Methods 40 (2006) 20–29

www.elsevier.com/locate/ymeth

Methods for nano-particle based vaccine formulation

and evaluation of their immunogenicity
Martha Kalkanidis a,b,1, Geoffrey A. Pietersz b,1, Sue D. Xiang a,1, Patricia L. Mottram a,c
,
Blessing Crimeen-Irwin a, Katie Ardipradja a, Magdalena Plebanski a,*
a
Vaccine and Infectious Diseases Laboratory, Burnet Institute at Austin, Studley Road., Heidelberg, Vic. 3084, Australia
b
Bio-Organic and Medicinal Chemistry Laboratory, Burnet Institute at Austin, Studley Road., Heidelberg, Vic. 3084, Australia
c
Helen Macpherson Smith Inflammatory Diseases Laboratory, Burnet Institute at Austin, Studley Road., Heidelberg, Vic. 3084, Australia

Accepted 12 May 2006

Abstract

Nano- and microparticles have long been used for the delivery of drugs and are currently being evaluated as vaccine delivery systems.
Particulates can elicit potent immune responses, either by direct immuno-stimulation of antigen presenting cells (APC) or/and by deliv-
ering antigen to specific cellular compartments and promoting antigen uptake by appropriate stimulatory cell types. Herein, we describe
a detailed method for the preparation of a novel nanoparticle-based antigen delivery system which induces strong cellular and humoral
immune responses in mice and sheep. This simple system is based on the use of 40 nanometer (nm) inert solid carrier beads to which
antigen is covalently coupled before injection. Covalent conjugation of antigen to the nanobeads, assessment of conjugation efficiency,
characterisation and measurement of in vivo immunogenicity by cytokine ELISPOT (to measure antigen-specific T-cell responses) and
ELISA (to measure antibody titers), are described. Emphasis is placed on providing trouble-shooting advice to enable the reproducible
production of soluble nano-size formulations that do not suffer from common problems such as aggregation, as well as understanding the
causes and thus avoiding a range of prevalent technical problems that occur when using immune response detection assays, such as the
cytokine ELISPOT assay and ELISA.
 2006 Elsevier Inc. All rights reserved.

Keywords: Nano; Micro; Beads; Nanometers; Particles; Covalent linkage; Aggregation; ELISPOT; ELISA; Interferon-gamma; IL-4; Antigen presenting
cells; Dendritic cells

1. Introduction immunization. It is therefore imperative to develop novel

During the last two centuries a number of vaccines have
been used for the treatment and control of infectious dis-
eases such as smallpox [1–3], diphtheria and polio [4,5].
However, effective vaccines to eliminate entrenched patho-
gens of human populations such as malaria, measles, acute
respiratory infections and HIV, are still to be developed. It
has become apparent that cancer, as well as neurodegener-
ative and autoimmune diseases such as Alzheimer and dia-
betes, can be prevented or treated by antigen specific
*
Corresponding author. Fax: +61 3 9287 0643.
E-mail address: mplebans@burnet.edu.au (M. Plebanski).
1
These authors contributed equally to this work.
vaccine formulations to promote the right type of protec-
tive immunity against these sophisticated pathogens and
diseases.
Traditional vaccines consist of either whole inactivated
or live attenuated microorganisms. However, these can be
unsafe and can produce some side effects, such as inflam-
mation [6]. On the other hand, vaccines that include
purified or recombinant macromolecules of pathogens,
such as surface proteins or polysaccharides, seem to have
minimal side effects [7]. Particulate delivery systems, such
as liposomes, emulsions, immunostimulatory complexes
(ISCOMs) and virus-like particles, enhance antigen-specific
immune responses [8]. Although a number of these new
adjuvants are currently in clinical trials, at present only

1046-2023/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2006.05.018
 M. Kalkanidis et al. / Methods 40 (2006) 20–29 21

aluminium salts (alum) are used as a vaccine adjuvant in A COOH + CH3CH2N C N CH2CH2CH2NMe 2 1
North America [9]. Additionally, another particulate deliv-
OH
ery system, the microemulsion MF59, was introduced in
N
Europe in combination with an influenza vaccine. Biode- O O

gradable microparticles such as albumin microspheres

O
[10] and particles prepared from poly(lactide co-glycolide) SO3Na 3
C O C N CH2CH2CH2NMe2 2
(PLG) have also been developed, with the latter vaccine
NHCH2CH3
delivery system successfully used to target human growth
hormone [11] as well as carriers for DNA vaccines [12,13].
O
Non-biodegradable nanoparticles, such as latex, poly- O SO3Na O
styrene [14], gold [15] and silica [16], have been examined H2N protein 5 H
C O N 4 C N protein 6
as adjuvant candidates. As an alternative to other virus-like
particles, these particulate systems have advantages in sta- O
bility and controllability of formulation. For example,
polystyrene nanoparticles can be manufactured with a vari- B O O
H H
C N protein N C 1
ety of functional groups (i.e., amino, aldehyde, hydroxyl,
sulfate and carboxyl) on their surface, to allow effective
O
conjugation to a variety of antigens. Polystyrene beads H
H2N protein C N protein 2
are potent antigen carriers for the induction of immunity
in experimental models [17–19]. The antigen is usually Fig. 1. Conjugation of antigen to nanoparticles via active ester. A. EDAC
mixed with the adjuvant to provide a potent immunogen. (1) reacts with the carboxylic acid groups to form an intermediate
In this case, the antigen is only adsorbed onto the surface o-acylisourea (2). This intermediate then reacts with NHS (3) to form an
of the bead. However, when the antigen is covalently cou- acylamino ester (4). The N-hydroxysulfosuccinimide activated bead will
then react with amines (5) (from protein) to form a stable covalently linked
pled to the bead, it induces higher cellular and humoral conjugate (6). B. In the absence of NHS, unwanted crosslinking is feasible
responses than when the antigen is adsorbed onto the bead and can generate side products such as the following: (1) bead–protein–
[18]. These polystyrene nanoparticles can be coupled via bead (1) and (2) protein–protein (2).
their surface carboxyl end-groups to the amino groups of
the antigen. This linking is achieved by chemical processes
as described below. (Fig. 1) for use in immunisation. Simple quality control
Nanobeads in the viral size range, covalently linked to (QC) procedures for the vaccine and subsequent assess-
antigen, elicited a broad immune response, activating anti- ment of immunogenicity are also described. The antigen
gen specific CD4 and CD8 T cells as well as antibodies [18]. used was ovalbumin (OVA). We also describe the problems
Immunization with these particles linked to specific anti- that may arise during the conjugation of the antigen to the
gens, was also protective against tumours [18]. The mecha- polystyrene particles.
nism underlying their potent immunogenicity appears to be
by the preferential localization to dendritic cells (DC) in the 2. Nanoparticle vaccine formulation
draining lymph node. The novel nano-vaccine formulation
of antigen coupled to carboxyl groups on the polystyrene 2.1. Conjugation of OVA to carboxylate microspheres
nanoparticles has been investigated with a range of pep-
tides [19] and proteins [18]. This formulation was protective Carboxylated polystyrene microspheres, ranging in size
against cancer in mice and has now been confirmed as a from 0.04 to 0.05 lm (40–50 nm, supplied as 4% w/v solids),
novel adjuvant in sheep [17]. are pre-activated in 2-N-morpholino-ethanesulfonic acid
Conventional conjugation methods involve the use of a (MES) (50 mM final, pH 6.2), N-hydroxysulfosuccinimide
crosslinker, i.e., carbodiimide, glutaraldehyde, N-hydroxy- (NHS) (50 mM final) and 1-ethyl-3-(3-dimethylaminopro-
succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), imi- pyl)carbodiimide (EDAC) (4 mg/ml final) for 2 h at room
nothiolane, and others [20,21]. Several problems can arise temperature (RT) on a rotating wheel (the pH of the mix-
during conjugate preparation. Usually, these involve having ture should be around 5.5–6.5). For example, to prepare
a mixture of conjugated and unconjugated antigen. Further- 500 ll (final volume) of conjugated beads: 125 ll of beads
more, the most common problem is the formation of large (1% w/v solids final), 50 ll of 500 mM MES, 16.5 ll of
aggregates and different size products in the same mixture. 1.5 M NHS and 20 ll of 100 mg/ml EDAC are mixed with
The antigen–nanoparticle conjugate must be stable and sol- 238.5 ll of sterile water, then rotated for two hours.
uble in aqueous solutions. Moreover, the conjugation prep- OVA (Grade III) is then added to the mixture (for 500 ll
aration should give a high yield of conjugated product and conjugates add 50 ll of 10 mg/ml OVA) to ensure a final
enable scalability. It is therefore difficult to standardize con- concentration of 1 mg/ml. The mixture is then incubated
ditions to obtain a product with consistent characteristics. for a further 1 hour at RT on a rotating wheel. Glycine
In this paper we describe a method for the optimal con- is then added (17.5 ll for a 500 ll conjugation) to a final
jugation of carboxyl polystyrene beads to an antigen concentration of 7 mg/ml (100 mM) for 30 min before
22 M. Kalkanidis et al. / Methods 40 (2006) 20–29

2.3. Specific binding (covalent) versus non-specific binding

OVA loaded onto bead surface

10
(adsorbed)
8
2
area, μg/μm

6 The amount of protein specifically bound to beads and

4 the amount adsorbed to beads in a conjugation mixture
2
0 SDS-PAGE gel and an electric field is applied, the beads
0 1 2 3 4 5
OVA added, mg/ml
Fig. 2. Conjugation of OVA to nanoparticles. The maximum loading
capacity of beads can be achieved by increasing the amount of OVA in the
conjugation mixture. The conjugation mixture consists of OVA, 1%
carboxyl beads (49 nm), 4 mg/ml EDAC, 50 mM NHS and 50 mM MES.
overnight dialysis in cold phosphate buffered saline (PBS)
using 12–14 kDa molecular weight cut-off (MWCO) dialy-
sis tubing. The efficiency of the conjugation can be calculat-
ed by determining the amount of un-conjugated material
using the bicinchoninic acid (BCA) assay following manu-
facturer’s protocol. The degree of specific binding can be
determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS–PAGE). Conjugated beads can be
stored at 4 C for up to 6 months and sonicated for
5 min before use.
For the preparation of conjugates, all reagents should be
made fresh with sterile endotoxin free water, or filter ster-
ilized. Some reagents, such as MES can be made as a stock
solution and stored at 4 C for up to 1 month; the pH
should be checked before use. For dialysis, the appropriate
molecular weight cut-off (MWCO) of the membrane should
be chosen according to the molecular weight of the specific
protein. As a general rule, it is best to choose a MWCO
that is half the molecular weight of the product to be
retained.
The use of NHS during the pre-activation step is useful
because it generates an active ester which can selectively
react with the amino group of OVA, and therefore it pre-
vents unwanted cross-linking (Fig. 1A and B).
The amount of protein (e.g., OVA) that can be loaded
onto the beads has also been investigated. We have found
that 1 mg of OVA is optimal for 1 ml of 1% beads. Howev-
er, the amount of OVA can be increased to saturate the
binding capacity of the beads as shown in Fig. 2.
can be determined using SDS–PAGE. When a sample of
the conjugation mixture is loaded onto the non-reducing
do not migrate onto the gel, protein not covalently coupled
but only adsorbed onto the beads will migrate into the gel.
Thus, if a protein band appears after gel staining, it indi-
cates the non-specific adsorption of the protein antigen to
the beads. Conversely, the absence of a protein band on
the gel reveals that the protein is covalently bound onto
the beads. When the conjugation mixture is prepared in
the presence of EDAC (as described above), there is
100% binding between OVA and the beads since no protein
band is apparent at the expected molecular weight for OVA
(43 kDa) (Fig. 3). On the other hand, by simply mixing
OVA with the beads in the absence of EDAC, causes
non-covalent binding of OVA to the beads, the gel reveals
the presence of the OVA band (Fig. 3).
2.4. Characterisation of nanoparticles by size and charge
In order to determine the final size and charge of the
particles after conjugation, a small aliquot (5 ll) of the con-
jugation mix is diluted in 500 ll of distilled water, and
loaded into a disposable cuvette. The size of the nanopar-
ticles is measured by a dynamic light scattering (DLS) par-
ticle sizer following manufacturer’s instructions. All
procedures are carried out at RT (25 C). In order to
obtain an accurate reading, information such as refractive
43 kDa

2.2. Determination of conjugation efficiency

The conjugation efficiency can be measured by a BCA

assay of the supernatant from the final conjugation mix-
ture. After the conjugation mixture has been dialyzed,
80 ll is collected and spun in the ultracentrifuge at
60,000 rpm for 20 min at 4 C. The supernatant is then col-
lected and analyzed using the BCA assay following the
manufacturer’s instructions for the microplate procedure.
If the beads are greater than 100 nm in diameter, the super-
natant can be obtained by spinning the mixture at
13,000 rpm instead of 60,000 rpm.
EDAC NO EDAC
Fig. 3. Analysis of specific binding of OVA by SDS–PAGE. Conjugation
mixtures with and without EDAC (4 mg/ml) can be analyzed by SDS–
PAGE electrophoresis to determine the amount of non-specifically bound
OVA. The conjugation mixture consists of OVA (1 mg/ml), 1% carboxyl
beads (49 nm), 50 mM NHS and 50 mM MES. Lane 1, conjugation
mixture with EDAC (4 mg/ml)—covalent binding. Lane 2, conjugation
mixture without EDAC—non-specific binding.
 M. Kalkanidis et al. / Methods 40 (2006) 20–29 23

index (RI) of the particle material (RI = 1.59 for polysty-
rene particles), and diluents (distilled water) should be
pre-entered into the program. After the reading is com-
plete, the particle size is calculated by the program and
reported as volume/intensity versus size graphs (Fig. 4A).
The Z-average value reported is the mean diameter of the
particles.
The charge of the particles can also be determined with
the aid of the dynamic light scattering instrument. Five
microlitre of the sonicated conjugation mixture is placed
into a disposable cuvette containing 500 ll distilled water.
The cuvette is then placed into the cell holder in the instru-
ment. When the reading has finished, intensity versus zeta
size graph is obtained. The charge reported is the Z-poten-
tial (mV) value (Fig. 4B).
2.5. Storage and immunization
Sterile antigen–nanoparticle formulations can be stored
at 4 C for up to 6 months. Please note that such formula-
tions can demonstrate various degrees of aggregation dur-
ing storage and aggregate formation also depends on the
pH of the mixture. We have shown that at pH 8.5, the for-
mulation has smaller size aggregates (2 to 3-fold) than at
pH 7.4 (data not shown). We strongly recommend sonicat-
ing the formulation before immunization of mice. Mice are
immunised by injecting 100 ll of above formulation (usual-
ly 1.55 · 1013 particles/mouse) intradermally at the base of
tail. For enhanced responses, mice are usually primed with
one dose (100 ll) and boosted with another (100 ll) dose 2
weeks later.

Fig. 4. Light scattering analysis of conjugates. A representative example of the size (A) and charge (B) distributions of the nanoparticle–OVA formulation
can be determined by light scattering.
24 M. Kalkanidis et al. / Methods 40 (2006) 20–29
3. Assessment of immune response
The induced cellular and humoral responses can be mea-
sured by an enzyme-linked immunospot assay (ELISPOT
assay) and an enzyme-linked immunosorbent assay
(ELISA), respectively
3.1. ELISPOT assay
The ELISPOT is a sensitive immunoplaque assay that
can detect the protein-secreting activity of individual cells
[22]. The cellular responses induced in mice after vaccina-
tion can be measured by the secretion of cytokines such
as interferon gamma (IFNc) and interleukin-4 (IL-4),
which are representative of TH1 and TH2 responses,
respectively. A general protocol for ELISPOT assay is
discussed here, and troubleshooting for the assay is listed
in Table 1. All reagents used in this assay are also listed
in Appendix A.
3.1.1. Preparation of antibody coated membrane plates
Generally, 96-well sterile filtration plates (Millipore-
IPVH-membrane plates for IFN-c and Millipore-HATF-
membrane plates for IL-4) are coated with 100 ll per well
of anti-mouse IFNc monoclonal antibody or anti-mouse
IL-4 monoclonal antibody respectively (MAb at 5 lg/ml
in sterile PBS, pH 7.4). Please note that prior to coating
with antibody, IPVH-membrane plates should be pre-wet-
ted with 50 ll per well of pure methanol for 3–5 min and
then rinsed with 200 ll per well of sterile PBS 5–6 times
before the methanol evaporates (ensure that as much of
the PBS is removed by tapping plates onto a paper towel).
All procedures should be carried out in a sterile environ-
ment( such as a Biological safety cabinet class II or laminar
flow workstation). Plates should then be completely sealed
(in cling wrap or parafilm) to avoid evaporation, and incu-
bated at 4 C overnight (or 37 C for 2 h, or at RT for a
minimum of 4 h). After coating, plates are washed 5 times
with 200 ll per well of PBS containing 0.05% Tween-20,
followed by washing another 5 times in PBS. Following
this, plates are blocked with 200 ll per well of heat-inacti-
vated serum (usually 0.5–2% syngeneic naı̈ve mouse serum,
or 2% fetal calf serum) in PBS for 2 h at 37 C, or overnight
at 4 C (fully sealed to prevent evaporation).
3.1.2. Addition of cells and stimulus
Splenocytes from mice should be freshly isolated and
prepared as a single-cell suspension in complete RPMI-
1640 medium, supplemented with 10% heat inactivated
FCS, 2 mM glutamine, 100 lg/ml streptomycin and 100
units/ml penicillin, under sterile conditions. Red blood cells
should also be lysed since the red blood cells can interfere
with the formation of a cell monolayer in the ELISPOT
plates. Wash plates as described above after coating, then
add 50 ll of cells (1–2 · 107 cells/ml) to each well (final
0.5–1 · 106 cells/well). Various protein or peptide cell stim-
uli, at 50 ll per well, are also added to the cells at predeter-
mined concentrations. In general, whole proteins are used
at a higher concentration than immunodominant epitopes.
Positive and negative controls should also be included and
assays performed in triplicate for each stimulus. Concanav-
alin A (ConA) is used at 1 lg/ml as a positive control to
induce maximal cytokine production. This mitogen should
always be added last to minimize cross-contamination
between wells. Media alone is used as a negative control.
It is important to take into account that the stimuli are
diluted 1:1 with the cells and therefore should be made at
twice their final concentration. Plates are then incubated
in a humidified 37 C incubator with 5% CO2 for 18–24 h
(it usually takes up to 24 h for IL-4 secretion). However,
the length of incubation is dependent on cell types and
the stimulus, as each cytokine will have its own kinetics
of secretion. If an incubation period is too short, insuffi-
cient spot numbers will result, whereas if incubation is
too long, background responses will be increased. During
this incubation period, avoid stacking the plates on top
of each other, as this may result in uneven cell distribution
on the membrane.
3.1.3. Development
After cells are incubated with the stimulus (peptide/
protein) at 37 C, the cells are discarded. From this point
onwards, sterile conditions are no longer necessary. Plates
should be washed 4–5 times in distilled water and allowed
to incubate in the distilled water for 5 min at RT to lyse
any cells adhered to the plate membranes, which may
result in the formation of membrane precipitates during
development. After washing the plates with PBS/Tween-
20 and PBS as described above, 100 ll per well of second-
ary antibodies (anti-mouse IFNc-biotin or anti-mouse
IL-4-biotin respectively) at 1 lg/ml in sterile-filtered
PBS/0.5% FCS are added and plates are incubated for
1–2 h at RT. After washing plates as described above,
add 100 ll per well of streptavidin-alkaline phosphatase
at 1lg/ml (to detect IFN-c secretion), or ExtrAvidintex-
tsuperscript-alkaline phosphatase at 1/3000 dilution (to
detect IL-4 secretion) in sterile-filtered PBS/0.5% FCS.
Incubate plates at RT for another 1–2 h. Prior to colori-
metric development, plates should be washed 4–5 times
in distilled water to remove phosphates, which interfere
with and retard the substrate reaction and color develop-
ment. Cytokine production can then be visualized by
developing the plates using a colorimetric AP kit follow-
ing the manufacturers’ instructions. Developing time is
dependent on the rate of color development, where
10–30 min is usually optimal, but can be developed from
5–60 min. Stop color development by washing extensively
with cold tap water. While washing, remove the plate
underdrain and continue to rinse. Blot plates on clean
cloth or paper towels to remove excess liquid and tap
dry the backs of the wells with an absorbent wipe. This
will ensure that the substrate has been completely
removed from the membrane. Allow plates to dry over-
night in the dark at room temperature. Spot intensity

Table 1
Troubleshooting for ELISPOT
Problem
After substrate development and
rinsing the microplate with water,
the dark-blue background color
of filter membrane makes spots
difficult to see
Too many spots and the density
of the spots make it difficult to
quantify them
All spots are concentrated on one
side of the wells or around the
edge of wells
Too many spots in negative
control wells (such as wells
containing culture media only)
Uneven size of spots in the same
well (big spots and small spots)
The number of spots in the wells
is high but their contrast as well
as intensity of staining is low
M. Kalkanidis et al. / Methods 40 (2006) 20–29
Possible cause
Wet membrane
Too many cells were added to the wells
Stacked the plates unevenly
Improper dispensing cells to wells during assay setup
Or improper dispending antigens to the wells during
assay setup
Contaminated reagents (e.g., endotoxin, LPS etc.)
Ongoing immune response from immunised mice
Cell clumps
Precipitant, particularly when spots
are small and sharp
Underdevelopment
25
Solution
ELISPOT plate reader and its software
cannot analyze the spots accurately until
PVDF filter membranes are completely
dry. Dry the filter membrane (in the wells)
in the dark at RT (60–90 min or
overnight) or 37 C (15–30 min)
Make dilutions of cells (i.e., 1 · 106,
5 · 105, 1 · 105, 5 · 104, 1 · 104 cells per
well) to determine the optimal number of
cells that will result in formation of
distinct spots
Avoid stacking the plates during
incubation with cells
Ensure the cells are evenly dispensed to
wells
Ensure gentle dispending antigens into
wells, to avoid cells dispersal in the
middle of the wells. It is good idea to add
antigen first, and add cells last
Ensure all reagents are filtered before
adding to wells
Do the ELISPOT assay later, i.e. after 14
days or 20 days, which will reduce
background
Ensure the cells are in single suspension
before adding to the wells
Filter all reagents before use
Develop the plates longer before stopping
the color development by washing plates
with water
(continued on next page)
26 M. Kalkanidis et al. / Methods 40 (2006) 20–29

Table 1 (continued)
Problem
Developer reagents are too cold
The number of spots in the test wells is
lower than expected but the positive
control wells turned black-blue
No real spots at all
Membrane fell off from the wells
Possible cause Solution
Bring reagents to RT before adding to the wells
Cell stimulation problem Ensure the stimuli are appropriate for the cytokine release
from the cells
Use a higher concentration of the immunodominant
epitopes than whole proteins
Incubation time after adding cells and For IFN-c production, cells are usually cultured for 8–
antigens are not long enough 12 h; for IL-4 production, cells are usually cultured for 18–
24 h
Too few cells added to the wells Increase the number of cells added per well
Missing steps during assay setups Check whether cells were added
Check whether antigens were added
Check whether secondary antibodies were added. You can
always go back and repeat the steps (after adding cells and
antigens) even after developing
Plates are too old Check the expiry date for the ELISPOT plates

may decrease with exposure to light (therefore, store
plates in the dark to prevent spot color bleaching, espe-
cially for long term storage). Spots can then be counted
using an ELISPOT Reader and analyzed with the associ-
ated software. It is vital that the software parameters
selected to count spots should take into account differenc-
es between ‘‘real’’ spots, with a perimeter containing a
concentration gradient, or artifactual ‘‘precipitates’’,
which have a sharp perimeter and clearly lack a concen-
ing reagents, plates should be properly sealed to prevent
evaporation during the incubation period. The choice of
incubation also depends on the characterization of the
coating antigen, if the coating antigen is temperature sensi-
tive, avoid incubating the plates at RT or 37 C, incubate
the plates at 4 C for a longer period of time instead, such
A 2000 Media
1800 SIINFEKL
SFU/milion cells +/- SEM

tration gradient. Interestingly, the size and intensity of 1600 OVA

the spots can also be qualitative and can be used to dis- 1400 OVA Helper
criminate between cells that secrete different concentra- 1200
tions of protein. ConA data is used to assess the 1000
viability of the cells in the assay. Samples that do not pro- 800
duce cytokines in response to ConA are not viable and 600
are excluded from that data. Fig. 5 shows typical IFNc 400
and IL-4 responses in mice after immunization with nano- 200
particle-OVA formulations. 0
40nm-OVA Naïve

3.2. ELISA B 50

The enzyme-linked immunosorbent assay (ELISA) is a

highly sensitive and quantitative serological assay used to 40 Media
SFU/milion cells +/- SEM

evaluate specific antibody activity of polyclonal sera [23]. OVA

A general protocol for ELISA will be discussed here, and 30 OVA Helper
some troubleshooting for the assay is listed in Table 2.
All reagents used in this assay are also listed in Appendix
20
A. Generally, coat 96-well micro-titre plates with 100 ll
per well of specific capture antigen (eg, OVA or specific
antigen used for immunization) at 5 lg/ml (or optimal con- 10
centration for a specific antigen) in 50 mM sodium bicar-
bonate buffer (pH9.6) by incubating at 37 C for 2 h (or
0
at 4 C overnight). Other coating buffers, such as PBS 40nm-OVA Naïve
(pH 7.4) and 20 mM Tris–HCl (pH 8.5) can also be used.
The choices of coating buffers are dependent on the coating Fig. 5. Analysis of IFN-c and IL-4 responses by ELISPOT assay.
ELISPOT data showing typical antigen specific IFN-c (A) and IL-4 (B)
antigen. It is important to investigate the antigen of interest
responses by CD4 and CD8 T cells in mice (n = 3) immunised with OVA.
at the beginning of assay development. Theoretically, it is Spleen cells were stimulated with media, OVA protein, or peptides
best to use a buffer with a pH 1–2 units higher than the containing the CD8 epitope (SIINFEKL) or CD4 epitope (OVA-helper).
pI of the protein being coated [24]. After adding the coat- Data presented as Spot Forming Units (SFU)/million cells ± SEM.
 M. Kalkanidis et al. / Methods 40 (2006) 20–29 27

Table 2
Troubleshooting for ELISA
Problem Possible cause Solution
Poor precision Incomplete washing and aspiration of wells Ensure that wash apparatus is working correctly
Wells should appear dry after aspiration
Unequal mixing of reagents Ensure adequate mixing
Pipetting error Check tips/channels, recalibrate the pipette if
necessary
Check and proper fitting of the tips
Edge effects Uneven temperatures around work surface Adhere to recommended incubation periods and
temperatures
Avoid incubating plates in areas where environmental
conditions vary
Use plate sealers
Inadequate fixing of plate cover, leading to evaporation Ensure correct use of plate cover
Poor signal across the plate Incorrect wavelengths Check filters/reader
Insufficient development time Increase development time
Coated plates are old Coat new plates
Capture antibody did not bind to the plate Use an ELISA plate (not a tissue culture plate)
Check compatibility of coating buffer
Buffer mismatch Check compatibility of buffer with enzyme
High background Insufficient washing Increase number of washes or add a 30 sec soak step in
between washes, or soak the plates for 5 min after final
wash
No blocker Used a neutral protein blocker
Detergent before block Use detergent only after blocker
Wrong surface Using the appropriate plastic surface according to
manufacture’s instruction
Cross-reaction Check reagents for compatibility
Too much signal Insufficient washing/washing step skipped Check protocol or add a 30 sec soak step in between
washes or soak plates for 5 min after final washing
step before additional reagent added
Substrate Solution mixed too early Substrate Solution should be mixed and used
immediately
Too much of Enzyme conjugated antibodies Check dilution, titrate if necessary
Cross reactivity Check compatibility of reagents, e.g., blocker protein
same animal source as antibody
Buffers contaminated with metals Make fresh buffers
Wells dry out Fill wells with buffer when not in use
No stop solution, or visualization time too long Add stop solution and adjust time
No signal Reagents omitted Check protocol
Reagent deteriorated Check storage conditions, and use fresh reagents
Not enough antibody used Increase concentration
Preservative in buffer Check compatibility of reagents
Capture antibody did not bind to plate Check plates, make sure to use an ELISA plate (not a
tissue culture plate)
No signal when a signal is expected Sample matrix is masking detection Dilute samples at least 1:2 in appropriate diluent, or
preferably, do a series of dilutions to look at recovery
Unexpected low readings in a few wells The piston or tip bearing cone of a pipette is Check whether there is acid in pistons or bearing,
contaminated by acid (due to acid vapour in piston) clean it if necessary
Poor duplicates Insufficient washing If using an automatic plate washer, check that all ports
are clean and free of obstructions; add a 30 s soak step
Use appropriate wash buffer
Uneven plate coating due to procedural error or poor Check coating and blocking volumes, times and
plate quality (can bind unevenly) method of reagent addition
Check plate used
Variable incubation time Work systematically and rhythmically in the same
sequence for all additions
Use a stop solution before reading
Evaporation Use plate sealers
Pipetting error Check tips/channels, calibrate the pipette if necessary
Check and proper fitting of the tips
Wet tip in reagent before dispensing
(continued on next page)
28 M. Kalkanidis et al. / Methods 40 (2006) 20–29

Table 2 (continued)
Problem Possible cause Solution
Poor assay to assay Insufficient washing If using an automatic plate washer, check
reproducibility that all ports are clean and free of obstructions
Variations in incubation temperature Adhere to recommended incubation temperature
Avoid incubating plates in areas where
environmental conditions vary
Variations in protocol Adhere to the same protocol from run to run
Buffers contaminated Make fresh buffers

as overnight. The coated plates should then be washed 5–6 3.5

times with PBS/0.05% Tween-20, and non-specific binding 3
sites blocked by adding 200 ll per well of 2% (w/v) bovine
serum albumin (BSA) and incubated at 37 C for 2 hours 2.5

OD (450nm)
(or 4 C overnight). Wash plates again as described above 2
and thoroughly tap dry to remove excess washing buffer
1.5
that may affect the subsequent serial titration of the test
solutions. 1
To determine the specific antibody activity (or titer) of
0.5
test sera, each serum sample can be serially diluted across
the plate (usually starting at 1/100 dilution in PBS/0.05% 0

50

100

200

400

800

1600

3200

6400

12800

25600

Tween-20, however, it will vary depending on the number
of repeat vaccinations, or if a high sera titre is expected).
The final volume of each diluted serum sample should be
100 ll per well. Positive and negative controls should also
be included. Test samples are usually run in duplicate.
After sampling, plates should be properly sealed to prevent
evaporation and subsequently incubated at 37 C for 2 h
(or at 4 C overnight). Again, the choice of incubation
method will depend on the physiological properties of the
substance to be measured in the serum. In some cases,
enzymatic activities in the serum may effect the levels of
the substance, therefore, incubation at 37 C should be
avoided [25]. Following incubation, wash the plates again
as described above, and add 100 ll per well of the
enzyme-conjugated secondary antibody (total Ig, such as
rabbit anti-mouse IgG+A+M conjugated to horseradish
peroxidase) at 1/2000 dilution (or optimal concentration
as stated by the manufacturer) to each well. Plates are then
incubated at 37 C for a further 2 h (or at 4 C overnight).
Prior to development, wash the plates again as described
above, and add 100 ll per well of 3,3 0 ,5,5 0 -tetramethylbenz-
idine (TMB) developing buffer (or other enzyme/substrate
system as required). To stop TMB hydrolysis, an equiva-
lent volume of 1 M HCl is added to each well. The time
of stopping the developing reaction is dependent on the
color development of background wells and the most opti-
mal contrast between positive test solutions and naı̈ve solu-
tions. It is important to always aim to add TMB or HCl to
plates in the same sequence and in the same time frame to
ensure that all plates are allowed to react for the same
length of time. Absorbance for each plate is read at
450 nm (or other wavelengths required by the enzyme/sub-
strate system) on a microplate reader (see Appendix A for
detail). Fig. 6 shows a typical antigen specific antibody
response in mice after immunization with nanoparticle-
OVA formulations.
51200
Dilution factors
Fig. 6. Analysis of antibody responses by ELISA. ELISA data showing a
typical antigen specific antibody response in mice (n = 3) after immuni-
zation with nanoparticle-OVA formulation, compared with naı̈ve mice
(n = 3). Data is presented as absorbance at 450 nm at various serum
dilutions.
4. Concluding remarks
Numerous studies have shown that microparticle-
based technologies represent promising vaccine candi-
dates. In our study, we have found that nano-particles
in the range of 40–50 nm have the potential to induce
potent cell mediated (CD4 and CD8 T cells) as well as
humoral immune responses. The novel nano-vaccine for-
mulation described in this paper has been used with a
range of antigens associated with cancer in the form of
recombinant proteins [17,18] and peptides [19]. The next
important step in the development of such nanoparticle–
antigen formulation is to evaluate its effectiveness in
human clinical trials.
Appendix A. Reagents, equipment and suppliers list
A.1. Materials and reagents
Carboxylated polystyrene microspheres are obtained
from Polysciences, Warrington, PA, USA.
All ELISPOT plates (96-well filtration plates) are
obtained from Millipore, Billerica, MA, USA.
96-well Nunc-Immuno micro-titre plates (MaxiSorp)
are obtained from Nunc, Roskilde, Denmark.
12–14 kDa membrane cut-off dialysis tubing is obtained
from Viskase, Willowbrook, USA.
 M. Kalkanidis et al. / Methods 40 (2006) 20–29 29

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