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4G8
4G8 + AβOligomer
Paul Szabo, Diana M. Mujalli, Matthew L. Rotondi and Norman Relkin
Weill Medical College of Cornell University, New York, NY
Poster No. 3104
We have found natural human antibodies in plasma, CSF and intravenous immunoglobulin
(IVIG) that bind to soluble oligomers of Aβ and other amyloid forming proteins but not to
monomers (1). These antibodies may be part of an innate immune response to toxic
oligomers and could prove relevant to the therapeutic actions of IVIG in Alzheimer’s disease.
To further explore the properties of these antibodies, we have developed sensitive assays for
quantifying anti-Aβ oligomer antibodies in biological fluids. One obstacle to accurately
quantifying anti-oligomer antibodies is the presence of proteins in plasma that interfere with
anti-Aβ antibody measurements by ELISA. In addition, there appears to be a sub-population of
natural antibodies that bind to polystyrene and other surfaces to yield absorbance signals
nearly comparable to anti-Aβ oligomer antibodies. Binding of these antibodies is not blocked
by conventional blocking agents. This background binding complicates standard ELISAs in
which with Aβ oligomers bound to plates are detected by antibodies in plasma or purified
IgG’s. We have explored several approaches to address this issue.
ELISA: ELISAs were performed as previously described for Anti-Aβ monomer (3). Maxisorp
ELISA plates [Nunc, Roskilde, Denmark] were coated with 0.1 ml of 0.1M, pH 9.6 NaHCO3
buffer containing 0.1 mg total Aβ, either monomer or oligomer, for 1 hour at 37o
C. The
plates were washed 3 times with 0.2 ml of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, pH 7.4) containing 0.05% Tween 20 (PBST). The plates were then blocked using
0.2 ml/well of 1% BSA in PBST for 1 hour at 37o
C and then washed 3 times with PBST as
described above. After addition of 0.1 ml of primary antibody or plasma to be tested, the
plates were incubated for 1 hr at 37o
C, washed 3 times with 0.2 ml PBST, and incubated
with 0.1 ml of a 1:10000 dilution of HRP-conjugated goat, affinity-purified anti-human IgG
antibody [Biosource International, Camarillo, CA] in PBST for 30 minutes at 37o
C. After 4
washes with PBST, they were developed by adding 0.1 ml/well TMB [Invitrogen, Carlsbad,
CA] for 15-30 minutes at room temperature. At the first indication that the negative control
wells were changing color, 0.1 ml of 1M HCl was added to each well to terminate the
reaction.
Anti-Aβ oligomer antibody assay: To assay for the presence of anti-Aβ oligomer
antibodies, we mixed antibodies with biotinylated Aβ 12mer in PBST and captured the
resulting antigen/antibody complexes using commercially-available streptavidin plates
[Pierce, Rockford, IL]. Approximately 10µg/ml of biotinylated Aβ 12mer was incubated with
variable amounts of either IVIg, human plasma or IgG, purified from human plasma by
Protein G chromatography. Incubation was for 1hr at RT and the resultant mixture of
antibodies, antigens and antibody/antigen complexes was then incubated on streptavidin-
coated plates for 2 hrs at RT to capture antigen and antibody/antigen complexes. The wells
were then washes 3 times with 0.2 ml of PBST.
Avidity of anti-Aβ oligomer antibodies: Avidity of human serum anti-Aβ antibodies was
measured by determining the molarity of the chaotropic salt ammonium thiocyanate
necessary to dissociate 50% of the human anti-Aβ antibodies bound to Aβ-coated ELISA
plates (3). Briefly, an Aβ oligomer- coated ELISA plate was prepared and incubated with
1:10 dilution of IVIg diluted in PBST and incubated at 37o
C for 1 hour. The wells were
emptied and 0.2ml of 0.1M phosphate buffer (pH 6.8) or phosphate buffer containing 0.5,
1.0, 2.0, 3.0 or 4.0 M ammonium thiocyanate was added to each set of wells; quadruplicate
incubations were done for each concentration. The plates were incubated with for 30
minutes at 25o
C, washed three times with PBST
Titer determinations: For all ELISAs, the OD at 405 nm was measured using a Synergy HT
ELISA reader [Bio-Tek, Winooski, VT]. Anti-Aβ antibody titers were calculated using KC4
Signature software to fit a four parameter sigmoid curve to the ODs obtained from the serial
dilutions of plasma or IVIg. The titer of the anti-Aβ antibody in a sample was taken as the
reciprocal of the dilution at which the OD was equal to one-half the maximum OD.
ResultsObjective
To develop assays that permit the identification and accurate quantitation of anti-Aβ oligomer
antibodies in human plasma, cerebrospinal fluid (CSF) and IVIG.
Result: Purified IgG (IVIG) showed significantly greater
binding than pooled plasma samples, suggesting that
other proteins in plasma interfere with the ELISA.
Purification of IgG eliminated effects of competing
proteins but did not eliminate all binding of human
antibodies to empty ELISA wells. On average, the titers of
IVIg bound to blank wells were about 10% of the titers for
Aβ oligomer plates and about 3-5% of the titer for Aβ
monomer plates.
1. Relkin et al. 2006. Alzheimer’s Dementia 3:S196. 3. Weksler, et al. Exp Gerontol. 2002.37:943-8.
2. Barghorn, et al. J.Neurochem. 2005. 95:834-47. 4. Pullen, et al. J Immunol Methods. 1986. 86:83-7.
WEILL
CORNELL
MEMORY
DISORDERS
PROGRAM
DEPARTMENT OF NEUROLOGY
AND NEUROSCIENCE
WEILL CORNELL MEDICAL
COLLEGE
This work was supported by grants from Baxter and the Appel, Citigroup, Litvin, and Koplow Foundations.
Material support in the form of IVIg was provided by Baxter. The authors acknowledge the advice of Drs
Hans Peter Schwarz, Alfred Weber, David Morgan and Brian O’Nuallain on various aspects of this study.
Human plasma and IVIg bind to the wells of ELISA plates bearing no Aβ peptides
Human antibodies against Aβ oligomers have higher avidity than antibodies to the ELISA plate
Increasing ELISA specificity by addition of a Thiocyanate wash
Primary Antibody
IgG Fab Fab2' Fc
CorrOD450nm
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2.0
2.5
Blank Plate
Aβ42 Oligomer Coated Plate
Aβ42 Monomer Coated Plate
IVIg Treatment
Undepleted Polystyrene Agarose Polystyrene+Agarose
Half-maxTiters
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40
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Aβ Monomer Plate
Blank Plate
[Ammonium Thiocyanate] (M)
0 1 2 3 4
%MaximalBinding
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60
80
100
Blank Plate
Aβ Oligomer Plate
To measure avidity, IVIg was bound to ELISA plates bearing
Aβ42 oligomers or plates bearing no antigen. Quadruplicate
wells were then incubated with increasing concentrations of
the chaotropic salt ammonium thiocyanate and the amount
of bound human antibody determined by standard ELISA
methods. The results are an average of two independent
determinations. Antibodies binding to Aβ oligomers have a
greater avidity for their ligand than those that bind to empty
wells.
Result: The pre-absorption of IVIg by passage over
either polystyrene or agarose columns increased the
relative titers (anti-amyloid : blank) from 15:1 to about
50:1. The drawback to this procedure was a significant
reduction in total signal from anti-Aβ-binding antibodies
in the sample. This makes this approach less desirable
for biological samples that have small volumes, low
titers and contain polyclonal mixtures of antibodies.
.
a.
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b.
Sample Dilution
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c.
Sample Dilution
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A
B
C
D
A with Aβ Oligomer
B with Aβ Oligomer
C with Aβ Oligomer
D with Aβ Oligomer
To test whether the binding of anti-Aβ antibodies with biotinylated Aβ oligomers in solution
and the capture of the resultant immune complexes on streptavidin plates would permit us to
assay anti-Aβ oligomer antibodies, we first tested whether the binding of the monoclonal
antibody 4G8, known to bind to Aβ 12mers, could be detected using this system.
Anti-Aβ oligomer antibody
titers in plasma and purified
IgG at 1 mg/ml from four
individuals were tested using
this method. While plasma
failed to show adequate
separation of signal from noise
(compare panels A and B).
The use of Protein G purified
IgG resulted in approximately
10-fold increase in signal to
noise along with substantially
higher signal per well.
The binding of purified human antibodies to
wells bearing Aβ oligomers (a and c) was
compared to empty wells (b and d). Assays
were performed on plasma (a and b) and
purified IgG from 4 individuals at 1 mg/ml (c
and d). With the exception of individual A,
4M thiocyanate treatment did not alter titers
measured in the plasmas
In contrast, purified IgG samples show both
lower and more consistent binding to empty
wells. Blank plate binding of puriified IgG is
reduced by ~80% by 4M thiocyanate
treatment (d), as was observed with IVIg. In
contrast, the binding to wells bearing Aβ42
oligomers (c) was reduced by approximately
20% by 4M thiocyanate .
Methods
Aβ Oligomer Preparation: To produce Aβ oligomers, 0.3 mg of dried,
HFIP-treated Aβ monomer was dissolved in 0.02 ml dry DMSO with
sonication, 0.2 ml of 1 x PBS and 0.02ml of 2% SDS were added and the
mixture incubated at 37o
C for 6 hours. The mixture was then diluted with
0.6 ml of water and incubated at 37o
C for 18 hours (2). SEC and
SDS:PAGE analysis demonstrated that the bulk of the Aβ in this
preparation was a stable 12mer. Biotin was coupled to the Aβ 12mer
using EZ-Link NHS-PEG12-biotin [Pierce, Rockford, IL] as specified by
the manufacturer; the molar excess of biotinylation reagent to
Aβ oligomers was 20 to 1. After dialysis against PBS, Western blots of
the product were probed with HRP-conjugated streptavidin to confirm
that the Aβ 12-mers were biotinylated [Figure 1].
d.
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A
B
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D
A + 4M Thiocyanate
B + 4M Thiocyanate
C + 4M Thiocyanate
D + 4M Thiocyanate
c.
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a.
Dilution
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b.
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A - Oligomer Plate
B - Oligomer Plate
C - Oligomer Plate
D - Oligomer Plate
A - Blank Plate
B - Blank Plate
C - Blank Plate
D - Blank Plate
1. Measurement of anti-Aβ oligomer titers in human plasma is subject to interference from
plasma proteins as well the binding of other antibodies to polystyrene and other ELISA plate
materials.
2. Separation of IgG using protein G largely eliminates interference from other plasma proteins.
3. Use of a liquid phase assay, in which anti-Aβ oligomer antibodies are incubated with
biotinylated Aβ oligomers in solution followed by capture of immune complexes on streptavidin
plates, reduces artifacts from binding of antibodies to plastic surfaces.
4. Improvements in specificity can be achieved in a standard ELISA format by washing with 4M
thiocyanate to dissociate the lower-affinity antibodies that bind to ELISA wells. This leads to a
10-20% reduction in total signal but a 2-3 fold increase in signal to noise.
Since the antibodies of interest can be identified by their higher binding affinity for oligomers,
we are currently investigating the application of Surface Plasmon Resonance techniques to the
measurement of anti-amyloid oligomer antibodies in biological samples.
A Method for Measuring Anti-Beta Amyloid (Aβ) Antibodies
in Human Plasma and Intravenous Immunoglobulin
Background
Human plasma samples (n=28) were assayed by ELISA with blank plates and plates bearing Aβ
peptide. The resultant absorbance values were compared.
Background binding of antibodies to blank wells is Fab mediated.
To determine whether the binding to blank wells is mediated by the antigen recognition site on Fab
or the through the Fc region, intact IgG, Fab, Fab2’ and Fc from a pool of 5 individuals were used for
ELISAs to blank, Aβ oligomer or Aβ monomer plates. The bound IgG, Fab and Fab2’ fragments
were detected using anti-human k chain secondary
antibody; the Fc, using anti-human IgG antibody. The
results were corrected for differences in the specific
activities of the two secondary antibodies.
Result: The binding to blank plates as well as antigen-
bearing wells was primarily through the Fab antigen
recognition site, suggesting that the observed binding to
blank plates most likely results from a normal IgG-
antigen interaction.
Blank plate binding can be partially eliminated by pre-incubation with polystyrene beads
We tested an approach to reducing the binding of IVIg to blank ELISA wells by passage of IVIg over
columns of polystyrene or agarose to deplete IgG molecules that readily bound to these substrates.
Liquid Phase assay using biotinylated Aβ oligomers and streptavidin capture plates.
Result: 4G8 (serial 3-fold dilutions starting at 1 mg/ml)
alone did not bind to the streptavidin plate but
incubation of 4G8 with Aβ 12mers resulted in the
capture of 4G8/Aβ 12mers immune complexes by the
streptavidin plate. The addition of 10-fold excess
unlabeled Aβ 12mers reduced the signal on
streptavidin plates.
Conclusions
Literature Cited
Using this procedure, as shown on the right, the signal
to noise ratio for the ELISA using purified human IgG
(1mg/ml) is substantially increased (approximately 3
without 4M thiocyanate treatment to about 7- to 10-
following this treatment). Thus, it appears possible to
define a set of conditions that permits the
measurement of anti-Aβ oligomer antibodies in
mixtures of purified human antibodies without the
interference from antibodies that bind to empty ELISA
wells.
0
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Plasma Specimen
Beta Amyloid
Blank Plate