figure 2 A-C: Direct ELISA Detection of Human IgG (150 kDa)
Optical density values and coefficients of variation of different formats of Greiner Bio-One ELISA microplates. Human IgG was boundto the plate surface (5 μg/ml) and detected colorimetrically with a goat anti-human IgG-HRP conjugate (3.3 ng/ml) and TMB.
Figure 2A: 655061 (high-binding F-bottom / standard 96 well microplates); Figure 2B: 705071 (high-binding C8 Single-break strip plates);Figure 2C: 762071 (high-binding F8 strip plates).

www.gbo.com

3 / 16

FORUMN° 9

2.0

CV [%]OD 450 nm

3A: HIGH-BINDING MICROPLATES. STANDARD 96 WELL. F-BOTTOM (ITEM NO. 655061)
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10

Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 102.0

CV [%]OD 450 nm

3B: HIGH-BINDING C8 SINGLE-BREAK STRIP PLATES (ITEM NO. 705071)
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10CV [%]OD 450 nm

3C: HIGH-BINDING F8 STRIP PLATES (ITEM NO. 762071)
2.0
1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
Lot 1

Lot 2

Lot 3

Lot 4

Lot 5

Lot 6

Lot 7

Lot 8

Lot 9

Lot 10Figure 3 A-C: Indirect ELISA Detection of Insulin (5,8 kDa)
Optical density values and coefficients of variation of different formats of Greiner Bio-One ELISA microplates. The microplatesurface was coated with the peptide hormone insulin (800 ng/ml). A monoclonal anti-insulin antibody (mouse, 80 ng/ml) in
combination with an anti-mouse IgG-HRP conjugate (300 ng/ml) was used for colorimetric detection with TMB in an indirect ELISA.
Figure 3A: 655061 (high-binding F-bottom /standard 96 well microplates);Figure 3B: 705071 (high-binding C8 Single-break strip plates);Figure 3C: 762071 (high-binding F8 strip plates).

4 / 16www.gbo.com

N° 9

3/ PERFORMANCE OF GREINER BIOONE HIGH-BINDING MICROPLATES
3.1/ EXEMPLARY ELISAS/

The binding of the glycoprotein hormone erythropoietin(EPO) to the plate surface was detected in an indirect
ELISA with an anti-EPO antibody from rabbit and an antirabbit IgG-HRP conjugate. EPO regulates the red blood cell
production. It is an acidic 34 kDa polypeptide consistingof 165 amino acid residues. Carbohydrate side chainsaccount for approx. 40% of the molecular mass of EPO.

A set of different immunoassays (direct and indirect
ELISAs) was established in our laboratory on high-binding
microplates (Fig. 4). Working concentrations were determined by performing checkerboard titrations. The working
concentrations used are shown in Table 2.
/

The 150 kDa glycoprotein IgG (human) was bound tothe microplate surface and detected with a goat antihuman IgG-horseradish peroxidase (HRP) conjugate
in a direct ELISA.

/

Binding of the peptide hormone insulin (5.8 kDa,51 AA)
was detected with a monoclonal mouse anti-insulin
antibody (AB) and an anti-mouse IgG-HRP conjugate in
an indirect ELISA.

/

Microplates were coated with high-density lipoproteins(HDL). HDL forms a class of lipoproteins that carry fatty
acids and cholesterol in the blood from the body’s tissues
to the liver. HDL has a hydrophobic core.
Binding of HDL was detected with an anti- HDL antibody
from goat and an anti-goat IgG-HRP conjugate in an
indirect ELISA.

FORUM

Substrate

AB-HRP conjugate

AntigenFigure 4A: Direct ELISA

Substrate
Secondary AB
conjugate

Coating
concentration

Primary
antibody

Secondaryantibody

IgG

5 μg/ml

3.3 ng/ml

x

Insulin

800 ng/ml

80 ng/ml

300 ng/ml

HDL

800 ng/ml

300 ng/ml

1,25 μg/ml

EPO

400 ng/ml

600 ng/ml

300 ng/ml

Molecule

Table 2:

Established working concentrations

Primary AB
AntigenFigure 4B: Indirect ELISA

www.gbo.com

5 / 16

N° 9

2.0

CV [%]OD 450 nm

FORUM

1.8
1.6
1.4

5 .0
4.5
4.0
3.5

1.2

3.0

1.0

2. 5

0.8

2.0

0 .6

1.5

0.4

1.0

0.2

0.5

0.0

0.0
IgG

Insulin

HDL

Epo

IgG

InsulinHDL

Epo

Figure 5: Optical density values and coefficients of variations
for exemplary ELISAs established on Greiner Bio-One high-binding microplates.

CONCLUSION:
Substrate

Greiner Bio-One high-binding microplates demonstrated
excellent performance in all tested immunoassays. Reproducible protocols with coefficients of variation lessthan 3% were established for all assays (Fig. 5).

Streptavidin-HRP
conjugatebiotinylated
protein3.2/ BINDING OF BIOTINYLATED PROTEINS

Figure 6A: Binding of biotinylated proteins and detection
with streptavidin-HRP streptavidin-HRP

OD 450 nm

Binding to a solid surface based upon passive adsorption
is highly dependent on both the surface characteristics
and the properties of the molecule to be bound (e.g. size,lot). Binding characteristics may vary between different
molecules on the same solid surface. To illustrate this potential variability, four different biotinylated proteins werebound to a 96 well high-binding plate and detected with astreptavidin-HRP conjugate (8 ng/ml). 0.05 M carbonate
buffer, pH 9.6 was used as the coating buffer (Fig. 6A).

2.0
1.8

Protein A

1.6

IgG

1.4

Albumin

1.2

Insulin

1.0
0.8

/

Protein A is a 42 kDa surface receptor protein of thebacteria Staphylococcus aureus. It contains little orno carbohydrate, is devoid of cysteins and rich inaspartic and glutamic acids.

/

Albumine (66 kDa, 584 AA) contains a lot of sulphureous amino acids.

/

Glycoprotein IgG (human)/

Peptide hormone insulin

6 / 16www.gbo.com

0 .6
0.4
0.2
0.0
1000

600

400

200

100

60

40

20

10

5

2

1

Protein concentration [ng/ml]

Figure 6B: Binding curves on a 96 well standard
high-binding microplate

N° 9

Binding curves for Protein A and IgG look very similar,
whereas the curves for albumin and insulin differ significantly (Fig. 6B). Taking into account the impact of molecule properties on binding, it is advisable to compare theperformance of different surfaces when developing a
new assay.

4/ MICROPLATE FORMAT AND WELLPROFILE
Microplates for ELISA applications are available in a large
variety of formats: standard 96 well plates, strip plates
and so-called single-break strip plates. The employmentof strip plates or single-break plates offers the advantage
of more flexibility. Individual strips or wells can be removed from the support frame so that the number of tests
to be performed can be adjusted to the actual numberof samples and is not predetermined by the microplate
format used.
For some ELISA applications a reduction of the sample
volumes can be an important aspect. Usually, this is doneby moving from a 96 well microplate to a 384 well microplate. However, this implies significant changes in plate
handling and the adaption of laboratory equipment.
96 well half area microplates offer an interesting alternative. This plate format allows a significant reduction of
the sample volume within the accustomed 96 well plate
format (Fig. 7, Table 3).

FORUM

96 wellstandard

96 wellhalf area

Covered surface area

92 mm282 mm2

Corresponding liquid height

3 mm

4.7 mmVolume coating solution

100 µl

75 µl

Washing Buffer

350 µl150 µlAnti human IgG-HRP conjugate

100 µl75 µl

OD*450 nm

1.2

1.3

Table 3:Comparison standard – half area microplates,* After adaption to 1 cm pathlength.

7A: F-BOTTOM CHIMNEY WELL

7B: HALF AREA

Figure 7: Well profiles
7A F-bottom/chimney well, 7B half area

www.gbo.com

7 / 16

FORUM

N° 9

8A: U-BOTTOM

9A: C-BOTTOM SINGLE-BREAKSTRIP PLATES: C8

8B: V-BOTTOM

9B: U-BOTTOMSTRIP PLATES: U8, U16

8C: F-BOTTOM STANDARD

9C: F-BOTTOMSTRIP PLATES: F8, F16

Figure 8: Different well profiles of 96 well ELISA microplates

Figure 9: Different well profiles of 96 well ELISA strip plates

Different well profiles – U-, V-, F-, C-bottom – are available(Fig. 7,8,9). In general, immobilisation of biomolecules is
not affected by the well profile. However, the fact that thecovered surface area and the resulting area-to-volume
ratio may vary should be taken into account (Table 4).

well bottom edges, enabling liquid to be removed in entirety. V-bottom plates feature a conically tapered wellbottom and are ideally suited for applications in which
the total sample volume must be pipetted off.

F-bottom plates demonstrate excellent optical properties
and are therefore ideal for precise optical measurements.
They are additionally compatible with common microplate
washers and readers. U-bottom wells allow the removalof liquid without recess. The C-bottom is a flat-bottom
profile with rounded corners. It combines the excellent
optical properties of the F-bottom profile with rounded

8 / 16www.gbo.com

FORUM

N° 9

Liquid volume (μl)

Covered surface
area (mm2)

Liquid height (mm)Area / volume
ratio (cm2 / cm 3)

96 well microplate U-bottom

100

84.6

4.2

8.5

96 well microplate V-bottom

100

88.6

5.3

8.9

96 well microplate F-bottom / standard

100

92.0

3.0

9.2

96 well microplate F-bottom / chimney well

100

93.0

2.9

9.3

96 well microplate F-bottom / half area

100

103.6

6.2

10.4

C8 Strip Plate

100

88.6

3.6

8.9

F8 Strip Plate

100

93.5

2.85

9.4

U8 Strip Plate

100

84.0

4.2

8.4

U16 Strip Plate

100

84.0

4.2

8.4

U16 Strip Plate

100

94.3

3.0

9.4

Table 4: Volume-dependent wetting of immunological platesMore detailed data are listed in the technical appendix of our catalogue or in the data sheets on our website www.gbo.com.

www.gbo.com

9 / 16

N° 9

5/ TECHNICAL APPENDIXBLOCKING / BUFFER

1

2

3

4

5

6

7

8

9 10 11 12
1:1

A
B

1:2

C

1:4

D

1:8

E
F
G
H
1:1 1:2 1:4 1:8
Secondary antibody

2.0

Primary antibody

Blocking buffers are commercially available as well.
Washing steps are included into the assay protocol toremove unbound reagents after incubation steps. Insufficient washing may result in a high background, whereas
excessive washing may lead to debonding of adsorbed
assay components, thereby decreasing the sensitivity of
the assay. Often the wash buffer is the same as used forblocking to which detergent has been added. The detergent
supports the removal of non-specifically bound moleculesand subsequent minimisation of background. A commonlyused detergent is Tween-20 in a concentration of 0.05%.

When developing an ELISA optimal working concentrations
for all assay components must be determined empirically.
This is done by performing a so-called checkerboard titration. In such an experiment the concentration of two assay
components is systematically varied. From the resulting data
a combination of concentrations of the tested substances
can be chosen that results in optimal optical density values and ensures a consistent performance of the ELISA.

2.0
1 .5
1 .0

The use of detergents should be avoided before blocking as this may cause a reverse effect and result in an
increased background. Therefore, the most effective
buffers to use for a given assay depend on the featuresof the assay components and the most suitable buffer
system should be determined empirically. In general,phosphate buffer interferes with alkaline phosphatase(see Detection).

10 / 16www.gbo.com

Primary antibody

Widely used assay buffers are PBS or Tris buffer. During
the coating procedure ionic strength and pH of the bufferused can play an important role. The most common buffer
for coating plastic surfaces with proteins and glycoproteins is 0.05 M carbonate buffer at pH 9.6. Nevertheless,for some proteins a different pH value may be more favourable. Alternative coating buffers are 10 mM PBS pH 7.4
or an acetate buffer at pH 5.5. It is essential to block unoccupied binding sites on the solid surface in a blocking
step after coating to reduce non-specific binding andto avoid background problems during subsequent assay
steps. Commonly used blocking agents are BSA, casein
or non-fat dried milk powder in Tris or PBS buffer.

CHECKERBOARD TITRATION (FIG. A)

OD 450 nm

FORUM

0.5
0.0
1:2

1:4

1:8

1:16

1:32

1:64 1:128 1:256 1:512 1:1024 1:2048

Secondary antibody
1:1

1:2

1:4

1:8

1:16

Figure A: Checkerboard titration

1:32

1:64

1:128

N° 9

COMPETITIVE ELISA (FIG. B)

In a competitive ELISA a labelled and pretitrated assay
component is added simultaneously with the sample. If
the antigen is bound to the solid surface then a mixtureof sample and the labelled antibody is added in the incubation step. The antibodies contained in the sample
and the enzyme-labelled antibodies compete for antigen
binding. The more antibodies contained in the sample,
the less enzyme-labelled antibody is available to bind to
the antigen coating. The reduced binding of the labeled
antibody results in a decrease of the signal. Alternatively
the antibody can be bound to the surface, after which thelabelled antigen is added to compete with antigen contained in the sample during the immunological reaction.

Substrate

Enzyme-labelled AB

AB containedin the sample
AntigenFigure B: Competitive ELISA

FORUM

are free of the problems due to working with radioactive
material. The two most commonly used enzyme labels
are horseradish peroxidase (HRP) and alkaline phospatase (AP). Suitable substrates are commercially available
for both enzymes.
Colorimetric detection results in a coloured reaction
product which can be quantified with a spectrophotometer. Widely used chromogenic substrates are 3, 3’, 5,5’-tetramethylbenzidine (TMB) for HRP and p-nitrophenyl
phosphate (pNPP) for AP. For colorimetric detection clear
microplates are ideal. Black microplates and strips areused for fluorescent immunoassays (FIA) as black microplates reduce background autofluorescence, well-to-wellcross talk and light scattering. Fluorescence is a physicalprocess where molecules (e.g. a fluorescent substrate)are excited by the absorption of light of a certain wavelength. When the molecules return to their ground state
they emit light at a different wavelength. A fluorometer isneeded to view the results. Luminescent immunoassays(LIA) are very sensitive as the signal can be multipliedand amplified. In luminescent immunoassays an enzyme
catalyses the conversion of a substrate to a product thatemits light. White plates and strips are ideal for luminescence detection as the white wells enhance the signal byreflecting the light. Cross talk and background luminescence are minimised. The emitted light is measured by aluminometer.

DETECTION

Initially, the detection of the interaction between antigen
and antibody was based on radioisotope labelling. Today,
enzymes, biotin or fluorophores are widely used for labelling and detection is based on chromogenic, fluorescentor chemoluminescent substrates. The intensity of thereleased signal is correlated with the amount of analytein the sample. These detection methods are equally oreven more sensitive than the radiochemical approach but

www.gbo.com

11 / 16

FORUM

N° 9

DIRECT AND INDIRECT ELISA (FIG. C, D)

Assays can be divided into “direct” and “indirect” methods. In a direct ELISA the assay component bound to thesolid surface interacts with the enzyme-labelled second
assay component. Direct ELISAs are not widely used aslabelling of the antigen or the primary antibody is timeconsuming and expensive. In an indirect ELISA detection
is conducted with an enzyme-labelled secondary antibody. First, a primary antibody interacts with the antigen.
In a second incubation step a labelled secondary anti-

Substrate

AB-HRP conjugate

Antigenbody that recognises the primary antibody is used. If
primary antibodies from the same species are used indifferent assays, the same enzyme-labelled secondary
antibody can be used for detection. Additionally, sensitivity
is increased as several secondary antibodies can bind to
one primary antibody resulting in signal amplification.
Labelled secondary antibodies are commercially available
in a large variety.

SANDWICH ELISA (FIG. E)

A Sandwich ELISA is used when antigens can not be bounddirectly to the solid surface. Instead the solid surface iscoated with a specific antibody which is used to “capture”
the antigen. Therefore, sandwich ELISA is sometimes referred to as capture ELISA. In a second immunologicalreaction, a second specific antibody is added which is detected by an enzyme-labelled secondary antibody. As aresult, the antigen is bound between two antibodies. For
sandwich ELISA an antigen must contain at least two
epitopes which allow the parallel binding of two antibodies.

Figure C: Direct ELISA
Substrate

Substrate

Enzyme-labelled
secondary AB

Secondary AB
conjugate

Second speciﬁc AB
AntigenPrimary AB

Capture AB

AntigenFigure D: Indirect ELISA

12 / 16www.gbo.com

Figure E: Sandwich ELISA

N° 9

FORUM

TEMPERATURE

Temperature influences the timeframe for binding of molecules to the solid surface. Total will be the same after acertain point, however, the rate of binding depends on thechosen incubation temperature. An increased incubation
temperature allows the reduction of the incubation time.
Typical incubation temperatures for coating are 4 °C,
22 °C and 37 °C. Note that the enzyme-substrate reactionis also temperature-sensitive. A temperature gradient
within the plate during detection may result in a shift of OD
values between outer wells and center wells (edge-effect).
Therefore a temperature gradient should be avoided.

1. Coating
The wells are filled with 100 μl coating solution (800 ng/ml
insulin in 0.05 M carbonate buffer; pH 9.6) and stored in
an incubator overnight (about 18 h) at 23 °C. To avoid evaporation, a petri dish can be filled with water and placed in
the incubator. Alternatively, the plates can be sealed withplate sealers.
2. Washing
After incubation the plates are washed four times with
350 μl PBS-T buffer (0.14 M NaCl; 0.01 M PO4; 0.05%
Tween 20; pH 7.4) per well. After the last washing step any
remaining liquid has to be removed by tapping the plate
onto a paper towel.

TYPICAL PROTOCOL FOR AN INDIRECT ELISA (FIG. F)

The test protocol below describes an indirect ELISA in
a 96 well polystyrene high-binding microplate which isbased upon the adsorptive binding of insulin to the innersurface of the wells and the subsequent interaction of
the bound insulin with a specific monoclonal anti-insulin
antibody produced in mouse. For detection an anti-mouse
IgG-HRP conjugate is used.

3. Blocking
Unoccupied binding sites on the well surface are blockedwith BSA in order to avoid binding of antibodies in subsequent steps of the assay.
The plates are filled with 150 μl/well blocking solution(3% BSA in PBS-T) and incubated for 60 min. at 23 °C. Afterincubation, the solution is hand-decanted. Remaining liquid
is removed by rapping the plate onto a paper towel.

www.gbo.com

13 / 16

FORUMN° 9

4. Addition of primary antibody
During the immunochemical reaction, the anti-insulin antibody (primary antibody) is added and binds to the affixedinsulin molecules. The plates are filled with 100 μl/well ofprimary antibody solution (80 ng/ml monoclonal mouse antiinsulin antibody in PBS-T) and incubated at 23 °C for 60 min.
5. Washing
Unbound antibodies are washed away in a subsequentwashing procedure (see 2).
6. Addition of secondary antibody
For detection of the interaction between insulin and thespecific antibody an anti-mouse IgG-HRP conjugate(secondary antibody) is used. The plates are filled with
100 μl/well of secondary antibody solution (300 ng/ml

7. WashingSee 2.
8. Detection
The plates are filled with 100 μl/well substrate solution(1 part of TMB to three parts of citric acid/acetate, each
0.1M; pH 4.9) and incubated at 23 °C for 20 min. TMB is split
by HRP and a coloured substance is released. The colorimetric reaction is stopped and fixed by adding 100 μl
0.5 M sulphuric acid per well. The strength of the colour
reaction is measured by a microplate reader at 450 nm.
As the reaction is stable for a maximum of 1 hour, theplates should be measured within this timeframe.

1. COATING

2. AFTER WASHING

3. BLOCKING

4. ADDITION OF
PRIMARY ANTIBODY

5. AFTER WASHING

6. ADDITION OFSECONDARY ANTIBODY

7. AFTER WASHING

8. DETECTION

Figure F: Typical protocol for an indirect ELISA

14 / 16anti-mouse IgG antibody-HRP conjugate in PBS-T). The
plates are incubated at 23 °C for 60 min.

www.gbo.com

N° 9

FORUM

6/ 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
nameProductcolourWorking volume(well)

Qty. inner /outer650001

U-bottommed.-binding

MICROLON 200

clear

40 µl - 280 µl

10 / 40

650061

U-bottomhigh-binding

MICROLON 600

clear

40 µl - 280 µl

10 / 40

651001

V-bottommed.-binding

MICROLON 200

clear

40 µl - 200 µl

10 / 40651061

V-bottomhigh-binding

MICROLON 600

clear

40 µl - 200 µl

10 / 40655001

F-bottom/ST

med.-binding

MICROLON 200

clear

25 µl - 340 µl

10 / 40655061

F-bottom/ST

high-binding

MICROLON 600

clear

25 µl - 340 µl

10 / 40655080F-bottom / Chimney Well

med.-binding

MICROLON 200

clear

25 µl - 340 µl

10 / 40655081

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

Plate design

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

Well profile

Bindingcharacterisitc

Binding brand
name767070

U-bottommed.-binding

MICROLON 200

clear

strip plate 12x8

50 µl - 280 µl

10 / 100

767071

U-bottomhigh-binding

MICROLON 600

clear

strip plate 12x8

50 µl - 280 µl

10 / 100

762070F-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

762075F-bottommed.-binding

LUMITRAC 200

whitestrip plate 12x8

20 µl - 350 µl

10 / 100

762074

F-bottomhigh-binding

LUMITRAC 600

whitestrip plate 12x8

20 µl - 350 µl

10 / 100

762076

F-bottommed.-binding

FLUOTRAC 200

black

strip plate 12x8

20 µl - 350 µl

10 / 100

762077

F-bottomhigh-binding

FLUOTRAC 600

black

strip plate 12x8

20 µl - 350 µl

10 / 100

756070F-bottommed.-binding

MICROLON 200

clear

strip plate 6x16

20 µl - 350 µl

10 / 100

756071

F-bottomhigh-binding

MICROLON 600

clear

strip plate 6x16

20 µl - 350 µl

10 / 100

754070

U-bottommed.-binding

MICROLON 200

clear

strip plate 6x16

50 µl - 280 µl

10 / 100

754061

U-bottomhigh-binding

MICROLON 600

clear

strip plate 6x16

50 µl - 280 µl

10 / 100

Product colour

www.gbo.com

15 / 16

FORUM

N° 9

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
Binding characterisitc

Binding brand nameColour stripe

Colour coding well rim

Qty. inner / outer

705070med.-binding

MICROLON 200

clear

10 / 100

705071high-binding

MICROLON 600

clear

10 / 100

705063

med.-binding

MICROLON 200

clear

red

10 / 100

705073high-binding

MICROLON 600

clear

red

10 / 100

705074high-binding

MICROLON 600

clear

blue

10 / 100

705065

med.-binding

MICROLON 200

clear

green

10 / 100

705075high-binding

MICROLON 600

clear

green

10 / 100

705066

med.-binding

MICROLON 200

clear

yellow

10 / 100

705076high-binding

MICROLON 600

clear

yellow

10 / 100

F073004 [rev. 10.2022]

Item no.

Devices of Greiner Bio-One are to be used by properly qualified persons only in accordance with the relevant Instructions for Use (IFU), whereapplicable. For more information contact your local Greiner Bio-One sales representative or visit our website (www.gbo.com).
All information is provided without guarantee despite careful processing. Any liability, warranty or guarantee of Greiner Bio-One GmbH is excluded.
All rights, errors and changes are reserved. If not stated otherwise, Greiner Bio-One GmbH has all copyrights and/or other (user-)rights in thisdocuments, in particular to signs such as the mentioned (word-picture-)brands and logos. Any use, duplication or any other use of the rights of Greiner
Bio-One GmbH is expressly prohibited. Media owner: Greiner Bio-One GmbH / Represented by Managing Directors Jakob Breuer and Heinz Schmid.
The company is registered in the Commercial Register at the first instance court in Stuttgart, HRB 224604 / VAT Number: DE812585719.

Greiner Bio-One GmbH Frickenhausen, Germany
PHONE +49 7022 948-0 / FAX +49 7022 948-514 / E-MAIL info@de.gbo.com
Greiner Bio-One is a global player.
Find the contact details of your local partner on our website.