Statistical analysis. The PowerPoint presentations provided below are annotated with notes.
The first provides guidance on interpretation of raw data for NA activity and IC 50 assays. It covers the following topics: determining standard virus dose; validation and interpretation of NA activity and IC 50 analysis; how to calculate IC 50 values, and examples of fluorescence and chemiluminescence data.
Interpretation of raw data. The second addresses a number of the issues encountered when undertaking statistical analysis of IC 50 data. It covers the impact of using different methods for determining IC 50 values; how to identify outlying results above the seasonal normand how to monitor trends in IC 50 data:. Statistical analysis of IC 50 s. Examples of both correct and incorrect curve fitting and point-to-point graphs for both sensitive and resistant isolates are provided below.
The files contain graphical examples and accompanying explanatory notes to aid interpretation. Frequently asked questions on IC 50 data analysis. Susceptibility to NI drugs is not absolute. There are typical ranges of IC 50 values that differ between influenza subtypes and between oseltamivir and zanamivir for each subtype.
Therefore this subtype and drug specific data should not be compared. Influenza B viruses tend to have IC 50 values fold higher than influenza A viruses. This is normal for influenza B and this lower susceptibility does not appear to have a significant clinical impact.
IC 50 values for influenza B viruses tend to be higher for oseltamivir than zanamivir. IC 50 values for H1N1 viruses tend to be higher for zanamivir than oseltamivir and IC 50 values for H3N2 viruses tend to be higher for oseltamivir than zanamivir. Also, values generated by fluorescence and chemiluminescence methods should not be compared. Typically, values generated by chemiluminescence are lower than those for the same virus and drug in the fluorescence test. IC 50 values generated from the different assay methods should not be directly compared.
Raw data relative fluorescence or luminescence units are plotted against the drug concentration. IC 50 values can be determined in two ways; using curve fitting software, or by a point to point analysis.
CDC have developed a curve fitting software which is available from them directly. Point to Point: This method uses in house excel templates. Guidance on how to undertake point-to-point analysis is provided above in the IC 50 analysis section. Is this normal? There is no firm definition of a resistant IC Commonly used criteria to identify isolates that outside the normal range are either:.
A value greater than 3SD from the mean or median value for the given subtype and drug.The concept of an absolute IC50 is not standard, and many find it not to be useful. But if you do, it is not hard to fit a curve to determine it. This EPA documentgive the needed equation which I have generalized a bit, so not require that the data already be normalized.
Note the distinction between the parameter Bottom and Baseline. Bottom is the Y value of the bottom plateau of the curve itself.
You'll definitely want to constrain Baseline to be a constant value based on controls. You may also want to constrain Top. Download the Prism file that fits that equation to make the graph shown above. When fitting data to that equation, don't forget to constrain Baseline and Top to appropriate values determined by controls.
These data are fit to a simpler equation where Baseline is set to equal zero, and Top is set to equal These are hard wired into the equation, so you don't have to remember to constrain those two parameters to constant values. Here is an alternative approach you can use if your data are normalized. It does not require entering a user-defined equation. Make sure that your data are normalized to some controls.
At the bottom of the data table, add a new row of data. Enter 50 into each Y column. Leave X blank for this row. Use nonlinear regression to fit the data to the log inhibitor vs. On the first fit tab of the nonlinear regression dialog, check the option: " Interpolate unknowns from standard curve. On the Constrain tab, consider constraining Top to have a constant value of The IC50 reported as part of the main results table will be the relative IC This is the absolute IC All rights reserved.
This guide is for an old version of Prism. Browse the latest version or update Prism. Fitting a dose-response curve to find the absolute IC50 The concept of an absolute IC50 is not standard, and many find it not to be useful. An alternative approach for normalized data Here is an alternative approach you can use if your data are normalized.
Scroll Prev Top Next More.A new web-server tool estimates K i values from experimentally determined IC 50 values for inhibitors of enzymes and of binding reactions between macromolecules e.
This converter was developed to enable end users to help gauge the quality of the underlying assumptions used in these calculations which depend on the type of mechanism of inhibitor action and the concentrations of the interacting molecular species.
Additional calculations are performed for nonclassical, tightly bound inhibitors of enzyme-substrate or of macromolecule-ligand systems in which free, rather than total concentrations of the reacting species are required. Required user-defined input values include the total enzyme or another target molecule and substrate or ligand concentrations, the K m of the enzyme-substrate or the K d of the target-ligand reaction, and the IC 50 value.
Assumptions and caveats for these calculations are discussed along with examples taken from the literature. Some analyses of networks, pathways and metagenomics focus on identifying key proteins or polynucleic acids as targets for inhibitory compounds.
Typically, high-throughput screening assays are initially used to compare and down-select potential inhibitors of enzymatic activity or macromolecule-ligand binding.
However, the IC 50 value depends on concentrations of the enzyme or target moleculethe inhibitor, and the substrate or ligand along with other experimental conditions. What is required is an accurate determination of the K i value, an intrinsic, thermo-dynamic quantity that is independent of the substrate ligand but depends on the enzyme target and inhibitor. Thus, comparisons can be more readily made among different laboratories to characterize the inhibitors.
While these more time-consuming assays are usually done with the most promising candidates, accurate, initial estimates of K i values for more of the candidates would be beneficial. A much discussed problem in the literature 1—8 is converting IC 50 to K i values because even the simplest types of inhibitory mechanisms e. Additional calculations are performed for tightly bound inhibitors of enzyme-substrate reactions in which free, rather than total, concentrations of the molecular species are calculated for nonclassic Michaelis—Menten kinetics.
Similar calculations can be performed for target molecule-ligand systems. User-defined input values include total concentrations of the enzyme or target molecule and substrate or ligandthe K m of the enzyme-substrate or the K d of the target-ligand reaction and the IC 50 value.
The outputs include tabulations of the K i values under different kinetic schemes, extensive tabulations of the results, summary histograms and the corresponding equations. Help buttons are available for Background, Assumptions, Literature, Links and Equations along with examples taken from the host database-server that contains kinetic information on neurotoxin inhibitors. An example calculation is included here for a tight-binding inhibitor of an enzyme—substrate reaction, while other enzyme inhibitor and protein—ligand—inhibitor examples are also provided.
Our rationale for creating this converter is to enable end users to judge the quality of the underlying assumptions for these calculations and to help facilitate research and the development of potential therapeutic products.
The website cited in 9 served as an initial design template for our IC 50 -to- K i converter. Equations 1—4 were adapted from refs. The analytic expressions for K i that are shown below were verified numerically by methods used in a previous kinetic analysis The derivations for converting IC 50 to K i values published by Brandt et al.
For tightly bound inhibitors, the equation for K i by Copeland et al. These equations are also relevant for protein—ligand—inhibitor P—L—I interactions that also adhere to the above assumptions. For noncompetitive inhibition 2. For total concentrations, E is replaced by P and S is replaced by L. Additional reaction schemes are located at this tool's website. As in classic enzyme—substrate systems the relation of K i and IC 50 in competitive inhibition is:.
For protein—ligand experiments with tight-binding inhibitors, the free rather than the total concentrations of the reactants need to be used as modified from ref. The concentration of the free inhibitor species is given by. For this study, we derived a corresponding value of K i for uncompetitive inhibition. Although in this study we use the term K d to quantify an antagonist's effect, the pharmacology-derived EC 50 value is more appropriate when functional experiments are performed It is assumed that all of the substrate- and inhibitor-binding reactions are reversible and that they all have a one-to-one stoichiometry, i.
It is also assumed that in the enzymatic reactions enzyme autocleavage did not occur and that when substrates for fluorescence resonance energy transfer were used, appropriate corrections for inner filter effects were performed. Comparison of K m or IC 50 values for a set of inhibitor candidates is only assumed to be valid when they are evaluated under identical experimental conditions.
In most experimental studies of enzyme kinetics, the total concentrations of substrate and inhibitor used are in excess of the enzyme concentration to make their free and total concentrations essentially the same 1.
Under the conditions of some ligand-receptor e. An internal link to the user-accessible converter is also located on the left side of the BotDB home page. The four required inputs for ESK m and IC 50 are indicated with default settings for several examples.Finney and later discussed in detail elsewhere Finney, ; Robertson et al.
In general, the data from bioassays mortality proportions and corresponding doses gives an S-shape curve. In order to make this curve linear, the proportions are transformed to probits and doses to log Furthermore, these programmes do not provide real-time results time is required for entering commands and processing.
Converting doses to log 10 doses x. Converting mortality to proportions. Converting corrected proportions p to empirical probits y.
Empirical probits less than 1 and more than 7 are ignored as they have little and no significance in the estimation of LD or LC Hayes, From the equation of the curve and log10 doses, the expected probits Yi are derived. From the expected probits Yiexpected mortality proportion followed by expected no. The original mortality Observed and derived mortality Expected are used to calculate the Chi-Square test with No.
If the Chi-square test is non-significant, it indicates good curve fitting. Z value is derived using the formula. The weighting coefficents are derived using the formula. The weighted coefficients were used to calculate the standard error. Working probits Yw are derived from the regression equation as follows. The LD or LC values are derived from the curve drawn using working probits and log doses. Antilog of the dose corresponding to respective probit value. Your comments and corrections are welcome to make this spread sheet better: alpharajm gmail.
Busvine J.Dose response curves tutorial 1 (Pharmacology 1 UTS Sydney)
A critical review of the techniques for testing insecticides. Commonwealth Agricultural. Finney, D.The half maximal inhibitory concentration IC 50 is a measure of the potency of a substance in inhibiting a specific biological or biochemical function.
IC 50 is a quantitative measure that indicates how much of a particular inhibitory substance e. IC 50 values are typically expressed as molar concentration. IC 50 is commonly used as a measure of antagonist drug potency in pharmacological research. IC 50 is comparable to other measures of potency, such as EC 50 for excitatory drugs. Due to the minus sign, higher values of pIC 50 indicate exponentially more potent inhibitors. The IC 50 terminology is also used for some behavioral measures in vivo, such as a two bottle fluid consumption test.
The IC 50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist on reversing agonist activity. IC 50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist.
IC 50 values are very dependent on conditions under which they are measured. In general, the higher the concentration of inhibitor, the more agonist activity will be lowered. IC 50 value increases as agonist concentration increases. Furthermore, depending on the type of inhibition other factors may influence IC 50 value; for ATP dependent enzymes IC 50 value has an interdependency with concentration of ATP, especially so if inhibition is all of it competitive.
In this type of assay, a single concentration of radioligand usually an agonist is used in every assay tube. The ligand is used at a low concentration, usually at or below its K d value. The level of specific binding of the radioligand is then determined in the presence of a range of concentrations of other competing non-radioactive compounds usually antagonistsin order to measure the potency with which they compete for the binding of the radioligand. Competition curves may also be computer-fitted to a logistic function as described under direct fit.
IC 50 is not a direct indicator of affinity although the two can be related at least for competitive agonists and antagonists by the Cheng-Prusoff equation. Alternatively, for inhibition constants at cellular receptors: . Whereas the IC 50 value for a compound may vary between experiments depending on experimental conditions, e.Analyze, graph and present your scientific work easily with GraphPad Prism.
No coding required. Home Support. Prism can easily fit a dose response curve to determine the IC Note that the X values are logarithms of concentration. Prism offers built-in equations designed to handle X values as either concentration OR log concentration. Be sure you select the correct equation when performing nonlinear regression!
If you'd like to convert concentration values to log concentration values - or vice versa - you can use Prism's Transform analysis to convert the X values.
Also note that this sample data set includes unknown values. Prism can interpolate these X values.
Click Analyze and then Nonlinear regression. Or click the Nonlinear Regression shortcut button just above the Analyze button. On the Nonlinear regression dialog, open the "Dose-Response -- Inhibition" family of equations, and choose "log inhibitor vs.
At the bottom of the dialog, check the option to "Interpolate unknowns from standard curve".
How to determine an IC50
Click OK and view the results. View the graph. More information: More tutorials on curve fitting Learn about the equation used to fit to the data.
Interpreting nonlinear regression results How exactly is the IC50 defined? Relative vs. Explore the Knowledgebase. Try for Free.Will be a great help for me many thanks vg.
If you have someone to show you how to do non-linear regression you can set up the calculations on MSexcel or any other spreadsheet. Not as refined as graphpad but the results are the same. I hope this reply will benefit a lot of students trying to learn how to perform a cytotoxicity experiment and calculate the IC You should never use Excel for IC50 estimation. Your viability data is not linear or fits simple log transformation.
For accurate calculation of IC50, a nonlinear curve fitting program that uses the Four Parameter Model is needed. A paid program such as GraphPad Prims is great, but there is a free online program BioDataFitwhich produces the same accuracy as GraphPad and it is easier to use. To learn how to prepare the raw data for inputting into an IC50 program, read here. To learn what to do if your experimental data does not fit the Four Parameter Model, which can be very frustrating to new users, read here.
Good luck and hope you all will smoothly get your cytotoxicity experiments done and get publication quality IC50s from these tips. DRT may be right. Will be a great help for me many thanks vg -vitalgene.
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