DESC:FIELD OF THE INVENTION
The present invention relates to a method for detecting anti-drug antibodies in a biological
sample, generated against biological therapeutics with minimal interference from a soluble
target.
BACKGROUND OF THE INVENTION
Monoclonal antibodies are an increasingly promising class of biological therapeutics for the
targeted therapy of several diseases including cancer. Administration of therapeutic
monoclonal antibodies can elicit immunological responses in the recipient with possibly
adverse consequences. One such response is that of the generation of anti-drug antibodies
(ADA), which could have neutralizing or non-neutralizing effects on the action of the drug.
Due to the heterogeneity in any biological response as well as the variety in the titre, type and
affinities of ADAs generated, immunogenicity testing for the ADAs needs to be optimized
based on drug and disease-specific factors, among others during development of any drug.
Assays for the detection and quantification of ADA mostly utilize their specific binding
properties to the study drug itself. However, the said detection is complex due to the presence
of interference factors in the samples (with varying modes of binding). Soluble target is an
interference factor, which could result in a false read-out. Interference ultimately results either
in underestimation of the levels of ADA present in the biological sample or detection of false
positives, affecting the performance and sensitivity of the assay. Hence high emphasis is
required on mitigating such interferences while optimizing a given immunogenicity testing
format.
The objective of the invention is to develop a specific immunogenicity assay, with minimal
interferences, for detecting anti-drug antibodies generated against IL-6 receptor anatgonists.
SUMMARY OF THE INVENTION
The present invention discloses an ELISA based method for detecting IL-6 receptor anatgonists
in a serum sample having or with a possibility of having interference due to specific or nonspecific
factors. The method employs use of antibodies or antibody fragments specific to the
said IL-6 receptor antagonist as coating and detection reagents, wherein the methodology in
entirety significantly reduces non-specific interactions and/or the interferences in the assay. In
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addition, the method combines cost-effectiveness, interference mitigation, desirable sensitivity
and target tolerance when compared to existing methods is the art.
Examples are directed towards a biotherapeutic drug, which is an IL-6 receptor anatgonist. The
method possesses acceptable accuracy and precision and qualifies for its robustness and
consistency as per the validation requirements of regulatory agencies.
In addition, by optimizing the dilution factor of the sample, working concentrations of detection
reagent and by use of cost-effective detection modes in an ELISA format, the disclosed method
provides a cost-effective assay methodology that also mitigates interference substantially.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates mean absorbance of positive control sample when compared to samples at
different levels of downstream processing stages
DETAILED DESCRIPTION OF THE INVENTION
Present invention discloses a method for detecting and quantifying anti-drug antibody against
IL-6 receptor antagonist in a blood or serum sample, wherein the sample has or is suspected to
have interference factors, the method comprising the steps of:
a) diluting the sample in a suitable assay diluent to a minimum required dilution
b) immobilizing antibody against the IL-6 receptor antagonist onto a solid support
c) contacting the diluted sample of step a) with the solid support, such that IL-6 receptor
antagonist present in the sample is bound to the immobilized antibody of step b)
d) eluting the bound IL-6 receptor antagonist of step c)
e) immobilizing the eluted IL-6 receptor antagonist of step d) onto a second solid support
f) diluting a labelled antibody against the IL-6 receptor antagonist in a suitable assay
diluent
g) contacting the immobilized IL-6 receptor antagonist of step e) with the diluted antibody
of step f, to form a complex
h) colorimetrically detecting the complex of step g, using standard protocols of enzyme
linked immunosorbent assay such that the signal-to-noise ratio detected is above 3 at
450-630nm
i) interspersing steps a to h with wash cycles;
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wherein, the method reduces interference substantially.
In a further embodiment, the labelled antibody of step e is a horse radish peroxidase labelled
antibody.
In yet another embodiment, the IL-6 receptor antagonist is tocilizumab.
The invention describes a method for detecting anti-drug antibody generated against an IL-6
receptor antagonist in blood samples, wherein the sample dilution factor, assay diluent buffer,
contact antibodies and wash program are selected to reduce non-specific interferences from the
sample, leading to enhanced sensitivity and substantially high target tolerance.
The method uses 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate of horse radish
peroxidase for the detection of the second order complex, thereby detecting the anti-TNF-a
antibody. However, the labeled antibody of step f can be labeled with any commercially
available suitable labelling agent (such as but not limited to ruthenium, iodine etc.,) and shall
be detected using appropriate substrate using the principle of enzyme linked immunosorbent
assay.
Definitions
The term ‘sample’ or ‘biological matrix’ as used herein the invention refers to blood sample or
serum sample isolated from the blood of healthy volunteers or patients who had been
administered with IL-6 receptor antagonist. The said sample has or is suspected to have nonspecific
interferences.
“Anti-drug antibody” refers to an antibody that is raised/formed/produced against the drug in
animals including humans.
“Sensitivity” of the assay is defined as the lowest concentration of standard sample drug
preparation which consistently provides signal in the assay.
“Lower limit of quantification (LLOQ)” is the lowest concentration of analyte that has been
demonstrated to be measurable with acceptable levels of accuracy and precision.
“Negative quality control (NQC)” is an unspiked sample (pooled serum/plasma obtained from
healthy volunteers) in which no anti-IL-6 receptor antibodies have been added.
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“Upper level of quantification (ULOQ)” is the highest concentration of analyte that has been
demonstrated to be measurable with stated levels of accuracy and precision.
Certain specific aspects and embodiments of the invention are more fully described by
reference to the following examples. However, these examples should not be construed as
limiting the scope of the invention in any manner.
EXAMPLES
During development of an ELISA based assay for detecting anti-IL-6 receptor antagonist
present in patient’s sample, various parameters were optimized to achieve acceptable
sensitivity and interference mitigation. One such critical parameter was the optimal dilution of
the test sample such that the signal-to-noise ratio is enhanced substantially. Other parameters
optimized are constitution of blocking buffer, wash program and, the choice of assay diluent
for dilution of sample and detection antibody.
Example 1: Sample Preparation
Serum samples were obtained from volunteers who had been administered with the IL-6
receptor antagonist. Standard samples are human serum samples obtained from healthy
individuals that has been externally spiked with IL-6 receptor antagonist.
Various concentrations of IL-6 receptor antagonist were spiked to prepare the standard sample.
Some of the standard samples were not spiked with IL-6 receptor antagonist so as to maintain
the blank sample. Further, the spiked and unspiked samples were diluted in a suitable assay
diluent buffer.
Example 2: Detection of antibodies against IL-6 receptor antagonist
Individual wells of 96 micro titer plate were coated with recombinant anti-IL-6 receptor
antibody. This is the coating antibody, which is diluted in 1X phosphate buffered saline (pH
7.4) and the plate was sealed and incubated overnight at 2-8 ºC. After incubation, the plate was
washed to remove uncoated antibody. Followed by this, plates were blocked with 5% BSA.
Plates were washed following which, sample, standard sample and blanks were added to each
well and incubated under shaking conditions. Plates were then washed to remove unbound
material. Bound anti-IL6 receptor antagonist antibodies were then eluted and immobilized onto
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a fresh 96-well plates. Subsequently, horse radish peroxidase tagged recombinant anti-IL-6
receptor antibody (diluted in assay diluent buffer) was added to each well and the mixture was
incubated in the dark. Followed by incubation, the plates were washed and bound anti-IL-6
receptor antagonist was detected using tetramethylbenzidine (TMB) substrate. Stop solution
was added to stop the reaction and absorbance was measured at 450 nm using a micro plate
reader. During each wash step, the plate was washed with 0.1 % PBST, five times. ,CLAIMS:We Claim:
1. A method for detecting and quantifying anti-drug antibody against IL-6 receptor antagonist
in a biological matrix sample, wherein the sample has or is suspected to have interference
factors, the method comprising the steps of;
a) diluting the sample in a suitable assay diluent to a minimum required dilution
b) immobilizing an antibody against the IL-6 receptor antagonist onto a solid support
c) contacting the diluted sample of step a) with the solid support, such that the IL-6
receptor antagonist present in the sample is bound to the immobilized antibody of step
b)
d) eluting the bound IL-6 receptor antagonist of step c)
e) immobilizing the eluted IL-6 receptor antagonist of step d) onto a second solid support
f) diluting a labelled antibody against the IL-6 receptor antagonist in a suitable assay
diluent
g) contacting the immobilized IL-6 receptor antagonist of step e) with the diluted labelled
antibody of step f, to form a complex
h) colorimetrically detecting the complex of step g, using standard protocols of enzyme
linked immunosorbent assay such that the signal-to-noise ratio detected is above 3 at
450-630nm
i) interspersing steps a to h with wash cycles;
wherein, the method reduces interference substantially.
2. The method as claimed in claim 1 wherein the labelled antibody is a horse radish
peroxidase labelled antibody.
3. The method as claimed in claim 1 wherein the IL-6 receptor antagonist is tocilizumab.
4. The method as claimed in claims 1-3 wherein the interference factors are non-specific
interference factors.