== Computing Ghrelin calibration curve using R ==

===This article describes creating calibration curve, and measuring the total-ghrelin concentration ===

The calibration curve can be created using R, and libracy "drc". Optionally, one can use library "sfmisc" for formatting of the labels on plot axis.

We will assume a 4-parameter log-logistic model:

[[Image(LL4.png, 300px)]]

The R code is attached below.

The example assumes the data to be available in file "ghrelin_conc std_a std_b avg.csv"

The measured data:

|| '''Ghrelin (ng/ml)''' || '''Standard a''' || '''Standard b''' ||
|| 1000000 ||-0.040596823||-0.052699697 ||
|| 100000 ||0.136105144||0.119766263 ||
|| 10000 ||0.61356354||0.606906959 ||
|| 1000 ||0.846543873||0.839887292 ||
|| 100 ||0.887693646||0.88345764 ||
||0||0.896770802||0.896165658 ||


##### Install libraries

##### Read the data
hormone.data <- read.csv("ghrelin_conc std_a std_b avg.csv")
hormone.data <- hormone.data[,1:3]
colnames(hormone.data)[1:3] <- c("Concentration","Response_1", "Response_2")

##### Reorganize the data
hormone.data <- reshape(hormone.data, varying=c("Response_1","Response_2"), direction="long", v.names=c("Response"))
hormone.data <- hormone.data[,c("Concentration", "Response")]

##### Fitting the model (4-parameter log-logistic function)
hormone.data.model <- drm(Response ~ Concentration, data = hormone.data, fct = LL.4())


The resultant parameters of a log-logistic equation are:

Model fitted: Log-logistic (ED50 as parameter) (4 parms)
Parameter estimates:
Estimate Std. Error t-value p-value
b:(Intercept) 9.5057e-01 2.2294e-02 4.2638e+01 0
c:(Intercept) -7.6010e-02 6.9075e-03 -1.1004e+01 0
d:(Intercept) 8.9163e-01 3.3216e-03 2.6843e+02 0
e:(Intercept) 2.5221e+04 7.7727e+02 3.2448e+01 0

The calibration curve can be plotted using the commands below:

##### Plotting a nice plot
par(pty="s", mar=c(5,5,1,1))
plot(hormone.data.model, type="confidence", cex.lab=2, axes=F, xlim=c(-10,10^6))
axis(side=1, at=hormone.data[1:6,1], labels=pretty10exp(hormone.data[1:6,1]), cex.axis=1.2)
axis(side=2, at=seq(0,1,0.2), labels=seq(0,1,0.2))
plot(hormone.data.model, type="all", add=T, pch=21, col="red", lwd=1, cex=2, bg="green")


The parameters of the eqution can be plugged into the formula below (an inverse of the model), and used in Excel, or other spreadsheet program.

[[Image(LL4-inv.png, 230px)]]

However, the concentration can be also easily estimated in R using "ED" function of the "drc" library. The code below demonstrates the concentration estimated from the response of 0.1, assuming alpha=0.05. The code returns the estimation, the error, and the condfidence interval.

##### Computing the concentration from the response, for instance for a response=0.1, and alpha=1-0.95
ED(hormone.data.model, respLev=0.1, interval="delta", type="absolute", level=0.95)