Validation Report/Data Anti-Trastuzumab ELISA

Calculation of Results:

Determine the mean O.D. for each pair of duplicate wells.

Performance Characteristics:

Please note that this validation is performed in our laboratory and will not necessarily be duplicated in your
laboratory. This data has been generated to enable the user to get a preview of the assay and the
characteristics of the kit and is generic in nature. We recommend that the user performs at the minimum; the
spike and recovery assay and the dilutional linearity assay to assure quality results. For a more
comprehensive validation, the user may run the protocols as suggested by us herein below to develop the
parameters for quality control to be used with the kit.

1. Sensitivity:

a) Limit Of Detection: It is defined as the lowest detectable concentration corresponding to a signal of

Mean of „0‟ standard plus 2* SD.
10 replicates of „0‟ standards were evaluated and the LOD was found to be 15.63 ng/ml.

b) Limit of Quantitation: It is defined as the lowest concentration for which Coefficient of Variation is
<20%. The LOQ is found to be <62.5 ng/ml.

2. Precision:

Precision is defined as the percent coefficient of variation (%CV) i.e. standard deviation divided by the
mean and multiplied by 100.
Assay precision was determined by both intra (n=5 assays) and inter assay (n=5 assays) reproducibility
on two pools with low (250 ng/ml) and high (4000ng/ml) concentrations. While actual precision may vary
from laboratory to laboratory and technician to technician, it is recommended that all operators achieve
precision below these design goals before reporting results.

Pool Intra Assay %CV Inter Assay %CV

Low 7.85% 6.82%
High 4.57% 3.68%

3. Recovery by Spiking:

In spike and recovery, a known amount of analyte is added (spiked) into the natural test sample matrix
and its response is measured (recovered) in the assay by comparison to an identical spike in the
standard diluent.
 Mean Standard
Serum Spiking Abs. Deviation SEM %C.V. Observed Expected % Recovery
neat (serum
spiked with
1000ng/mL anti-
trastuzumab) 1.361 0.0184 0.0130 0.96 1005.78 1000 100.578
1:5 dilution 0.172 0.0092 0.0065 3.79 240.56 200 120.28
1:10 dilution 0.112 0.0035 0.0025 2.24 0.005 100 0.005
1:50 dilution 0.131 0.0269 0.0190 14.50 0.004 20 0.02
1:100 dilution 0.113 0.0141 0.0100 8.85 0.068 10 0.68
1:1000 dilution 0.118 0.0035 0.0025 2.13 0.0078 1 0.78

5. Standard Curve Characteristics:

3.000
R² = 0.9887
2.500
Absorbance (450nm)

2.000

1.500

1.000

0.500

0.000
0 250 500 1000 2000 4000
Anti-Trastuzumab Standard Concentration
(ng/ml)

5 replicates (n=5) of standard curve were evaluated and the standard curve characteristics were

Correlation Coefficient Intercept Slope

(r) (A) (B)

-0.997 2.878 -1.840

6. Dilution Linearity: Serial Dilutions were carried out using 4000 ng/ml as topmost Standard, linearity was
observed till 62..5ng/ml.

Mean Standard
Std conc. Abs Deviation SEM %C.V. Observed Expected % Recovery
0 0.117 0.0042 0.0030 2.56 0 0
1:128 dilution 0.147 0.0035 0.0025 1.71 32.45 31.25 103.84
1:64 dilution 0.145 0.0071 0.0050 3.45 64.59 62.5 103.344
1:32 dilution 0.155 0.0141 0.0100 6.45 128.98 125 103.184
1:16 dilution 0.164 0.0014 0.0010 0.61 260.48 250 104.192
1:8 dilution 0.568 0.0028 0.0020 0.35 515.76 500 103.152
1:4 dilution 1.343 0.0035 0.0025 0.19 1009.62 1000 100.962
1:2 dilution 1.873 0.0318 0.0225 1.20 2023.75 2000 101.1875
4000 2.598 0.0148 0.0105 0.40 4025.95 4000 100.64875

7. High Dose Hook Effect:

The high dose hook effect refers to measured levels of antigen displaying a significantly lower
absorbance than the actual level present in a sample. This appears when a simultaneous ELISA assay is
saturated by a very high concentration of sample antigen binding to all available sites on both the solid
phase antibody as well as the detection antibody and thereby preventing the sandwich-formation. The
antigen-saturated detection antibodies in solution will be washed off giving a falsely low signal. A “hook”
is observed in the curve when data is plotted as a signal versus antigen concentration .

A high dose hook is indicated in the plotted curve when the assay is saturated by high antigen concentrations.

Increasing concentrations >125 ng/ml were assayed as unknowns. The hook capacity yielding an
absorbance reading less than the 125 ng/ml standard was ~0.1 mg/ml.

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