Application Note

The New Gold Standard for Gene Therapy
Protein Expression Potency Measurements

Simple Western™: Analytical-Grade Protein Expression Quantification in a
Multi-Attribute Platform Approach
Gene Therapy Potency Measurements Are in
Need of Potent Analytical Methods

Get the Flexibility of Western Blots with theAnalytical-Grade Quantitation of ELISA

A major challenge preventing transformative genetherapies from reaching patients is the lack of fit-forpurpose potency assays, which are some of the mostimportant assays in an analytical package but are often
the most complex and high-risk.1-5 Defined as “thespecific ability or capacity of the product, as indicated byappropriate laboratory tests or by adequately controlled
clinical data obtained through the administration of
the product in the manner intended, to effect a given
result,” potency assays are used for process development
testing, analytical characterization, QC release testing,stability studies, and comparability studies.2 Therefore,
potency assays must be quantitative, dose-responsive,
and well-controlled. Because of their complex mechanismof action, potency assays require a matrix approach ofmultiple complementary assays, incorporating infectivity,transgene mRNA and protein expression, and biologicalactivity.

Simple Western™ is a capillary electrophoresis (CE)
platform that pairs molecular weight (MW) or isoelectric
point separation with a quantitative immunoassay readout and represents the next-generation gold standard forquantitative, reproducible, and highly specific analyticalgrade protein expression potency assays. By pairing
the flexibility and specificity of Western blots with theanalytical-grade quantitation of ELISA, Simple Westernis ideal for making specific measurements in complex
sample types like tissue homogenates and analyzing
intracellular protein expression without matrix effects thatcan confound ELISA read-outs.

ELISA is a workhorse for protein expression potency
analysis,6 but limitations include interference bymatrix effects, challenging assay development, andoverestimation of analytes due to cross-reactivity.
Western blot is still used in clinical trials despite poor
reproducibility, limited quantitation, large sample volume
requirements, and low throughput.7 Flow cytometryis limited by background interference,8 and the lack ofvalidated antibodies can complicate GMP requirements.
Finally, PCR methods are not fully predictive of protein
expression.6
As a result, new fit-for-purpose protein expression
potency assays are needed, ideally in a platform approachthat will enable efficient, simple, and inexpensive
upstream and downstream process development.9

As a platform technology that needs only a single
validated antibody and offers rapid 3-hour runtimes,
Simple Western lowers the custom assay development
hurdles of ELISA and accelerates drug development frompre-clinical phases and scaling to GMP. Plus, Simple
Western is a multi-attribute method used in gene therapy
development, beyond protein expression potency assays,for quantitative CQA monitoring of purity, titer, empty/
full capsid content, and capsid protein ratio.10-15
In this Application Note, we demonstrate a Simple
Western relative potency assay with parallel line analysis(PLA) for AAV-mediated gene delivery in cultured humancells. Specifically, we demonstrate:• Relative protein expression potency between AAV
serotypes with high specificity and <5% CVs• Absolute protein expression measurements in AAVtransduced cells using a recombinant standard curve• Detection of residual intracellular capsid proteins in
AAV transduced cells as an infectivity surrogate

By measuring GFP normalized to total protein directly intransduced cells, we show that AAV2 is significantly morepotent than AAV9 with highly reproducible quantitationthat is 10X more sensitive than flow cytometry, withimproved specificity and less matrix effect than ELISA.16

Materials and Methods
Relative Protein Expression Potency Assay
The materials used for the relative potency assay are listed
in TABLE 1. Serotypes AAV2 and AAV9 were used totransduce the human liver cancer cells (the HepG2 liver
cell line), which has been used for in vivo potency assays
for AAV gene therapy.17 These vectors were engineered
to deliver and express GFP driven by the CMV promoter,an established reporter of AAV transduction in HepG2
cells,17 by random genomic integration with no homology
sequences flanking the CMV-GFP transgene.

AAV Transduction of Human Liver Cells
HepG2 cells were transduced in duplicate with AAV2 or
AAV9 vectors with CMV-GFP genetic payloads in a 7-point
titration of 1 x 106, 2 x 105, 4 x 104, 8 x 103, 1.6 x 103, 3.2 x102, and 6.4 x 101 vector genomes per cell (vg/cell). Empty
AAV2 and AAV9 were included as negative controls. At 48
hours post-transduction, 6 x 106 cells were collected fromeach vial and control treatment and split into 3 Eppendorf
tubes (2 x 106 cells per tube). Cells were spun at 1000 x g
for 2 minutes, the supernatant was removed, and the cells
were washed with 1 mL of PBS. Cells were spun again at
1000 x g for 2 minutes and the supernatant was removed,
and the tubes were placed on dry ice for 10 minutes toflash freeze the cells and stored at -80 °C.

Simple Western Analysis
All samples were prepared as described in the 12-230
kDa Separation Module product insert. The anti-GFP
antibody was diluted to 20 µg/mL in Milk-Free Antibody
Diluent. All samples were analyzed on Simple Western’s

Jess™ instrument with default settings. Relative potency
was determined by three Simple Western runs withindividually prepared samples at a concentration of
0.1 mg/mL. For this study, AAV9 was selected as thereference material and AAV2 as the test material. GFP and
total protein were detected in Probe 1 and Probe 2 of the
RePlex™ assay, respectively.

Parallel Line Analysis (PLA)
Raw data and data normalized to total protein were used
to determine relative potency using the PLA method. See
the Appendix for more information on the PLA method
using GraphPad Prism.

Absolute Protein Expression Measurements
To quantify absolute GFP protein expression levels, a
3X serial dilution series of recombinant GFP (rGFP) was
prepared from 5 ng/mL to 0.06 ng/mL and analyzed
in the same Simple Western run as the AAV2 or AAV9transduced cells. The peak areas resulting from rGFP
analysis were plotted by rGFP concentration, and a log-log
regression with 1/y2 weighting was applied to create astandard calibration curve that was used to quantify
absolute GFP expression in transduced cells. All samples
were analyzed in duplicate.

Detection of Intracellular AAV VP1/VP2/VP3
The materials used for intracellular AAV detection arelisted in TABLE 2. All samples were prepared as describedin the 66-440 kDa Separation Module product insert,
except the sample denaturation was performed at 70 °C for
10 minutes. The anti-VP1/VP2/VP3 antibody was diluted
to 20 µg/mL in Antibody Diluent 2 and the anti-mouseHRP secondary antibody was diluted to 1X in Antibody
Diluent 2. All samples were analyzed in duplicate on
Simple Western’s Jess instrument with default settings,
except the sample load time was adjusted to 6 seconds.

TABLE 1. Materials used for relative potency analysis

TABLE 2. Materials used for VP1/2/3 detection

ItemPart Number

Item

12-230 kDa Separation Module

SM-W004

66-440 kDa Separation Module

SM-W008

EZ Standard Pack 2

PS-ST02EZ-8

Goat-Anti-Mouse-HRP Antibody

040-655

Anti-Goat Detection Module

DM-006

Streptavidin-HRP

Total Protein Detection Module

Vendor

Bio-TechnePart Number

042-414

DM-TP01

Luminol-S

RePlex™ Module

RP-001

Peroxide

Recombinant Jellyfish GFP Protein

NBC1-22949

Antibody Diluent 2

Anti-GFP Antibody

AF4240Anti-AAV VP1/VP2/VP3 Antibody

PROGEN

690058S

N/A

AAV2 vector stock

Virovek

N/A

AAV2/9 with CMV-GFP and empty

Bio-Techne

VendorVirovek043-311043-379042-203

Results of the Simple Western Relative Protein Expression Potency Assay
Finding the Log-Log Linear Range of the Assay

Whole-cell lysates of HepG2 cells transduced with a
5X 7-point serial dilution series from 1 x 106 to 6.4 x 101
AAV2 or AAV9 were analyzed at concentrations of 1, 0.1,
and 0.01 mg/mL. When these samples were analyzed on
Jess using an anti-GFP antibody, the results showed that
AAV2-transduced GFP expression reached saturation
at 1 mg/mL (FIGURE 1, top panel), and the GFP signal
was very low in AAV9-transduced cells at lower dosages(FIGURE 1, middle panel). At a concentration of 0.1 mg/
mL, GFP expression was linear from 6.4 x 101 to 8 x 103 vg/
cell of AAV2 and from 1.6 x 103 to 1 x 106 vg/cell of AAV9(FIGURE 1, bottom panel).
Based on these results, relative potency was determinedat a final whole-cell lysate concentration of 0.1 mg/mL
containing cells transduced with AAV2 or AAV9 with
5-point viral titrations (vg/cell) of 6.4 x 101 to 4 x 104 and1.6 x 103 to 1 x 106 for AAV2 and AAV9, respectively.

Measuring Relative Potency of AAV2 to AAV9Using the sample concentration of 0.1 mg/mL establishedabove, Simple Western analysis of transduced liver cells
showed that the maximum GFP expression for AAV2
occurred with titers >4 x 104 vg/cell, and the maximum
AAV9 GFP expression occurred with titers >1 x 106
vg/cell (FIGURE 2A). These results were consistent
with flow cytometry analysis of cells from the same
sample timepoint, with AAV2 transduction resulting insignificantly higher GFP expression than AAV9 (FIGURE
2B). However, Simple Western was more sensitive thanflow cytometry because GFP was detectable in cells
transduced with AAV9 titers as low as 1.6 x 103 vg/cell,while flow cytometry required AAV9 titers of at least 4.0 x
104 vg/cell. Thus, detection by Simple Western was 10-foldmore sensitive than flow cytometry under the conditions
tested here.
We used the RePlex feature on Jess to perform sequential
immunoassay and total protein analysis using an antiGFP antibody in Probe 1 (FIGURE 3, green overlays) andtotal protein detection in Probe 2 of RePlex (FIGURE 3,
blue overlays). This method was repeated 3 times withindependently-prepared samples to calculate relative
potency using the PLA method described below.

FIGURE 1. Finding the log-log linear range of
the Simple Western relative potency assay.

7
6

AAV2

mg/mL
1.0
0.1
0.01

5
4

7 AAV9GFP Area (log)

Initial sample evaluation was performed to determine
the concentration of the reference and test sample with alog-log linear range of detection. In this study, AAV2 wasconsidered the test sample and AAV9 as the reference.

mg/mL
1.0
6 0.1
0.01

5
4
3
2
7
6
5
AAV2
AAV9

4
3

0.1 mg/mL

1

2
3
4
5
AAV vg/cell (log)

Whole-cell lysates of HepG2 cells transduced with AAV2 (circles)
and AAV9 (squares) were analyzed at final concentrations of 1 mg/
mL (filled shapes), 0.1 mg/mL (shaded shapes), and 0.01 mg/mL(open shapes). Peak areas of transgene GFP expression were
measured using an anti-GFP antibody.

6

FIGURE 2. (A) Simple Western and (B) flow cytometry analysis of AAV-mediated GFP transgene expression

A

B

7

100
80

GFP+ %GFP Area

6
5
4
3
1

60
40
20
0

2
3
4
5
6
AAV2
AAV9 vg/cell (log)

1

2
3
4
5
6
AAV2
AAV9 vg/cell (log)(A) GFP expression was measured by Simple Western in whole-cell lysates (0.1 mg/mL) of cells that were flash-frozen at 48 hours post-transduction with AAV2 (filled
circles) and AAV9 (outlined squares). (B) Percent GFP-positive cells (GFP+ %) were measured by flow cytometry from the same 48-hour sampling time.

FIGURE 3. Simple Western analysis of AAV-mediated GFP transgene expression with total protein detection

2

n
ei
Pr
ot
al

12

To
t

4.0 x 104
8.0 x 103
1.6 x 102
3.2 x 102
6.4 x 101

10
8
6
4

1

2

0

4
3
2

n
ei
al

Pr
ot

1.0 x 106
2.0 x 105
4.0 x 104
8.0 x 103
1.6 x 103

To
t

5

AAV9
vg/cell

FP

6

0

G

GFP Chemi. (RLU x 100,000)

3

16
14

1
0
12

40
MW (kDa)

66

116

16
14
12
10
8
6
4
2
0
180

Total Protein Chemi. (RLU x 100)

4

FP

5

AAV2
vg/cellG

6

The overlaid electropherograms resulting from Simple Western analysis of whole-cell lysates of liver cells transduced with serial dilutions series of AAV2 (top panel)and AAV9 (bottom panel) to deliver CMV-GFP genetic payload. Samples were probed with an anti-GFP antibody in Probe 1 of RePlex (green overlays) and total
protein detection was performed in Probe 2 of RePlex (blue overlays).

Parallel Line Analysis (PLA)a mean relative potency of 1452 and 1651 for raw and total
protein-normalized values, respectively (TABLE 3). Whennormalization of protein expression data is required, total
protein is more reliable than using a housekeeping protein
as the denominator.18 Furthermore, the quantificationof relative potency by Simple Western was highly
reproducible, with inter-assay CVs of 2.0% and 4.8% for
raw and normalized values, respectively (TABLE 3).

The relative potency assay was calculated from Simple
Western measurements described above (FIGURE 3) using
the PLA method (FIGURE 4). A minimum of 3 points per
sample is required to determine parallelism. The top point
for the AAV2 series fell above the upper range limit of ourassay based on initial assay development and therefore
had to be removed from the analysis. Comparing theaccepted data points, the slopes between the two linear
regressions were not significantly different, enabling
parallel line analysis of relative potency (FIGURE 4). See
the Appendix for more information on how to perform the
PLA method with GraphPad Prism.

TABLE 3. AAV2 potency relative to AAV9

The relative potency of AAV2 to AAV9 was calculated
for each run, and the mean and inter-assay coefficientsof variability (CV) were calculated across all three runs
(TABLE 3). Consistent with reported results,17 Simple
Western established AAV2 as a significantly more potent
serotype than AAV9 for transduction of HepG2 cells, withRaw

Normalized

Run 1

1423

1597

Run 2

1451

1614

Run 3

1481

1743

Mean

1452

1651

%CV

2.0%

4.8%

Relative potency was determined from three Simple Western runs withindependently-prepared samples. Raw and total protein-normalized values
were calculated by the PLA method using GraphPad Prism (see Appendix).

FIGURE 4. Simple Western parallel line analysis for calculating relative protein expression potency

8
R2 = 0.9843

6

R2 = 0.9931
RP

4
GFP Area (log)

Raw

Raw

R2 = 0.9913

R2 = 0.9913

Corrected

Corrected

28
R2 = 0.9841

6

R2 = 0.9951
RP
R2 = 0.9882

R2 = 0.9882
4

2

1

2

3

4

5

6
AAV2

7 1
2
3
AAV9 vg/cell (log)

4

5

6

GFP peak areas were plotted by the log AAV titer for raw values (top) and total protein-normalized values (bottom). When the highest two points of the
AAV2 titration series were removed, the resulting slopes were not significantly different according to GraphPad analysis (see Appendix).

7

Absolute Protein Expression Measurements in AAV-Transduced Cells
To quantify absolute protein concentrations of transgene
GFP expression in AAV-transduced cells, we prepared a
serial dilution series of purified recombinant GFP (rGFP)
that was analyzed in the same run as the test samples oftransduced cells. Log-log regression with 1/y2 weighting
was applied to the rGFP peak areas plotted by rGFP
concentrations to create a standard calibration curve,
which was used to quantify absolute GFP concentrationsin AAV transduced samples (FIGURE 5B). As expected,
significantly higher absolute GFP concentrations were
measured with AAV2, as high as 3913 ng/mL (TABLE
4 and FIGURE 5C-D). We also calculated absolute GFP
expression per cell, which ranged from 196 fg/cell down
to 1 fg/cell at the highest and lowest AAV2 viral loads,
respectively (TABLE 4 and FIGURE 5D).

TABLE 4. Absolute GFP expression measurements
AAV Titer

AAV2-Transduced Cells

AAV9-Transduced Cells

vg/cell

GFP ng/mL

GFP fg/cell

GFP ng/mL

GFP fg/cell

1.0 x 106

3913

196

239

12

2.0 x 10

5

3579

179

63

3

4.0 x 104

3593

180

14

1

8.0 x 10

0

1950

97

3

1.6 x 103

697

35

ND

3.2 x 102

134

7

ND

6.4 x 101

30

1

ND

3

GFP expression was not detectable (ND) in cells transduced with AAV9 oftiters ≤ 1.6 x 103 vg/cell.

FIGURE 5. Absolute quantification of GFP expression levels in AAV-transduced liver cells

rGFP
ng/mL
5.0
1.67
0.56
0.19
0.06

8
6
4
2
0
12

40
66 116
MW (kDa)

1

5

4

-1
0
rGFP (log ng/mL)

D

1

2

6

5

10

10

10

10

10

x

x

x

x

x

8

4

2

1

4

3

3

10

1.
6

2

-1

x

7

1

3
4
5
6
AAV2 AAV9 vg/cell (log)

10

2

0

3.
2

-1

1

x

0

-2
1

AAV2 R² = 0.9739
AAV9 R² = 0.9858

3
-2

230

GFP fg/cell (log)

GFP ng/mL (log)

C

6

6.
4

Chemi. (RLU x 10,000)

10B
rGFP Area (log)

A

AAV2

AAV9 vg/cell

GFP expression was not detectable in cells transduced with AAV9 of titers ≤ 1.6 x 103 vg/cell. Error bars represent standard errors of the means.

Screening for Intracellular AAV Capsid Proteins in Transduced Cells as an Infectivity Surrogate
We tested if Simple Western could detect the persistenceof AAV capsid proteins in cells post-transduction. As apositive control, a serial dilution series of purified AAV2stock was prepared in a HepG2 lysate background andanalyzed using an anti-VP1/2/3 antibody. Clear signals
corresponding to VP1/2/3 appeared that decreased withdecreasing vector concentration (FIGURE 6, top panel).
To create a standard curve, the VP3 peak area from eachsample was plotted by AAV2 concentration, and log-log
regression with 1/y2 weighting from 1.3 x 109 to 5.0 x 1010
VP/mL (FIGURE 6, top panel inset). Next, cells transducedwith the 4 highest AAV2 titers (FIGURE 1, 8 x 103 to 1 x 106
vg/cell) were analyzed with the anti-VP1/2/3 antibody,
revealing a similar VP1/2/3 separation profile as the
AAV2 stock control (FIGURE 6, bottom panel). Finally,
we quantified the absolute concentrations of AAV2

in transduced cells using the VP3 peak areas and thestandard curve, resulting in AAV2 concentrations of 1.67 x
1010 to 8.41 x 1011 VP/mL (adjusted for dilution) that directly
correlated with the viral loads used for transduction(FIGURE 6, bottom panel inset).
TCID50 assays are used to measure infectivity, but they
can be unreliable and are not easily amenable to scale. To
our knowledge, this is the first time Simple Western wasused for absolute quantification of AAV vectors directlyin transduced cells as a surrogate for AAV infectivity
and a potential rapid, multi-attribute alternative to the
TCID50 assays. The persistence of AAV in vivo is clinicallymeaningful as residual AAV capsid proteins may be
immunogenic and can impact patient safety following theadministration of an AAV gene therapy product.

FIGURE 6. Intracellular detection of VP1/2/3 in transduced liver cells as a surrogate for AAV infectivity

7

AAV2
VP/mL
5.0 x 1010
2.0 x 1010
88.0 x 109
3.2 x 109
1.3 x 109
4
Empty lysate

VP3

VP3 Area (log)

AAV2 Stock

6
R2 = 0.9877

5
4

VP2

9
VP1

10
AAV2 (log VP/mL)

110

Intracellular AAV2
Transduced Cells

15
10
5

AAV2 (VP/mL x 1011)

Chemi. (RLU x 10,000)

12

AAV2
vg/cell1 x 106
2 x 105
4 x 104
8 x 1038
6
R² = 0.9984

4
2
0
0

5
10
AAV2 Dose (vg/cell x 105)

066
MW (kDa)

116

Simple Western analysis of AAV2 vector stock prepared in an untreated HepG2 whole-cell lysate background diluted 1:5 (top panel) and HepG2 whole-celllysates diluted 1:20 of cells transduced with AAV2 (bottom panel) using an anti-VP1/2/3 antibody. The VP3 peak area of the AAV2 stock was plotted bystock concentration to create a standard curve (top panel inset), and used to quantify AAV2 in transduced cells, which was adjusted by dilution and plottedby dosage (bottom panel inset). Cell transduced with AAV9 did not give a detectable signal using the anti-VP1/2/3 antibody (data not shown).

A Fit-For-Purpose Potency Assay

References

Here, we established Simple Western as a specific,
highly reproducible, and quantitative tool for AAV
relative potency measurements. The relative potency
between AAV2 and AAV9 serotypes was determinedby measuring GFP transgene expression normalized tototal protein, which is detected automatically in the same
sample capillary using Simple Western’s RePlex feature.
Ultimately, Simple Western conclusively demonstrated
AAV2 as a significantly more potent serotype than AAV9,which is consistent with reported results.17 The assay was
highly quantitative and reproducible, with a CV <5%across three independently prepared samples and runs.

1. The Role of Analytics in Reproducibility and Regulatory Approval of Celland Gene Therapies (2020) Bio-Techne Phacilitate Report.

The results obtained with Simple Western which showed
more potent transduction and GFP expression by AAV2
compared to AAV9 were consistent with flow cytometry
analysis. However, Simple Western was more than ten
times more sensitive than flow cytometry under theconditions tested here. Simple Western has the added
advantage of detecting intracellular proteins, which
can be challenging by flow cytometry. Furthermore,
Simple Western provided absolute protein expression
measurements directly in cells using a standard curve.
Simple Western is a fit-for-purpose tool for quantitative
gene therapy potency assays to quantify vector-mediatedprotein expression because Simple Western can work witha variety of complex sample matrices with minimal matrix
effects.16 Compared to traditional methods like ELISA
and Western blot, Simple Western requires much smaller
protein loads, preserving precious tissue samples. Simple
Western also provides increased automation, which allows
for better standardization across multiple sites, as wellas higher throughput, which will allow for increased
biological and technical replicates for increased accuracy.

A Multi-Attribute Platform Approach for Viral
Vector Characterization and Manufacturing
As an open immunoassay platform, any antibody may be
used for detection with the advantage of needing only one
target-validated antibody to develop custom assays. Thisimmunoassay flexibility enables multi-attribute analysis
in the same Simple Western instrument and even within
the same Simple Western run. With a single platform,
Simple Western measures several CQAs for vector
characterization. In addition to potency, these include:• Purity and identity• Empty/full capsid content ratio• AAV capsid protein ratio (VP1:VP2:VP3)

2. Guidance for Industry, Potency Tests for Cellular and Gene Therapy
Products (2020) US FDA.
3. FDA Calls for More Data on Sarepta Gene Therapy for DMD (2020)
BioPharma-Reporter.
4. Sarepta Therapeutics Provides Program Update for SRP-9001, its
Investigational Gene Therapy for the Treatment of Duchenne Muscular
Dystrophy (2020) Sarepta Therapeutics Press Release.
5. FDA Wants More Details on Sarepta Gene Therapy, Potentially Delaying
Final Test (2020) BioPharma Dive Brief.
6. De et al (2016) Molecular Therapy 24 S186.
7. Schnell et al (2019) 15 40–6.
8. Aronson et al (2020) 18 250-258.
9. Accelerating The Time To Market Of Gene Therapies Through Use
Of A Platform Approach To Characterization And Safety Testing (2019)
MilliporeSigma White Paper
10. Tabebordbar et al (2016) Science 351 407-411.
11. Bäck et al (2019) Neuron 102 05-119.e8.
12. Shyam et al (2022) Ophthalmol Sci 2 100084.
13. Costa-Verdera et al (2021) Nat Commun 12 6393.
14. Bezzerides et al (2019) Circulation 140 405-419.
15. Vector Characterization (2022) ProteinSimple Bio-Techne.
16. Stuck in the Matrix? Escape Matrix Effects in Complex Samples With
Simple Western (2023) ProteinSimple Bio-Techne App Note.
17. Ellis et al (2013) Virol J 10 74.
18. Eaton et al (2013) PLoS One 8 e72457.

Appendix

At a Glance: Relative Potency Determination

PLA Relative Potency with GraphPad Prism• Transform all data to log

Here, we provide step-by-step instructions on how todetermine relative potency by the PLA method using
GraphPad Prism analysis software. This PLA method may
be applied to calculate relative potency in applications
beyond gene therapy, like vaccine development.

• Apply simple linear regression fit• Determine linear regression equation for AAV9Reference and AAV2 Test
• Software determines if the slopes are equal, parallelism• Solve for X at an overlapping Y value• Determine relative potency from X value with thefollowing steps:
a. Transform log(X) values to 10x
b. Reference = Reference X-value/Reference X-valuec. Test = Reference X-value/Test X-value

Additional details and screenshots are provided below.

1. Create an XY file and select the appropriate Y option, click Create2. Under Data Tables, Data 1 paste the Reference and Test data

*Ensure that the replicates are stacked in the same column

3. After entering data, click on the Analyze button
Select Transform in the Analyze Data window.
Click OK

4. In the Parameters: Transform window, check the Transforms X and Y values using X and Y=Log(X or Y)
The New Graph – Create a new graph of the results box should also be selected
Click OK

5. The transformed data table will appear in the Results – Transform of Data 1 tab
Click on the Analyze button and select Simple Linear Regression
Click OK

6. In the Parameters: Simple Linear Regression window, click on the Compare option
Also Calculate boxes should already be checked
Click OK

7. The Simple linear regression results will appear in the Results – Simple linear
regression tab.
Under Tabular results, you will find the linear regression Equations needed todetermine the Relative Potency of the Test Sample

8. In the same Results folder, under the Are lines different? tab you will find whether the Reference and Test slopes areequal

9. Prism compares slopes of two or more regression lines if you check the option: “Test whether the slopes and intercepts
are significantly different”. If selected, a summary of this analysis will be presented on the results tab titled “Are lines
different?”

Comparing slopes
Prism compares slopes first. It calculates a P value (two-tailed) testing the null hypothesis that the slopes are all identical(the lines are parallel). The P value answers this question:
If the slopes really were identical, what is the chance that randomly selected data points would have slopes as different (ormore different) than you observed.

If the P value is less than 0.05
If the P value is low, Prism concludes that the lines are significantly different. In that case, there is no point in comparing
the intercepts. The intersection point of the two lines is:
X = Intercept1 - Intercept2
Slope2 - Slope1
Y = Intercept1 + Slope1 = Intercept2 + Slope2 · X

If the P value for comparing slopes is greater than 0.05
If the P value is high, Prism concludes that the slopes are not significantly different and calculates a single slope for alllines. Essentially, it shares the Slope parameter between the two data sets.

10. Using the Simple linear regression equations, calculate theRelative Potency by solving each equation for X by using a common
Y value(s) between the Reference and Test linear regression curves

7

Area (log)

63-Fold Titration
Reference
Test

5
4
3
-5.0

-4.5
-4.0
Dilution (1/X) (log)-3.5

11. Determine relative potency from X value with the following
steps:
a. Transform log(X) values to 10x
b. Reference = Reference X-value/Reference X-valuec. Test = Reference X-value/Test X-valuereference
Slope
2.097
Y-intercept
13.9
Rel Pot at log

test
2.036
13.79
5

log (X) -4.24416 -4.31729
power10 (X) 5.7E-05 4.82E-05
reference
Rel Potency
1.00

test
1.18

Bio-Techne® | R&D Systems™ Novus Biologicals™ Tocris Bioscience™ ProteinSimple™ ACD™ ExosomeDx™ Asuragen®
For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners.
STRY0309338_WBU_AN_Relative Potency Simple Western_CHH