Comparison of Curve Fitting Models for Ligand Binding Assays

2004, 59, S177S181

J. A. Little
Department of Bioanalysis, Huntingdon Life Sciences Ltd., Woolley Road, Alconbury, Cambridgeshire PE28 4HS, UK; E-Mail: LittleJ@UKOrg.Huntingdon.com

Received: 5 September 2003 / Accepted: 18 November 2003 Online publication: 19 February 2004

Abstract
In the case of ligand binding assays the relationship between instrumental response and analyte concentration is non-linear, usually either hyperbolic or sigmoidal. As it is not possible to calibrate an assay at all the levels to be measured a suitable method of constructing a concentration-response relationship (the standard curve), based on a limited number of carefully spaced standards is required. The method should be robust and operator independent. The two main approaches that have been used involve empirical and mechanistic (theoretical) models. Empirical models utilise any mathematical function(s) that appears to have the characteristics of the experimentally derived assay data. Empirical models require no understanding of the principles of the assay. Mechanistic models make assumptions about the physico-chemical processes involved in the assay procedure. In practice either single function or spline models are used. The curve tting solution may be explicit (in the case of spline interpolation), or by least-squares curve tting regression methods, including polynomial and logistic equations, the latter involving many iterations. Examples of good curve tting selection are presented and contrasted with inappropriate models in a number of common assay formats.

Keywords
Immunoassays Ligand binding assays Comparison of curve tting Data processing

response, R) and ligand concentration (C) forms an inversely proportional rectangular hyperbola in the case of limited reagent assay (Fig. 1), and a directly proportional curve, reaching a plateau, in the case of immunometric methods (Fig. 2). In practice, ligand binding assays are calibrated with a set of carefully spaced standards, processed with each assay batch. The responses obtained are related to the standard concentrations by using a mathematical curve tting model and the sample concentrations determined by interpolation. These models have replaced the highly subjective practice of the analyst manually drawing the standard curve on graph paper. There are two types of curve-tting model: Mechanistic, based on assay theory Empirical, based purely on the measured responses

Mechanistic Models Introduction

The ligand binding assay is often the method of choice for the quantication of macromolecules. The most frequently
Presented at: 15th International Bioanalytical Forum. The Changing Role of Bioanalysis: Discovery to Market, Guildford, UK, July 14, 2003

used technique is the immunoassay which occurs in two main formats: the limited antibody immunoassay (e.g. radioimmunoassay; RIA) and the excess antibody immunometric assay (e.g. immunoenzymometric assay; IEMA). These are eectively two halves of the biphasic reaction of antibody with antigen (the ligand). The interaction (assay

Mechanistic models are based upon the Law of Mass Action. They take into account concentrations of reagents and binder anity for the ligand; assumptions usually made are that the reaction is at equilibrium, the interaction is univalent, no misclassication of bound and free fractions occurs, the binder reagent is uniform and there are no interfering components.

Original DOI: 10.1365/s10337-003-0182-8

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Point to Point (Interpolation) Methods

The simplest example is linear interpolation, in which straight lines are used to join the empirical data. In order to t a curve adequately, polynomial equations are used. There is an explicit solution to interpolation, made by calculus. There are two main types:

Spline
A single function equation cannot by itself t immunoassay standard curve data. Instead, sections of polynomial equations, usually cubic, are used to join adjacent points. The equations have the form: y a bx cx2 dx3 y = response x = concentration a, b, c and d are constants specic for each section of curve A danger of uncritical use of spline curve tting is that variable standard curve data can appear to give a good curve t with large nodal distortions going unnoticed, and in the extreme case gradient sign changes occurring.

Fig. 1. Immunoassay limited antibody reagent. Ag Antigen (analyte, ligand); Ab Antibody (binding reagent, xed limited concentration); Label Label for assay response generation

Smoothed Spline
A renement of the interpolated spline is smoothing, producing a monotonic single function equation over the full range of the standard curve. This is a mathematical smoothing process that in essence does not allow sharp changes in curve gradient and removes nodal distortions.

Fig. 2. Immunometric assay (2-site) excess antibody reagent. Ag Antigen (analyte, ligand); Ab Antibody (binding reagent, in excess relative to Ag); Label Label for assay response generation

An example of a single-site model to t RIA curves follows [1]: D p c:b q:R2 c:b 1 q p D:R c 0 p* = tracer concentration b =non-specic binding c =1/anity constant q = antibody concentration R =free/bound ratio D = analyte concentration The analyte concentration (D) for any sample can be derived from the response (R). Renements have been proposed including a multi-site model. In practice, however, mechanistic models can rarely be adequately dened or the assump-

tions fully met. As a result mechanistic models are rarely used and seldom appear in commercial data processing packages.

Regression Methods
A number of algorithms can be tted to standard curve data using regression analysis that are based on least mean squares in which the dierence between the observed and modelled response is minimised: X ^ y i y i 2 ^ y = the tted response y = the measured response The solution therefore has no explicit or exact solution, and is arrived at by an iter-

Empirical Models
The most appropriate approach to curve tting is to use a mathematical function that closely mimics the distribution of the standards measured as part of each assay batch. No appreciation of assay theory is required. There are two main categories of empirical curve tting methods: Point to point (interpolation) methods Regression methods

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ative process. Weighting, inversely proportional to the variance of the measured response may also be applied to the tting procedure so that imprecise data have reduced inuence.

Polynomial Regression
The constants a, b, c and d in the equation y a bx cx2 dx3 are obtained by the method of least squares and produces automatic smoothing (compare this with the smoothed spline). Often this approach will t observed data adequately, especially over limited concentration ranges.

Fig. 3. Interpolated Spline

Logistic Equations
Prior to computer calculation of data, manual plotting methods were developed that involved manipulation, or transformation, of the standard data, in an attempt to linearize the curve. The simplest reciprocation of the response data, developed into the Logit transformation: Logit Y Loge y =100 y y = response non specic response (this response may be normalised using the maximum response observed). A plot of logit Y against concentration yields a straight line over the central part of an immunoassay (limited antibody) curve. This approach has since been developed into the four parameter (4PL) logistic equation [2]: y a d =1 x=cb d y = response x = analyte concentration a = response at zero concentration b = slope factor c = ED50 (concentration at the true mid point of the curve) d = response at innite dose The logistic equation essentially describes an immunoassay standard curve (Fig. 1) in which a and d are the asymptotes, c is the mid point (corrected for non-specic binding) and b is the slope factor (a power function). The immunometric assay curve (Fig. 2) can be treated as an inverted rectangular hyperbola. In this case the lower section of the curve is usually a Original

Standard concentration Fitted duplicate concentration % dierence (duplicates)

37.6 37.6 37.6 0.00 0.00

75.20 67.71 150.0 )9.96 99.5

150.0 145.2 154.0 )3.22 2.65

301.0 302.8 299.2 0.61 )0.61

602.0 605.7 598.4 0.61 )0.61

1203 1239 1169 3.02 )2.80

2406 2304 2564 )4.24 6.59

Fig. 4. Cubic Regression

Standard concentration Fitted duplicate concentration % dierence (duplicates)

37.6 61.58 61.58 63.8 63.8

75.20 135.8 148.0 80.6 96.8

150.0 145.8 150.2 )2.80 0.10

301.0 300.0 296.3 )0.34 )1.57

602.0 600.0 591.5 )0.34 )1.74

1203 1672 1553 39.0 29.1

2406 2350 2360 )2.34 )1.91

straight line and the curve rised to a plateau. In order to accommodate this asymmetry, a fth adjustable parameter m is added, giving the 5PL logistic equation: y a d =1 x=cb m d

Goodness of Fit
Properly, curve-tting alternatives should be assessed during assay development. The goodness of t should be determined this requires a test of t, made up of two components:

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are considered acceptable whilst others are candidates for editing.

Selection of Curve Fitting Method

Which curve t should be used? Mechanistic models are rarely available and are usually inadequately dened - not recommended Interpolation methods - Spline methods may produce distorted data and apparently good ts of poor and imprecise data - generally not recommended Polynomial regression methods - may be adequate for limited concentration ranges Logistic Regression methods - robust, designed for the dynamics of ligand binding assays: i) 4PL logisitic recommended for limited reagent immunoassay (EIA, RIA) ii) 5PL logistic recommended for excess reagent immunoassay (IEMA, ELISA, IRMA)

Fig. 5. 4 Parameter Logistic

Standard concentration Fitted duplicate concentration % dierence (duplicates)

37.6 35.56 35.56 )5.43 )5.43

75.20 128.7 143.5 71.1 90.8

150.0 140.8 146.1 )6.10 2.62

301.0 308.7 305.0 2.57 1.33

602.0 595.0 587.1 )1.16 )2.48

1203 1645 1519 36.7 26.3

2406 2367 2379 )1.61 )1.143

Examples of Standard Curve Fitting Anomalies

An example immunometric standard curve with a defective standard point is shown tted by four commonly used methods (Fig. 36). This example curve is designed to illustrate the strengths and weaknesses of each method and demonstrate their fundamental characteristics. Spline (Fig. 3). A gradient sign change and nodes are evident that give the potential to produce ambiguous results whilst apparently tting all standards, including the second (defective) one. Cubic regression (Fig. 4). Reasonable tting for the central portion of the curve is obtained, missing the second highest and second lowest standard. 4 parameter logistic (Fig. 5). Second highest standard tted with > 20% error showing the inability to deal with curve asymmetry; the second lowest standard is not inuencing the curve t greatly and the rest of the curve is satisfactory. 5 parameter logisitic (Fig. 6). All standards apart from the defective second defective standard are tted correctly. Original

Fig. 6. 5 Parameter Logistic

Standard concentration Fitted duplicate concentration % dierence (duplicates)

37.6 35.56 35.56 )1.67 )1.67

75.20 128.7 143.5 62.1 82.3

150.0 140.8 146.1 )10.4 )6.84

301.0 308.7 305.0 4.13 2.81

602.0 595.0 587.1 )0.68 )1.87

1203 1645 1519 9.36 2.82

2406 2367 2379 )2.41 0.93

How far on average does the tted line deviate from the observed points (S1) The standard points replicate variance (S2) The ratio S1/S2 is the variance ratio; in general the lower this is, the better is the

t, judged for any one set of standard data. For this approach to be valid, the standard curve must be performed in high replication this is possible in assay development but not in routine usage, where a useful rule of thumb is that standards within 15% of the tted line

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Conclusions
In general the logistic methods are the most robust and least inuenced by anomalous standard data. The 5 PL method is able to cope with asymmetric curves. In practice, anomalies such as those shown in the example curves may

be slight and can go unnoticed by inexperienced analysts. When developing an immunoassay method attention should be given to the selection of the most appropriate and robust curve tting method for the type of immunoassay concerned in order to avoid future unnecessary analytical diculties.

References
1. Ekins RP, Newman B (1970) Acta Endocrinol (Suppl) 147:11 2. Rodbard D (1974) Clin Chem 20:1255

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