2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 40
Application Note
Development of a Biological Assay to Analyze Neutralizing Antibodies Directed against
Human IFN-β.
Jessica J. Esposito, Sara Crisafulli, Thomas B. Lavoie, Ph.D. and Ronald G. Jubin, Ph.D.
PBL InterferonSource, 131 Ethel Road West, Suite 6, Piscataway, NJ 08854
Introduction antibodies (1:100) and incubated them with varying concentrations of IFN-β.
Interferon beta (IFN-β) is the most commonly prescribed treatment for Multiple Again, PAb 31410-1 displayed most potent activity and low CV rate with MAb
Sclerosis (MS). By an unknown mechanism, this treatment reduces the 21460-1 exhibiting good NAb activity and a low CV rate.
damaging effects to the nervous system in relapsing-remitting and progressive To examine the presence of any off-target effects associated with a NAb
MS and thus slows the progression of disease. Treatment duration is essentially assay, we examined the Abs without IFN-β (Figure 3A) in an antiviral assay
for life since IFN-βis not a cure. (AVA) not displaying a dose-dependent alteration of antiviral activity. To further
As seen with other biotherapeutics, a certain percentage of the patients determine whether the Abs might affect type I IFN signaling, we performed a
develop antibodies toward IFN-β. The two types of antibodies produced are study using IFN-α in place of IFN-β (Figure 3B) with Abs not exhibiting any
defined in functional terms. Binding antibodies (BAbs) are any antibodies that dose-dependent inhibition of IFN-α in place of the IFN-β.
can bind to the IFN-β. Neutralizing antibodies (NAbs) are a subset of BAbs which To establish a rugged assay, it is critical that different operators obtain
have the ability to inhibit the biological activity of the IFN-β. NAbs are of particular similar results. The table below shows the results of one such study. As can be
concern since they may interfere with the therapeutic efficacy of the IFN-β. seen, the results are very comparable.
Interferon was originally identified as an antiviral protein and inhibition of Next, we calculated the neutralizing activity of each Ab to directly compare
viral-mediated lysis is still the standard assay used for IFN. When cells in efficacy (Table 2) with PAb 31410-1 displaying the highest neutralization capacity. In
culture are challenged with a virus they may be subject to death and lysis, addition, inter- and intra-assay precisions were evaluated for each Ab. Again, PAb
otherwise known as the Cytopathic Effect (CPE). By preventing viral killing, the 31410-1 displayed most potent activity and low CV rate with MAb 21460-1 exhibiting
interferon blocks the CPE. The assay we use is based on the inhibition of the good NAb activity and a low CV rate. Inter-operator performance (Table 1) was
CPE. When there are NAbs in a particular sample, the effectiveness of the evaluated with PAb 31410-1, showing little variation in final neutralization titers.
interferon is blocked and CPE is observed.
Conclusion
Results The results reported in this study have shown that a panel of anti-IFN-β
Interferon activity was measured using a cytopathic effect assay (CPE). The antibodies exhibited varying neutralizing capacities. PAb 31410-1 showed the
assay is based on the ability of Encephalomyocarditis Virus to kill the human lung most potent activity; however, PAb activity can shift over time as the immune
carcinoma cell line, A549. Cell density and EMC virus dilution were optimized to response to the antigen changes in the animal. Separately, we identified at
yield a good differential between the cell control (no virus) and the virus control least one MAb, 21460-1, which has potential use in these assays as well. As a
(no IFN). Under these conditions, IFN-β protects the cells in a dose dependent MAb, it targets a single epitope whereas PAbs are a complex mixture.
manner. Shown below are duplicates from the titration of IFN-βin the A549/EMCV Therefore, we cannot be certain of the actual anti-IFN-β antibody
CPE assay. The EC
50
for interferon generally ranges from 0.5 to 2 U/ml. concentration within the total protein levels. Calculation and comparison of
A series of Monoclonal (MAb) and Polyclonal (PAb) antibodies were examined for neutralization titers further demonstrate that PAb 31410-1 has the most
neutralization potential. In one format, antibodies were serially diluted and potent neutralization and MAb 21460-1 displays good NAb activity, both with
screened against a constant concentration of IFN-β at 10 U/ml. Several candidate low CV rates. Comparison of the assay with inter-operator performance
antibodies were identified by this approach. All Abs displayed neutralizing activities confirms a stable NAb assay with little variation in final neutralization titers.
in a dose-dependent manner with the PAb 31410-1 displaying best fit neutralization. The initial studies are promising and warrant further expanded studies with
Clearly, to have utility as a standard, the antibody chosen should work in MS patient serum samples to better determine the assay sensitivity, precision
multiple assay formats. In order to examine this potential, we diluted the and false positive rate.
CPE REPRESENTATION A549/EMCV CPE Assay for IFN-β
Inter-Operator Neutralization
Performance Anti Hu IFN-β Titer (U/ml)
120
Operator 1 (1:10) 2.1 x 10
4
100
(1:100) 2.5 x 10
4
i
o
n
ct
80
Operator 2 (1:10) 2.0 x 10
4
e
60
(1:100) 2.6 x 10
4
P
r
ot
%
40
20
Table 1: Inter-operator Study. To establish a rugged
assay, it is critical that different operators obtain similar
0
-2 -1 012
results. The table above shows the results of one such
study. The results are highly comparable.
[IFN] log (U/ml)
Figure 1A: CPE Representation. Figure 1B: CPE Assay.
PBLInterferonSource Toll Free: 1 877- PBL-8881
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