SAKSIN LIFESCIENCES PVT LTD

Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

I. PURPOSE:
To perform the method validation of inhouse developed ELISA using commercially available FAB,
Accentrix as the standard.
II. SCOPE:
This procedure applies to the method validation of inhouse developed ELISA using Accentrix as the
standard at R&D of Saksin Lifesciences Pvt Ltd, Module No: 306, TICEL Biopark 2, Taramani,
Chennai-600113.

III. ABBREVIATIONS:
µg -Microgram
µl -Microlitre
BSA -Bovine Serum Albumin
Conc: -Concentration
CV -Coefficient of Variation
ELISA -Enzyme Linked Immuno Sorbent Assay
FAB -Antibody fragment
h -Hours
H2SO4 -Sulphuric Acid
HQC -High Quality Control
HRP -Horse Radish Peroxidase
hVEGF -Human Vascular Endothelial Growth Factor
LLOQ -Lower Limit of Quantification
LQC -Lower Quality Control
ml -Millilitre
MQC -Middle Quality Control
ng -Nanogram
nm -Nanometre
OD -Optical Density
PBS(T) -Phosphate Buffered Saline (with Tween 20)
QA -Quality Assurance
QC -Quality Control
RT -Room Temperature
SD --Standard Deviation
Std -Standard
T20 -Tween 20
TMB -3,3',5,5'-Tetramethylbenzidine
ULOQ -Upper Limit of Quality Control
US FDA United States Food and Drug Administration

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

IV. PRECAUTIONS
In-house quality procedures were followed while carrying out the validation
V. HEALTH AND SAFETY INSTRUCTIONS
All safety procedures were followed when handling reagents and samples.

VI. RESPONSIBILITY:
1. Research executive: The Research executive has written the protocol, performed the experiments and
documented the report
2. Head of the Department: The HOD reviewed the protocol, monitored the experiments, reviewed the
results and the document.
3. Quality Control Executive: The QC executive reviewed the protocol and the validation document.
4. R&D Head: The R&D Head authorised the report.

VII. REFERENCES
Bioanalytical Method Validation: Guidelines for Industry- US FDA
VIII. CRITICAL STEPS IN VARIATION
Care was taken to avoid/ minimize manual errors with respect to pipetting and variability while
reading absorbance with respect to the standard dilutions.

IX. CHEMICALS, REAGENTS AND CONSUMABLES

PBS
BSA
TMB
H2SO4
T20
Accentrix
VEGF121/ 165
Goat Anti-Human Kappa HRP
Blocking Buffer-1% BSA in PBS (prepared as per SOP: SPL-BIO-035)
PBST (prepared as per SOP: SPL-BIO-035)
Dilution Buffer (0.2 % BSA in PBST): prepared as per SOP: SPL-BIO-035
Stop Buffer (2.72% H2SO4) prepared as per SOP: SPL-BIO-035

X. PROCEDURE
The procedure for the performance of ELISA was followed as per SOP: SPL-BIO-035. Briefly,
a. The Nunc 96 well plate was coated with 100 µl of hVEGF at final conc. of 50 ng/well and incubated
for 2 h at RT with mild shaking.

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

b. Excess hVEGF from the coated plates was removed and 200 µl of blocking buffer was added and
incubated for 1 h at RT with mild shaking.
d. The contents were removed, and the plate was damped dry on filter paper.
e. Then the plate was washed 3 times with 200 µl wash buffer /well.
f. Accentrix was prepared at a concentration of 100 ng/ml in Dilution Buffer from the stock of 10 µg/
ml. Subsequently, serial dilutions were made from the 100 ng/ml stock concentration as follows:
3.25, 1.56, 0.78, 0.39, 0.19 and 0.097 ng/ ml.
g. Also, the QC samples were diluted appropriately to attain the desired concentrations.
h. 100 µl of Accentrix standards, QC samples and the blank (dilution buffer) were added to the
respective wells in duplicates and incubated for 1 h at RT with mild shaking.
i. The plate was damped 3 times with 200 µl wash buffer/well
j. Subsequently, 100 µl anti-kappa light chain HRP (1:5000 in Dilution Buffer) was added into the
well and incubated for 1 h at RT.
k. The plate was again damped and washed 4 times with 200 µl wash buffer/well and 2 times with
200 µl PBS/well and damped dry on filter towel.
m. 100 µl TMB substrate was then added to each well and then incubated for 5 min in dark conditions
by covering the plate with an aluminium foil.
n. To stop the reaction, 100 µl 1N H2SO4 was added to each well.
o. The absorbance in each well was read at 450 nm using a Multimode Plate Reader (refer SOP:
SPL-EQP-022).
p. The standard curve was plotted using absorbance data from Accentrix standards against its
concentration.
q. The concentration of the samples was calculated from the equation derived from the standard
graph using Non-Linear regression (Four parameter equation). For routine analysis linear range of
the curve can be used for analysis. The value was multiplied with the dilution factor to obtain the
final concentration.

XI. CONSIDERATIONS FOR VALIDATION

The following parameters were considered for the method validation of the assay.
1. Calibration curve
2. Sensitivity
3. Accuracy
4. Precision

XII. RESULTS

1. Calibration Curve:
Six calibration standards ranging from 0.09765 ng/ml to 3.1248 ng/ml and 5 QC standards (LLOQ,
LQC, MQC & HQC, ULOQ) were used for establishing the calibration curve in 6 independent assay

3|Page
 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

runs in duplicates. The LLOQ and ULOQ points were taken from the calibration curve corresponding
to the lowest and the highest values, respectively. The LQC point was chosen approximately 3 times
the value of LLOQ. MQC value corresponded to the mid value in the calibration curve. Similarly, the
HQC value corresponded to a point lower than the ULOQ. The standard & QC points were made
from different working stocks of same master stock. Analyte concentration versus assay response
relationship was fitted with a 4-PLmodel (Gen5 software) and the back-calculated concentrations
were derived from the corresponding equation.
Acceptance criteria:
i. At least 75% of the non- zero standards should have accuracy and precision of back calculated
conc ≤ 20 % except at LLOQ where it should be ≤ 25%.
ii. Total error (sum of accuracy & precision) should not exceed 30% except at LLOQ.
iii. Chosen QC samples LLOQ, LOC, MQC, HQC, and ULOQ should be validated.
iv. Coefficient of determination (R2) should be ≥ 0.98

Results:

a. Batch 1: The calibration data and the graph are shown in Table 1 and Figure 1, respectively

Table 1: Calibration data for Batch 1

Mean
Conc Mean Conc1 Conc2 %CV % error Total Pass/
STD OD1 OD2 Conc SD
ng/ml OD (ng/ml) (ng/ml) (Precision) (Accuracy) Error Fail
(ng/ml)
Std1 0.09765 0.034 0.027 0.031 0.117 0.100 0.109 0.012 11.079 11.111 22.190 Pass
Std2 0.1953 0.064 0.060 0.062 0.195 0.185 0.190 0.007 3.722 2.714 6.435 Pass
Std3 0.3906 0.127 0.115 0.121 0.369 0.336 0.353 0.023 6.620 9.754 16.374 Pass
Std4 0.7812 0.242 0.223 0.233 0.722 0.660 0.691 0.044 6.345 11.546 17.891 Pass
Std5 1.5624 0.529 0.452 0.491 1.729 1.446 1.588 0.200 12.605 1.607 14.212 Pass
Std6 3.1248 0.950 0.878 0.914 3.444 3.134 3.289 0.219 6.665 5.255 11.919 Pass
LLOQ 0.097 0.038 0.032 0.035 0.128 0.113 0.121 0.011 8.802 24.227 33.029 Fail
LQC 0.3 0.087 0.093 0.090 0.255 0.272 0.264 0.012 4.562 12.167 16.729 Pass
MQC 0.9 0.257 0.280 0.269 0.771 0.847 0.809 0.054 6.643 10.111 16.754 Pass
HQC 2.4 0.675 0.705 0.690 2.296 2.418 2.357 0.086 3.660 1.792 5.452 Pass
ULOQ 3.125 0.835 0.875 0.855 2.951 3.120 3.036 0.120 3.937 2.864 6.801 Pass

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

Fig 1: Calibration curve for Batch 1

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= -0.00169, B= -0.977, C= 32.5, D= 8.73 and R2= 1

More than 75% of the standards satisfied the criteria that the back calculated concentrations had values
within 20% of the nominal concentrations. The LLOQ did not satisfy the % error for the accuracy which
exceeded 25% and the total error exceeded the limit of 30%. The rest of the QC points satisfied the
acceptance criteria. Taken together the calibration curve for batch 1 is validated.

a. Batch 2: The calibration data and the graph are shown in Table 2 and Figure 2.

Table 2: Calibration data for Batch 2

Conc1 Conc2 Mean
Conc Mean % CV % error Total Pass/
STD OD1 OD2 (ng/ (ng/ Conc SD
ng/ ml OD (Precision) (Accuracy) Error Fail
ml) ml) (ng/ ml)
Std1 0.09765 0.051 0.051 0.051 0.112 0.113 0.113 0.001 0.629 15.207 15.836 pass
Std2 0.1953 0.107 0.081 0.094 0.206 0.162 0.184 0.031 16.909 5.786 22.695 pass
Std3 0.3906 0.210 0.161 0.186 0.392 0.300 0.346 0.065 18.802 11.418 30.220 pass
Std4 0.7812 0.410 0.368 0.389 0.784 0.697 0.741 0.062 8.308 5.210 13.518 pass
Std5 1.5624 0.880 0.718 0.799 1.856 1.463 1.660 0.278 16.746 6.215 22.960 pass
Std6 3.1248 1.387 1.293 1.340 3.247 2.967 3.107 0.198 6.372 0.570 6.942 pass
LLOQ 0.097 0.038 0.041 0.040 0.091 0.097 0.094 0.004 4.513 3.093 7.606 Pass
LQC 0.3 0.143 0.161 0.152 0.269 0.300 0.285 0.022 7.705 5.167 12.872 Pass
MQC 0.9 0.351 0.374 0.363 0.664 0.710 0.687 0.033 4.735 23.667 28.401 Fail
HQC 2.4 0.914 0.987 0.951 1.941 2.129 2.035 0.133 6.532 15.208 21.741 Pass
ULOQ 3.125 1.275 1.215 1.245 2.917 2.745 2.831 0.122 4.296 9.408 13.704 Pass

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

Fig 2: Calibration curve for Batch 2

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= -0.0179, B= 0.934, C= 17.5, D= 8.16 and R2 = 1

All the six standards in the calibration curve for batch 2 satisfied the acceptability criteria. However,
among the 5 QC points used, the MQC did not satisfy the criteria for % error in accuracy wherein the %
error in accuracy was greater than 20. Rest of the QC standards satisfied the acceptability criteria and
thus the calibration curve for Batch 2 stands validated.

b. Batch 3: The calibration data and the graph are shown in Table 3 and Figure 3.

Table 3: Calibration data for Batch 3

Conc1 Conc2 Mean

Conc Mean % CV % error Total Pass/
STD OD1 OD2 (ng/ (ng/ Conc SD
ng/ ml OD (Precision) (Accuracy) Error Fail
ml) ml) (ng/ ml)
Std1 0.09765 0.033 0.031 0.032 0.114 0.109 0.112 0.004 3.171 14.183 17.354 pass
Std2 0.1953 0.068 0.065 0.067 0.206 0.199 0.203 0.005 2.444 3.687 6.131 pass
Std3 0.3906 0.135 0.162 0.149 0.368 0.430 0.399 0.044 10.988 2.151 13.138 pass
Std4 0.7812 0.338 0.289 0.314 0.824 0.715 0.770 0.077 10.016 1.498 11.514 pass
Std5 1.5624 0.629 0.644 0.637 1.466 1.499 1.483 0.023 1.574 5.114 6.688 pass
Std6 3.1248 1.356 1.403 1.380 3.191 3.314 3.253 0.087 2.674 4.087 6.761 pass
LLOQ 0.097 0.025 0.030 0.028 0.092 0.105 0.099 0.009 9.332 1.546 10.879 Pass
LQC 0.3 0.093 0.100 0.097 0.267 0.285 0.276 0.013 4.612 8.000 12.612 Pass
MQC 0.9 0.275 0.300 0.288 0.686 0.740 0.713 0.038 5.355 20.778 26.133 Pass

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

HQC 2.4 0.866 0.824 0.845 2.000 1.903 1.952 0.069 3.515 18.688 22.202 Pass
ULOQ 3.125 1.094 1.199 1.147 2.539 2.793 2.666 0.180 6.737 14.688 21.425 Pass

Fig 3: Calibration curve for Batch 3

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= -0.00492, B= 1.17, C= 9.64, D= 6.28 and R2 = 1

All the standards and the QC points have satisfied the acceptability criteria and thus the calibration
curve for batch 3 stands validated.

c. Batch 4: The calibration data and the graph are shown in Table 4 and Figure 4

Table 4: Calibration data for Batch 4

Mean
Conc Mean Conc1 Conc2 % CV % error Total Pass/
STD OD1 OD2 Conc SD
ng/ ml OD (ng/ ml) (ng/ ml) (Precision) (Accuracy) Error Fail
(ng/ ml)
Std1 0.09765 0.032 0.026 0.029 0.136 0.115 0.126 0.015 11.832 28.520 40.352 fail
Std2 0.1953 0.053 0.048 0.051 0.203 0.187 0.195 0.011 5.802 0.154 5.956 pass
Std3 0.3906 0.108 0.110 0.109 0.377 0.384 0.381 0.005 1.301 2.586 3.887 pass
Std4 0.7812 0.226 0.200 0.213 0.734 0.655 0.695 0.056 8.043 11.098 19.142 pass
Std5 1.5624 0.462 0.506 0.484 1.451 1.584 1.518 0.094 6.197 2.874 9.071 pass
Std6 3.1248 0.998 1.061 1.030 3.196 3.418 3.307 0.157 4.747 5.831 10.578 pass
LLOQ 0.097 0.031 0.032 0.032 0.131 0.134 0.133 0.002 1.601 36.598 38.199 Fail
LQC 0.3 0.096 0.097 0.097 0.341 0.341 0.341 0.000 0.000 13.667 13.667 pass
MQC 0.9 0.268 0.264 0.266 0.861 0.849 0.855 0.008 0.992 5.000 5.992 pass

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

HQC 2.4 0.715 0.706 0.711 2.245 2.218 2.232 0.019 0.856 7.021 7.876 pass
ULOQ 3.125 0.826 0.886 0.856 2.610 2.811 2.711 0.142 5.244 13.264 18.508 pass

Fig 4: Calibration curve for Batch 4

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= -0.013, B= 1.08, C= 14.5, D= 6.17 and R2 = 0.999

More than 75% of the standards satisfied the criteria that the back calculated concentrations had values
within 20% of the nominal concentrations. The LLOQ did not satisfy the % error for the accuracy which
exceeded 25% and the total error exceeded the limit of 30%. The rest of the QC points satisfied the
acceptance criteria. Taken together the calibration curve for batch 4 stands validated.

d. Batch 5: The calibration data and the graph are shown in Table 5 and Figure 5

Table 5: Calibration data for Batch 5

Conc1 Conc2 Mean

Conc Mean % CV % error Total Pass/
STD OD1 OD2 (ng/ (ng/ Conc SD
ng/ ml OD (Precision) (Accuracy) Error Fail
ml) ml) (ng/ ml)
Std1 0.09765 0.028 0.031 0.030 0.087 0.099 0.093 0.008 9.124 4.762 13.886 pass
Std2 0.1953 0.050 0.060 0.055 0.173 0.212 0.193 0.028 14.326 1.434 15.759 pass
Std3 0.3906 0.117 0.129 0.123 0.405 0.445 0.425 0.028 6.655 8.807 15.462 pass
Std4 0.7812 0.240 0.265 0.253 0.786 0.861 0.824 0.053 6.440 5.415 11.855 pass
Std5 1.5624 0.456 0.463 0.460 1.420 1.438 1.429 0.013 0.891 8.538 9.429 pass
Std6 3.1248 1.102 1.007 1.055 3.414 3.097 3.256 0.224 6.885 4.183 11.068 pass
LLOQ 0.097 0.029 0.027 0.028 0.090 0.080 0.085 0.007 8.319 12.371 20.690 Pass

8|Page
 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

LQC 0.3 0.081 0.091 0.086 0.287 0.321 0.304 0.024 7.908 1.333 9.242 Pass
MQC 0.9 0.272 0.280 0.276 0.881 0.906 0.894 0.018 1.978 0.722 2.701 Pass
HQC 2.4 0.833 0.827 0.830 2.543 2.526 2.535 0.012 0.474 5.604 6.078 Pass
ULOQ 3.125 0.922 0.992 0.957 2.822 3.048 2.935 0.160 5.445 6.080 11.525 Pass

Fig 5: Calibration curve for Batch 5

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= 0.0413, B= 1.22, C= 8.8, D= 4.57 and R2 = 0.999

All the standards and the QC points have satisfied the acceptability criteria and thus the calibration
curve for batch 5 stands validated.

e. Batch 6: The calibration data and the graph are shown in Table 6 and Figure 6

Table 6: Calibration data for Batch 6

Conc1 Conc2 Mean
Conc Mean % CV % error Total Pass/
STD OD1 OD2 (ng/ (ng/ Conc SD
ng/ ml OD (Precision) (Accuracy) Error Fail
ml) ml) (ng/ ml)
Std1 0.09765 0.030 0.029 0.030 0.109 0.107 0.108 0.001 1.309 10.599 11.909 pass
Std2 0.1953 0.069 0.063 0.066 0.211 0.198 0.205 0.009 4.495 4.711 9.206 pass
Std3 0.3906 0.128 0.111 0.120 0.373 0.328 0.351 0.032 9.078 10.266 19.345 pass
Std4 0.7812 0.297 0.250 0.274 0.855 0.716 0.786 0.098 12.513 0.550 13.063 pass
Std5 1.5624 0.528 0.472 0.500 1.560 1.385 1.473 0.124 8.404 5.754 14.158 pass
Std6 3.1248 1.035 1.033 1.034 3.302 3.296 3.299 0.004 0.129 5.575 5.703 pass
LLOQ 0.097 0.036 0.028 0.032 0.124 0.106 0.115 0.013 11.068 18.557 29.624 pass

9|Page
 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

LQC 0.3 0.090 0.079 0.085 0.268 0.240 0.254 0.020 7.795 15.333 23.128 pass
MQC 0.9 0.251 0.256 0.254 0.722 0.734 0.728 0.008 1.166 19.111 20.277 pass
HQC 2.4 0.788 0.623 0.706 2.420 1.865 2.143 0.392 18.317 10.729 29.046 pass
ULOQ 3.125 0.822 0.830 0.826 2.535 2.565 2.550 0.021 0.832 18.400 19.232 pass

Fig 6: Calibration curve for Batch 6

Note: Y = (A-D)/(1+(X/C) ^B) + D, where A= -0.0134, B= 0.975, C= 22.9, D= 7.97 and R2 = 0.999

All the standards and the QC points have satisfied the acceptability criteria and thus the calibration
curve for batch 6 stands validated.
Assay run summary:
The summary of the six independent assay runs is shown in the table 7 below:

Table 7: Summary of the six assays performed

Parameter Batch1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6
No: of calibration standards used 6 6 6 6 6 6
No: of calibration standards passed 6 6 6 5 6 6
(%) (100%) (100%) (100%) (80%) (100%) (100%)
No: of QC standards used 5 5 5 5 5 5
No:of QC standards passed 4 4 5 4 5 5
(%) (80%) (80%) (100%) (80%) (100%) (100%)

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

All the mentioned results highlight that the assay is calibrated for the range of 0.097 ng/ ml to 3.1248
ng/ ml of Accentrix concentration. The LLOQ and the ULOQ for the assay are declared to be 0.097 ng/
ml and 3.1248 ng/ ml respectively.
2. Sensitivity of the Assay:
The sensitivity of the assay was determined by spiking the matrix with low dilutions of the analyte and
the deriving the back calculated concentration of the analyte.

Acceptability criteria:
The lowest concentration with accuracy & precision ≤ 25% is taken as the assay sensitivity.

Results: The data for assessing the sensitivity is shown in table 8 below.

Table 8: Assay to determine the sensitivity

Mean
Conc Mean Conc1 Conc2 % CV % error Total Pass/
STD OD1 OD2 Conc SD
ng/ ml OD (ng/ ml) (ng/ ml) Precision) (Accuracy) Error Fail
(ng/ ml)
Std1 0.09765 0.034 0.027 0.031 0.117 0.100 0.109 0.012 11.079 11.111 22.190 pass
Std2 0.1953 0.064 0.060 0.062 0.195 0.185 0.190 0.007 3.722 2.714 6.435 Pass
Std3 0.3906 0.127 0.115 0.121 0.369 0.336 0.353 0.023 6.620 9.754 16.374 Pass
Std4 0.7812 0.242 0.223 0.233 0.722 0.660 0.691 0.044 6.345 11.546 17.891 Pass
Std5 1.5624 0.529 0.452 0.491 1.729 1.446 1.588 0.200 12.605 1.607 14.212 Pass
Std6 3.1248 0.950 0.878 0.914 3.444 3.134 3.289 0.219 6.665 5.255 11.919 Pass
Conc 1 0.048 0.018 0.015 0.017 0.080 0.072 0.076 0.006 7.443 58.333 65.777 fail
Conc 2 0.09765 0.030 0.029 0.030 0.108 0.107 0.108 0.001 0.658 10.087 10.745 Pass
Conc 3 0.1953 0.057 0.051 0.054 0.176 0.161 0.169 0.011 6.295 13.722 20.017 Pass
Conc 4 0.3906 0.111 0.102 0.107 0.323 0.298 0.311 0.018 5.693 20.507 26.200 Pass

The Conc 2 (0.09765 ng.ml) is the lowest concentration that satisfied the acceptance criteria. Thus
0.09765 ng/ ml is regarded to be the sensitivity of the assay. The experiment was repeated to confirm
the sensitivity of the and the data is shown in table 10.

Table 10: Confirmation of sensitivity of the assay

Mean
Conc Mean Conc1 Conc2 % CV % error Total Pass/
STD OD1 OD2 Conc SD
ng/ ml OD (ng/ ml) (ng/ ml) (Precision) (Accuracy) Error Fail
(ng/ ml)
Std1 0.09765 0.051 0.051 0.051 0.112 0.113 0.113 0.001 0.629 15.207 15.836 pass
Std2 0.1953 0.107 0.081 0.094 0.206 0.162 0.184 0.031 16.909 5.786 22.695 pass
Std3 0.3906 0.210 0.161 0.186 0.392 0.300 0.346 0.065 18.802 11.418 30.220 pass

11 | P a g e
 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

Std4 0.7812 0.410 0.368 0.389 0.784 0.697 0.741 0.062 8.308 5.210 13.518 pass
Std5 1.5624 0.880 0.718 0.799 1.856 1.463 1.660 0.278 16.746 6.215 22.960 pass
std6 3.1248 1.387 1.293 1.340 3.247 2.967 3.107 0.198 6.372 0.570 6.942 pass
Conc 1 0.048 0.022 0.017 0.0195 0.067 0.059 0.063 0.01 8.98 31.25 40.23 Fail
Conc 2 0.09765 0.036 0.036 0.036 0.088 0.089 0.0885 0.00 0.80 9.37 10.17 Pass
Conc 3 0.1953 0.077 0.083 0.08 0.155 0.166 0.1605 0.01 4.85 17.82 22.66 Pass
Conc 4 0.3906 0.187 0.165 0.176 0.348 0.308 0.328 0.03 8.62 16.03 24.65 Pass

The Conc 2 (0.09765 ng/ ml) is confirmed to be the lowest concentration that satisfied the acceptance
criteria. Thus 0.09765 ng/ ml is confirmed to be the sensitivity of the assay.

3. Accuracy and precision of the assay

Accuracy of the assay is calculated in terms of % error in accuracy; which is calculated for each back
calculated concentration of the QC samples of the six independent assays performed.
Calculation of the % error in accuracy is done as follows,
% error in Accuracy= Absolute value of [(calculated conc: - nominal conc:)/ nominal conc:] *100
Intra assay and inter assay is calculated in terms of %CV between the observations of the QC samples
within the duplicates in the assay and between the QC samples across the six independent assays.
Acceptance criteria:
For accuracy, the mean value of the back calculated conc for each QC sample should be ≤ 20% of
nominal conc: except at LLOQ where it should be ≤ 25% for at least 2/3rd samples.
For precision, the % CV should be ≤ 20% for all the QC samples within the assay and between the
assays except at LLOQ which should fall within ≤ 25%.
Results:
The % error in accuracy and the %CV for the six independent runs are shown in the Table 11

Table 11: Determination of Accuracy and Precision for the assay

QC SAMPLE LLOQ LQC MQC HQC ULOQ
Nominal Conc (ng/ ml) 0.097 0.3 0.9 2.4 3.125
Batch 1
OD1 0.038 0.087 0.257 0.675 0.84
OD2 0.032 0.093 0.28 0.705 0.88
Observed Conc 1 (ng/ ml) 0.128 0.255 0.771 2.296 2.95
Observed Conc 2 (ng/ ml) 0.113 0.272 0.847 2.418 3.12

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
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METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

Observed Mean Conc (ng/ ml) 0.12 0.26 0.81 2.36 3.04
SD 0.01 0.01 0.05 0.09 0.12
%CV 8.80 4.56 6.64 3.66 3.94
%error in Accuracy 24.23 12.17 10.11 1.79 2.86
Batch 2
OD1 0.038 0.143 0.351 0.914 1.275
OD2 0.041 0.161 0.374 0.987 1.215
Observed Conc 1 (ng/ml) 0.091 0.269 0.664 1.941 2.917
Observed Conc 2 (ng/ ml) 0.097 0.3 0.71 2.129 2.745
Observed Mean Conc (ng/ ml) 0.094 0.2845 0.687 2.035 2.831
SD 0.004 0.022 0.033 0.133 0.122
%CV 4.513 7.705 4.735 6.532 4.296
%error in Accuracy 3.093 5.167 23.667 15.208 9.408
Batch 3
OD1 0.025 0.093 0.275 0.866 1.094
OD2 0.03 0.1 0.3 0.824 1.199
Observed Conc 1 (ng/ ml) 0.092 0.267 0.686 2 2.539
Observed Conc 2 (ng/ ml) 0.105 0.285 0.74 1.903 2.793
Observed Mean Conc (ng/ ml) 0.0985 0.276 0.713 1.9515 2.666
SD 0.01 0.01 0.04 0.07 0.18
%CV 9.33 4.61 5.36 3.51 6.74
%error in Accuracy 1.55 8.00 20.78 18.69 14.69
Batch 4
OD1 0.031 0.096 0.268 0.715 0.826
OD2 0.032 0.097 0.264 0.706 0.886
Observed Conc 1 (ng/ ml) 0.131 0.341 0.861 2.245 2.61
Observed Conc 2 (ng/ ml) 0.134 0.341 0.849 2.218 2.811
Observed Mean Conc (ng/ ml) 0.1325 0.341 0.855 2.2315 2.7105
SD 0.00 0.00 0.01 0.02 0.14
%CV 1.60 0.00 0.99 0.86 5.24
%error in Accuracy 36.60 13.67 5.00 7.02 13.26
Batch 5
OD1 0.029 0.081 0.272 0.833 0.922
OD2 0.027 0.091 0.28 0.827 0.992
Observed Conc 1 (ng/ ml) 0.09 0.287 0.881 2.543 2.822
Observed Conc 2 (ng/ ml) 0.08 0.321 0.906 2.526 3.048
Observed Mean Conc (ng/ ml) 0.085 0.304 0.8935 2.5345 2.935

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

SD 0.01 0.02 0.02 0.01 0.16

%CV 8.32 7.91 1.98 0.47 5.44
%error in Accuracy 12.37 1.33 0.72 5.60 6.08
Batch 6
OD1 0.036 0.09 0.251 0.788 0.822
OD2 0.028 0.079 0.256 0.623 0.83
Observed Conc 1 (ng/ ml) 0.124 0.268 0.722 2.42 2.535
Observed Conc 2 (ng/ ml) 0.106 0.24 0.734 1.865 2.565
Observed Mean Conc (ng/ ml) 0.115 0.254 0.728 2.1425 2.55
SD 0.01 0.02 0.01 0.39 0.02
%CV 11.07 7.79 1.17 18.32 0.83
%error in Accuracy 18.56 15.33 19.11 10.73 18.40

The % CV and % AR satisfied the acceptability criteria for intra assay precision except %AR of MQC in
batch 2 and LLOQ in batch 4.
Inter assay precision is calculated similarly between the assay runs and is tabulated below in Table 12.

Table 12: Inter assay precision

Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Mean SD % CV
Mean LLOQ 0.1205 0.094 0.0985 0.1325 0.085 0.115 0.108 0.018 16.730
Mean LOQ 0.2635 0.2845 0.276 0.341 0.304 0.254 0.287 0.032 10.983
Mean MQC 0.809 0.687 0.713 0.855 0.8935 0.728 0.781 0.084 10.742
Mean HQC 2.357 2.035 1.9515 2.2315 2.5345 2.1425 2.209 0.214 9.697
Mean ULOQ 3.0355 2.831 2.666 2.7105 2.935 2.55 2.788 0.180 6.461

The summary of accuracy and precision is given in table 13

Table 13: Summary for the data for Accuracy and Precision
Parameter LLOQ LOQ MQC HQC ULOQ
Accuracy (Mean %AR) 16.065 9.278 13.231 9.840 10.784
Mean Intra assay precision (%CV) 7.273 5.430 3.478 5.559 4.415
Inter assay precision (% CV) 16.730 10.983 10.742 9.697 6.461

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

Intra- and inter-assay %CV of all 5 QC samples are within 20% and %AR of all 5 QC samples are
within 20%. This result validates the accuracy and precision of the assay.

SUMMARY:
The assay for the determination of quantification of FAB201 was performed with commercially
available Accentrix as the reference standard. The assay was validated using the validation
parameters, viz., calibration curve, sensitivity, accuracy and precision. The range of concentrations of
Accentrix standards for the calibration was established between 0.09765 ng/ ml to 3.125 ng/ ml and
the sensitivity of the assay was found to be 0.09675 ng/ml. The assay was also validated for intra
assay and inter assay precision using 5 QC standards.

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 SAKSIN LIFESCIENCES PVT LTD
Module No. 306, Floor, TICEL Bio-Park-II, No.5, CSIR Road, Taramani, Chennai-600 113
3rd
METHOD VALIDATION OF INHOUSE DEVELOPED ELISA USING ACCENTRIX AS
STANDARD FOR FAB201 QUANTIFICATION
DOCUMENT ID: SPL/REPORT 10

FINAL APPROVAL PAGE

The signature of the officials indicates that the validation study was executed as per the approved
validation protocol and the report truly reflects the raw data obtained in the study.

Executed By Reviewed By
(Research Executive- BIO) (Head of Department-BIO)

(Sign and Date) (Sign and Date)

Reviewed By Approved and Authorized By

(Quality Control Executive) (R&D Head)

(Sign and Date) (Sign and Date)

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