StandardCurvesAnalysis

Introduction
Astandardcurveisusedtocalibrateaninstrumentorassay.TheStandardCurvesmacroinSigmaPlot
providesfiveequationsthatmaybefittoyourdata.
Theserangefromastraightlineequationtotwodifferentfiveparameterlogisticequations.
TheXdatamayormaynotbelogarithmicand,ifnot,maystillbegraphedlogarithmically.MultipleY
replicatecolumnsmaybeused.
Predictedvaluesmaybeobtainedafterthecurvefitisperformed:YvaluesfromXs,XvaluesfromYs,
andECxxvaluesfromxxpercentages.
ThesepredictedvaluesmaybeaddedtoyourgraphassymbolswithdroplinestotheXandYaxes.
TheDynamicCurveFittingalgorithmmaybeusedtohelpsolvedifficultcurvefittingproblemsinvolving
localminima.
Thesearetypicallyencounteredwiththefiveparameterlogisticfunctions.

Usingthemacro

Themacropanelbelowshowsthevariousstandardcurveoptions.

Tousethismacro
1. PuteitherXandYcolumnsorXandmultiplereplicateYcolumnsintoyourworksheet.
Thesecolumnsmustbeadjacent.SeetheexampleintheYreplicatessection.Ifyou
plantocomputepredictedXsorYsfromthecomputedcurve,youwillalsoneedto
enteracolumnofthesourcevalues.
2. SelecttheequationfromtheEquationlisttousetofitthecurve.Youroptionsare(see
theequationdescriptionsintheEquationssection):
1. straightline,
2. quadraticequation,
3. Fourparameterlogisticequation
4. fiveparameterlogisticequation
5. fiveparameterlogisticequation2slopes

3. UsetheLogXaxisscalecheckboxtoselectwhetherornottoplottheXaxisusinga
commonlogscale.ThisoptionwillbeautomaticallyselectedifyouhaveXdatainlog
formatandyouselecttheLogformatXdatacheckbox.
4. IfyoufindthatyourdataisdifficulttofitthenselecttheDynamiccurvefitcheckbox.In
thiscase200curvefitswillbeperformedusinginitialstartingvaluesthatspanthe
parameterranges.Thebestfitwillbeselectedfromthe200results.
5. Ifthecurvefitdoesnotconvergethenthereissomebadrelationshipbetweenthe
equationselectedandthedatabeinganalyzed.Butthegoalofastandardcurveisto
obtainasmoothcurverepresentationtothedatasoitmaybeimportanttoachieve
convergence.Thebestwaytodothisistoincreasethecurvefittolerancefromthe
standard1e10to1e3,say,orsomevaluewhereconvergenceoccurs.Themaximum
numberoffititerationsmayalsobeincreased.
6. SelectthecolumnstousefortheXandYdatafromtheXdatacolumnandYdata
columndroplistboxes.IfyourXdataisalreadyinalogformat,makesureyoucheckthe
LogformatXdataoption.
7. IfyouhavereplicateYmeasurementsforeachXdatapointthenselecttheYreplicates
optionandthenselecttheLastYreplicatecolumn.
8. SelectthePredictunknownsoptiontocomputeresultsusingthesolutiontothefit.You
cancomputeacolumnofnewYvaluesfromgivenXs,orXsfromgivenYvalues.Ifyou
areusingthefourorfiveparameterlogisticequationsyoucancomputeECpercent
valuesforaspecifiedrangeofpercentvalues.Youcanalsoelecttoplottheresultsof
theseonyourstandardcurvebyselectingthePlotpredictedvaluescheckbox.
9. Whenfinished,clickOK.Astandardcurveiscreated,andifyouelectedtocompute
additionalvalues,theyarealsoplottedusingdroplinestoindicatetheXandYvalues.
Standard Curve
120
100

response

80
60
40
20
0
-20
10-6

10-5

10-4

10-3

10-2

10-1

100

101

concentration

103

102

Yreplicates

IfyouhavemultiplemeasurementsforeachXvalue,selecttheYreplicatesoptionandthen
selectthelastreplicatecolumn.TheYreplicatecolumnsmustbetotherightofandadjacentto
theXdatacolumn.

Logdataformat
IfyourXdatausessimpleintegers,especiallynegativenumbers,itisalreadyinlogformatand
youshouldselecttheLogformatXdataoption.Themacrowillautomaticallycreateanew
columnofequivalentnumericdata,andautomaticallyplotXonalogaxisscale.

Equations
LinearEquation

y = y 0 + ax
Astraightline,characterizedbytheslopeaandtheyintercepty0.

QuadraticEquation

y = y 0 + ax + bx 2
Thestandardparabolicequationwithquadraticcoefficientb,slopeaandintercepty0.

FourParameterLogisticEquation

y = min+

max- min
- Hillslope
x
1+
EC50

30000

25000

max

Response

20000

15000

10000

5000

min 0

100

101

102

103

104

105

EC50
Concentration

Thisisatypicaldoseresponsecurvewithavariableslopeparameter.Itissometimesabbreviatedas
4PL.Thefourparametersare:
Minbottomofthecurve
Maxtopofthecurve
5

EC50thexvalueforthecurvepointthatismidwaybetweenthemaxandminparameters.Itiscalled
thehalfmaximaleffectiveconcentration.EquivalentdefinitionsareED50(halfmaximaleffectivedose)
andforinhibitioncurvesIC50(halfmaximalinhibitoryconcentration).
Hillslopecharacterizestheslopeofthecurveatitsmidpoint.Largevaluesresultinasteepcurve
whereassmallvaluesashallowcurve.The4PLcurvewillincreasewithxiftheHillslopeispositiveand
decreaseifitisnegative.

FiveParameterLogisticCurve

y = min+

(max- min )
x - Hillslope s

1 + x

where

- 1 log2 (1s )- 1
)
(
x b = EC 50 10 Hillslope

120
s=1
s = 10
s = 0.1

100

Response

80
60
40
20
0

10-7

10-6

10-5

10-4

10-3

10-2

10-1

Concentration

100

101

102

103

ThisistheRichardsformulationofthefiveparameterlogistic.Itaddsanasymmetryparametersto
thefourparameterlogistic.Theasymmetryisshownabovewithlargechangesincurvaturewith
changesinsinthelowercurvebutrelativelysmallchangesintheuppercurve.
TheadditionalalgebraicequationforxbmaintainsEC50asthehalfmaximumyvalue.Theequationhas
beenwrittensothatapositiveHillsloperesultsinacurvethatincreaseswithx.
Fourofthefiveparametersarethesameasthoseinthefourparameterlogistic.
6

scontrolstheasymmetry.Ifs=1thenthisfunctionisthesameasthefourparameterlogistic.sless
than1decreasestheoverallslopeofthecurvewhereassgreaterthan1increasestheoverallslope.

FiveParameterLogisticTwoSlopes
TheRickettsandHeadequationwithtwoslopeparameters(theparametersactuallybetterdescribe
thetwodifferentcurvatures)isshownbelow.IthasadifferentshapethanRichardsequationandwill
fitsomedatasetsbetter.

y = min+

max- min
- Slope 1

1 + f x x
EC 50

- Slope 2
+ (1- f x ) x
EC 50

TheequationcanbewrittenintermsofSlopeContoforceSlope1andSlope2tobethesamesign.Ifthis
isnotthecasetheninraresituations,errorinthedatawillresultinafitwithslopesofoppositesign.In
thiscase,thefunctionattemptstofollowtheerrorwhichresultsinanirregularlyshapedcurve.

y = min+

- Slope

1 + f x x
EC 50

max- min

x - Slope SlopeCon
+ (1- f x )
EC 50

Where

Slope = Slope 1
Slope 2 = Slope SlopeCon,
fx =

AvSlope
1 + x
EC 50

AvSlope =

SlopeCon > 0

2 Slope SlopeCon
(1 + SlopeCon )

120
Slope1 = 1
SlopeCon=0.5
SlopeCon=1
SlopeCon=2

100

Response

80

60

40

20

10-1

100

101

102

103

Concentration

104

105

106

ThegraphshowstheincreasingasymmetrywithincreasingSlopeCon(andthereforeincreasingSlope2).
IfSlopeCon=1thenSlope1=Slope2andthecurveissymmetricandidenticaltothefourparameter
logisticcurve.

References
1. Richards,F.J.Aflexiblegrowthfunctionforempiricaluse.J.Exp.Botany10.pp290
300.
2. Ricketts,J.H.andG.Head.Afiveparameterlogisticequationforinvestigating
asymmetryofcurvatureinbaroreflexstudies.Am.J.Physiol.277(Regulatory
IntegrativeComp.Physiol.46).R441R454.1999.