Download (ECL) Method for the Quantitation of Calcitonin in Rat Serum

Validation of an Electrochemiluminescence (ECL) method for the
quantitation of calcitonin in rat serum
P. Pitsikas, C. Ronacher, V. Klypa, P. Khatcherian, S. Croteau, M. Djefel, M.-O. Pepin, and S. Cotton
Charles River, Montreal
4
Calcitonin is a hormone that is produced primarily by the human parafollicular cells of the thyroid, and
in many other animals in the ultimopharyngeal body. Calcitonin is involved in the reduction of
blood calcium (Ca2+), counteracting the effects of parathyroid hormone (PTH). Calcitonin can be
used as a marker for Paget’s disease, menopause osteoporosis, obesity, hypercalcaemia, Medullary
thyroid cancer (MTC) and bone metastases. The MSD® (Meso Scale Discovery) platform was used to
develop a more sensitive assay compared to the commercial kits available on the market. Therefore,
the development and validation of an Electrochemiluminescence (ECL) method for the quantitation of
calcitonin is a useful tool to support non-clinical studies where the endogenous level of this biomarker
will aid in study interpretation.
2
MATERIALS AND METHO DS
A study was undertaken to qualify and validate an assay using the electrochemiluminescence MSD®
platform for the quantitation of calcitonin in rat serum. Analytical qualification and validation
parameters, including precision, accuracy, selectivity and parallelism of the method were assessed.
Stability of calcitonin in rat serum was also validated under different conditions such as long term
storage, freeze and thaw cycles as well as storage at ambient room temperature and 4°C.
DISCUSSIO N
Higher levels of serum calcitonin in response to a high-fat diet have been observed and reported in
several published articles. Sera from obese rats were analyzed and detectable endogenous levels
were observed for calcitonin. (Figure 1)
Calcitonin conc. (pg/mL)
3 weeks on high-fat diet
140
3 weeks on regular diet
120
STD 9*
100
80
60
40
20
0
Figure 1 Levels of calcitonin in obese rats
Table 1 Stabilities. To be acceptable, the difference (%) had to be
within ±25% when the global mean concentration of the stability
sample was compared to the global mean concentration of the
control sample for at least 2 out of 3 lots.
Ambient RT
7 hrs 3 min
4°C
26 hrs 52 min
Freeze/thaw cycles
(-20°C and -80°C)
Long term Storage
(-20°C and -80°C)
4
232 days
AC
LLOQ
2604,17
STD 7
1302,08
STD 6
651,04
STD 5
325,52
STD 4
162,76
STD 3
81,38
STD 2
40,69
STD 1 (LLOQ)
25,43
STD 0
0,00
QC1
110
QC2
100
QC3
Precision
AC
23.0
LLOQ
18.0
13.0
8.0
140
QC1
QC2
QC3
ULOQ
tQC
AC
R² = 0.9988
600.0
AC
70
28.0
700.0
ULOQ
80
Selectivity in rat serum spiked with endogenous
calcitonin
Parallelism of calcitonin in rat serum
tQC
90
5208,33
STD 8 (ULOQ)
*Accessory standard
Relative Accuracy
120
%Recovery
5 weeks on high-fat diet
160
CO NCLUSIO NS
The validation data demonstrated that this ECL method is suitable to measure calcitonin in less than
130 µL for duplicate analysis of rat serum from non-clinical studies. In addition, the results show that
the method is sensitive enough to detect even subtle changes in these marker profiles and can be
applied as a biomarker in non-clinical studies. Preliminary data also show that the assay can quantify
calcitonin in mouse and monkey serum.
In order to define the lower and upper limit of quantitation of the assay, precision and accuracy
assessments of QC samples (LLOQ, low, mid, high, ULOQ and trending QC) were conducted. The
lower and upper limit of quantitation (25.43 pg/ml and 2604.17 pg/mL, respectively) met acceptance
criteria, thus showing a wide dynamic range of standard curves. (Table 2 and Figure 2)
%CV
180
5
Precision and Accuracy
130
Concentration
(pg/mL)
The selectivity assessment demonstrated no interference effect of the matrix when rat serum was
spiked with a lot of rat serum having a high endogenous level. (Figure 4)
Short-term stability of calcitonin in rat serum was proven up to 7 hours and 3 minutes at ambient RT
and in a refrigerator set to maintain 4°C for up to 26 hours and 52 minutes. Study samples can be
subjected to at least of 4 freeze-thaw cycles in freezers set to maintain -20°C and -80°C. Also, study
samples are stable for at least 232 days at both temperatures. (Table 1)
Table 2 Range of Curve
STD ID
Selectivity
Stability
140
Endogenous concentration of calcitonin
Parallelism assessments for calcitonin proved successful between undiluted samples and samples
diluted 8-fold, using 3 different lots of rat serum. (Figure 3)
Endogenous level
RE S ULTS
3
Parallelism
The MSD assay validation met acceptance criteria for all tested parameters, including intra/inter assay
precision and accuracy, selectivity and parallelism:
120
500.0
Serum lot 1
Serum lot 2
400.0
Serum lot 3
300.0
R² = 0.9854
100
% Recovery
ABS TRACT
Concentration (pg/mL)
1
80
60
200.0
40
100.0
20
0.0
0
0
0.2
0.4
0.6
0.8
1
1.2
1/Dilution Factor
AC
1
2
3
4
5
6
7
8
9
10
3.0
-2.0
Figure 2 Precision and Accuracy for calcitonin. To be acceptable, the %CV had to be
≤25% and the % Theoretical between 75-125% for at least 67% of the occasions
performed. For the LLOQ and ULOQ, a %CV ≤30% and a % Theoretical between 70-130%
were acceptable.
Figure 3 Parallelism performed with endogenous level of calcitonin. For a
dilution factor to be accepted, the %recovery had to be within 75-125%.
Figure 4 Selectivity performed by spiking rat serum lots with rat serum
with a higher endogenous level for calcitonin. No interference was
observed if at least 80% of spiked rat serum had a %recovery between
75-125%.