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<head>
<meta charset="UTF-8">
<meta http-equiv="X-UA-Compatible" content="IE=edge">
<meta name="viewport" content="width=device-width, initial-scale=1.0">
<title>Landing Page</title>
<link rel="stylesheet" href="MyFontsWebfontsKit/MyFontsWebfontsKit.css">
<link rel="stylesheet" href="help_page.css">
<script type="text/javascript" async
src="https://cdnjs.cloudflare.com/ajax/libs/mathjax/2.7.1/MathJax.js?config=TeX-MML-AM_CHTML">
</script>
<script type="text/x-mathjax-config">
MathJax.Hub.Config({
TeX: { equationNumbers: { autoNumber: "all" } }
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</script>
</head>
<div class="container-fluid mt-6 page-container">
<div class="row" style="margin-top:3%">
<div class="col-sm-12" >
<div class="text-center">
<img class="anabel-logo" src="Anabel_logo.png" alt="Anabel logo" style = "width:300px">
</div>
</div>
</div>
<div class="row">
<div class="col-sm-12">
<p id="analysis-text-centered">Anabel (Analysis of binding events) is a software designed for the analysis of binding curves with which
the interactions of biomolecules are evaluated. Currently, Anabel supports exported kinetic datasets from Biacore, BLI, Score and an
open data format. Furthermore, Anabel will help you fit a model to your kinetic data and will calculate all kinetic
constants. These include the association rate constant (kass),
the dissociation rate constant (kdiss) as well as the equilibrium dissociation constant (KD).</p>
</div>
</div>
</div>
<!-- Usage -->
<div class="container-fluid mt-6 page-container">
<div class="row" style="margin:5%;">
<div class="col-sm-12">
<!-- These are just text elements, with some different stylings -->
<h2 class="landing-page-h2">How to use Anabel</h2>
<h3 class="landing-page-h3">Step 1: Use your own data or use the example data</h3>
<p class="landing-page-p">The best way to get to know Anabel is to use our example data. Do so by clicking the checkbox "Or use our example data". If you go for this option, you can ignore the browse button.</p>
<p class="landing-page-p">If you want to do an analysis on your data, you have to make sure that you bring your data into the correct form. Currently, anabel is supporting .xlsx, .csv or .tsv files. The first column in your data MUST be the time column. Every other column can be columns that carry the corresponding response values. Example:
</p>
<table><thead>
<tr>
<th>Time</th>
<th>Analyte-A</th>
<th>Analyte-B</th>
<th>Analyte-C</th>
</tr></thead>
<tbody>
<tr>
<td>1</td>
<td>0</td>
<td>3</td>
<td>2</td>
</tr>
<tr>
<td>2</td>
<td>20</td>
<td>30</td>
<td>10</td>
</tr>
<tr>
<td>3</td>
<td>30</td>
<td>50</td>
<td>20</td>
</tr>
</tbody>
</table>
<h3 class="landing-page-h3">Step 2: Select the mode of analysis </h3>
<p class="landing-page-p"> The mode of analysis strongly depends on the experiment you performed. Here is a brief description and the major characteristics for each of the modes:
</p>
<p class="landing-page-p">
<b>Single Curve Analysis:</b> The 1:1 binding model is calculated individually for each of the supplied binding curves. This results in one kass, kdiss, KD ... value for each of the binding curves. The resulting values can only be rough estimations of the real binding kinetic values, since no range of Analyte concentrations were meassured. However, the analysis can be a good experimental starting point if one has no clue of the expected KD value.
</p>
<p class="landing-page-p">
<b>Single Cycle Kinetic:</b> This analysis method can be used, if multiple concentrations of analyte have been subsequentially meassured and the sensorgramm includes all binding steps one after another. Please be aware, that a binding step MUST consist of an association and a dissociation phase every time. The binding model is then applied to every supplied sensorgram in the data. All parameter (kass, kdiss) are calculated globally over all supplied bindingsteps. Rmax is fitted locally for every binding step. Hence, the user will receive one kass, kdiss and KD per binding curve and as many local Rmax values as there are binding steps (concentration steps) in the sensorgram.
</p>
<p class="landing-page-p">
<b>Multi Cycle Kinetic:</b> This analysis method can be used, if multiple sensorgrams have been recorded, each with a certain concentration of analyte. In this evaluation mode, all relevant parameter will be fitted globally. Please be aware, that every sensorgram MUST consist of an association and a dissociation phase every time.
</p>
<h3 class="landing-page-h3">Step 3: Supply all the additional information</h3>
<p class="landing-page-p">
All fields must be supplied in order to perfrom a binding curve analysis. in case of the Single Cycle kinetics (SCK) and Multi Cycle Kinetis multiple (MCK) values must be supplied per field. Please seperate the values using ",". Also have a look at the example data, if you are unsure on how to supply these information. It is important to point out, the the concentrations MUST be provided as "nM" and MUST be in the correct order. In case of the SCK the order is appearance in the binding curve. This is typically from small to large. In case of the MCK mode, the order is according to the appearance in your Dataset from left to right.
</p>
<h3 class="landing-page-h3">Step 4: Run anabel and Download the Results</h3>
<p class="landing-page-p">At this point, the data should be illustrated correctly. Next, hit the "Run Anabel" button. Thereafter, you can download an excel table that contains all relevant parameter and a pdf document containing the plot images together with the fits.</p>
</div>
</div>
</div>
<!-- Theoratical background -->
<div class="container-fluid mt-6 page-container">
<div id="theor_bg" class="row" style="margin:5%;">
<div class="col-sm-12">
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<h2 class="landing-page-h2">Theoretical background</h2>
<p class="landing-page-p"> In the following section we derive binding constants based on the assumption that a 1:1 binding occurs between a ligand L (on a surface) and an analyte A (in solution). </p>
<h3 class="landing-page-h3"> Determination of binding constants</h3>
<center>
<figure class="figure">
<img src="bc_phases.png" width="800" align="middle">
<figcaption class="figure-caption">Different regions of a binding experiment.</figcaption>
</figure>
</center>
<br><p class="landing-page-p"> The interaction of a ligand L with an analyte A, forming a ligand-analyte complex AL, can be described by the following chemical reaction <a href="#[1]">[1]–[3]</a>:</p>
<br>
<center>
<figure class="figure">
<img src="LA.png" width="400" align= "middle">
<figcaption class="figure-caption">Schematic chemical binding reaction of the analyte A to the ligand L.</figcaption>
</figure>
</center>
<br><p class="landing-page-p"> In this reaction, k<sub>ass</sub> and k<sub>diss</sub> are the rate constants for the association and dissociation of the analyte-ligand complex. A reaction with two molecules forming one complex is called a bimolecular reaction, whereas the disintegration of one complex into two molecules is a unimolecular reaction. The more AL complex is built, the higher are the concentrations of free molecules A and/or L and the higher is the rate constant of association k<sub>ass</sub> (1). In contrast, the higher the concentration of the complex (AL) and the higher the rate constant of dissociation k<sub>diss</sub> are, the more AL complex will decay into A and L (2). Therefore, the building rate of the analyte-ligand formation (d[AL]/dt) can be described by the following differential equation <a href="#[3]">[3]</a>:</p>
$$\frac{d[AL]}{dt}=k_{ass}\cdot{[A]}\cdot{[L]}$$
$$\frac{d([A]+[L])}{dt}=k_{diss}\cdot{[AL]}$$
$$\frac{d[AL]}{dt}=k_{ass}\cdot{[A]}\cdot{[L]}-k_{diss}\cdot{[AL]}$$
<p class="landing-page-p"> In the equilibrium state, equation (3) will become zero and can be rearranged to form the law of mass action equation for our biomolecular reaction. Subsequently, the association constant (K<sub>A</sub>) and the dissociation constant (K<sub>D</sub>), which is the reciprocal of K<sub>A</sub>, <a href="#[3]">[3]</a> can be described as follows:</p>
$$K_A=\frac{k_{ass}}{k_{diss}}=\frac{[AL]}{[A]\cdot{[L]}}=\frac{1}{K_D}$$
<p class="landing-page-p"> However, this model is only valid for a homogeneous system in which both the ligand and the analyte are in solution, are not limited by any transport processes or steric hindrances and react in a 1:1 manner. Yet, In many cases (e.g. SPR or BLI experiments), the ligand molecule is immobilised on a surface and the analyte is added at a certain concentraion in solution (Figure 3). With the assumption of a linear gradient of analyte, the velocity of the analyte-ligand complex formation on a solid surface can be described as equation (5). Details about the mathematical background and how to derive the equations can be obtained from <a href="#[1]">[1]–[3]</a>.
</p>
$$\frac{dΓ(t)}{dt}=k_{ass}\cdot{[A]}\cdot{(Γ_{max}-Γ(t))}-k_{diss}\cdot{Γ(t)}$$
<p class="landing-page-p"> The previous ligand concentration is replaced by the surface load capacity (Γ(t)). It describes the amount of analyte-ligand complexes per surface area at a given time point t. Hence, Γ<sub>max</sub> refers to the maximum amount of complexes per surface area unit. Subsequently, equation (3) can be rearranged to:
</p>
$$\frac{dΓ(t)}{dt}=k_{ass}\cdot{[A]}\cdot{Γ_{max}}-(k_{ass}\cdot{[A]}+k_{diss})\cdot{Γ(t)}$$
<p class="landing-page-p"> If the concentration of analyte [A] is kept constant during the experiment, this differential equation can be solved with the following function (mathematical derivation is shown in <a href="#[1]">[1], [2]</a>), which is refered to as binding model from here onward:
</p>
$$Γ(t)=Γ_{GG}-Γ_{GG}\cdot{e^{-k_{obs}\cdot{t}}}$$
<p class="landing-page-p"> With</p>
$$k_{obs}=k_{ass}\cdot{[A]}+k_{diss}$$
<p class="landing-page-p"> Γ<sub>GG</sub> describes the equilibrium surface load capacity where Γ(t) approximates to a constant value over time. Moreover, k<sub>obs</sub> represents the observable binding rate constant. Furthermore, k<sub>obs</sub> is dependent on the concentration of analyte [A] and the rate constants for association k<sub>ass</sub> and dissociation k<sub>diss</sub>.
</p>
<p class="landing-page-p"> This derived mathematical model can now be used for the fitting of the association and dissociation binding signals (Figure 1). Thereby, an observable rate constant k<sub>obs</sub> can be calculated for every exponential curve. The k<sub>obs</sub> value generally describes the curvature. In case of an association curve fit, k<sub>obs</sub> includes both the association and the dissociation rate constant (6). However, in case of a dissociation curve fit, the analyte concentration is zero. Neglecting avidity and rebinding effects, the mathematical equation can be simplified to (9):
</p>
$$k_{obs}=k_{diss}$$
<div class="further_reeds">
<h3 class = "landing-page-h3"> Further Reads </h3>
<p class="landing-page-p">
Please check out the <a href="http://www.biapages.nl/"> BIA-pages</a> or the <a href="https://www.sprpages.nl/"> SPR-pages</a> in case you want to understand more about the science of Biomolecular Interaction Analysis. Both websites are brilliant resources not only about the theory, but also about the practical experimentation. In addition to that, there is a fantastic community who will always help you out in case you have a question about your experiments or data!
</p>
</div>
<div class="references">
<h3 class = "landing-page-h3"> References </h3><ol>
<li id="[1]"><span>K. Laenge, "Application of flow injection analysis in label free binding assays." Universitaet Tuebingen, 2000.</span></li>
<li id="[2]"><span>M. Reichert and G. Gauglitz, "Affinitaetsreaktionen - Chemgapedia."[Online]. Available: http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/13/vlu/kinetik/affinitaet/affinitaetsre aktionen.vlu.html. [Accessed: 19-Oct-2015].</span></li>
<li id="[3]"><span>R. Karlsson, A. Michaelsson, and L. Mattsson, "Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system," J. Immunol. Methods, vol. 145, no. 1–2, pp. 229–240, Dec. 1991.</span></li>
<li id="[4]"><span>J. Berthier and P. Silberzan, Microfluidics for Biotechnology. 2010.</span></li>
</ol>
</div>
</div>
</div>
</div>
<!-- Publications -->
<div class="container-fluid mt-6 page-container-pub">
<div id="pubs" class="row" style="margin:5%;">
<div class="col-sm-12">
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<h2 class="landing-page-h2">Publications</h2>
<ul class="fa-ul">
<li class="landing-page-li" style="color:white;"><img src="Publication-paper-icon.svg" height = "40px" width = "40px" class = "float-left"></img>
<p><strong>KOFFI and Anabel 2.0—a new binding kinetics database and its integration in an open-source binding analysis software </strong><br>
Leo William Norval, Stefan Daniel Kraemer, Mingjie Gao, Tobias Herz, Jianyu Li, Christin Rath, Johannes Woehrle, Stefan Guenther, Günter Roth <br>
Database, Volume 2019, 2019, baz101. October 11, 2019. <br>
https://doi.org/10.1093/database/baz101</p>
</li>
<li class="landing-page-li" style="color:white;"><img src="Publication-paper-icon.svg" height = "40px" width = "40px" class = "float-left"></img>
<p><strong>Anabel: An Online Tool for the Real-Time Kinetic Analysis of Binding Events.</strong><br>
Stefan D Kraemer, Johannes Woehrle, Christin Rath, Günter Roth <br>
Bioinformatics and Biology Insights.<br>
January 9, 2019. https://doi.org/10.1177/1177932218821383</p>
</li>
</ul>
</div>
</div>
</div>
<!-- Download -->
<div class="container-fluid mt-6 page-container">
<div id="install" class="row" style="margin:5%;">
<div class="col-sm-12">
<!-- These are just text elements, with some different stylings -->
<h2 class="landing-page-h2">Install the package</h2>
<p class="landing-page-p">
ANABEL is available as an R package, <br>
to install ANABEL, start R (version "4.0") or newer and enter: <br>
<code>install.package("anabel") </code><br>
<strong>Documentation</strong><br>
To view documentation for the version of this package installed in your system, start R and enter:<br>
<code>browseVignettes("anabel")</code>
</p>
</div>
</div>
</div>