ANALYTICAL SCIENCES FEBRUARY 2005, VOL.

21 167
2005 © The Japan Society for Analytical Chemistry

Detection Limit Estimated from Slope of Calibration Curve:

An Application to Competitive ELISA
Yuzuru HAYASHI,*† Rieko MATSUDA,* Katsutoshi ITO,** Waka NISHIMURA,** Kazuhiro IMAI,***
and Masako MAEDA*

*National Institute of Health Sciences, 1-18-1 Kami-Yoga, Setagaya, Tokyo 153–8501, Japan
**School of Pharmaceutical Sciences, Showa University, Tokyo 142–8555, Japan
***Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo 202–8585, Japan

This paper theoretically derives a general rule that while the slope of the semi-logarithmic plot (Y vs. log X) of a
calibration curve varies depending on analyte concentration, X, the slope takes a specific value at the detection limit (LD).
This rule holds good irrespective of the shape of the calibration curve (linear or non-linear) and in this paper, is applied to
competitive ELISA (enzyme linked immunosorbent assay). The following relationship is deduced: slope of log-dose B/B0
at LD = [relative standard deviation (RSD) of blank responses] ÷ 0.13. The LD obtained from the above-mentioned slope
corresponds to the dose at which the RSD of dose estimates is 0.3 (= 30%). A commercial kit for 17α-hydroxyprogesterone
is taken as an example.

(Received September 9, 2004; Accepted October 27, 2004)

OHP), donated from Eiken Chemical Co., included the second

Introduction
The precision and detection limit are of great importance in
many disciplines of analytical chemistry, including ELISA as
well. The detection limit, LD, has been defined by international
organizations such as ISO, IUPAC and ICH. An example is:1
antibody coated on microplates, 17-OHP labelled with
horseradish peroxidase, rabbit anti-17-OHP antiserum, etc.
Solutions of standard 17-OHP, labeled 17-OHP and antiserum
were pipetted into a well of a microplate and incubated for 3 or
20 h. After the addition of the chromogen solution and stop
solution, the absorbance was measured at 490 nm.

sY
LD = 3.3 — (1)
a Theory
where sY denotes the standard deviation (SD) of responses, Y, Taking into account the differential, dY/dX, of the calibration
for blanks or around expected LD and a is the slope of a linear curve, we can change the equation of the LD definition (Eq. (1))
calibration line. as:
The above definition (Eq. (1)) is restricted within the cases of
linear calibration (a = constant). In competitive immunoassays, sY sY sY X
however, the calibration curves are curvilinear (a = variable) LD = 3.3 × = 3.3 × = 3.3 × (2)
dY dY 1 dY
and the definition is not useful without modification. While dX  d ln X  X  d ln X 
ancillary measures for LD, e.g., the dose when B/B0 = 80%
intercept, have been utilized,2 statistical approaches have also where the absolute value of the differential is used in case the
been published.2–5 slope is negative. Since the concentration, X, is restricted at LD,
A recent paper has proposed that LD is the dose at which the X is equal to LD. Therefore, the unknown variables, X and LD,
RSD of dose estimates, X, is 30%.6 This proposal is based on can be eliminated from Eq. (2) as:
the relationship: (sY/a)LD = 1/3.3 = 30%. However, a thorough
survey of uncertainty over a wide range is inevitable to find the dY
dose of 30% RSD. This paper derives an alternative method  d ln X  = 3.3sY (3)
which retains the probabilistic aspect but dispenses with the
wide-range survey. During the process of the derivation, a For practical purposes, we convert the natural logarithm into the
general rule for calibration curves at detection limit is developed. common logarithm, using the well-known relationship, ln X =
2.303log X:

Materials and Methods dY s

d log X = 0.13
Y
(4)
The details of experiments were described in a previous paper.6
A competitive ELISA kit for 17α-hydroxyprogesterone (17- where 1/(3.3 × 2.303) ≈ 0.13. The unknown quantity |dY/d log
X| can simply be given by Eq. (4).
† To whom correspondence should be addressed. Equation (4) is a general rule for calibration curves at
168 ANALYTICAL SCIENCES FEBRUARY 2005, VOL. 21

Equation (7) can be written as:

dB/B ρM
d log X0 = 0.13 (9)

Equation (9) is the specific equation for competitive ELISA.

By a similar derivation, we can obtain the relationship
between the RSD, ρX, of dose estimates and the slope of a semi-
logarithmic B/B0 curve. The equations for the total conversion
from the response SD, sY, to ρX can be found in the literature:7

sx sY sY
ρx = =
dY
=
dY
(10)
X X dX   d ln X 

where sX denotes the SD of dose estimates. Noticing Eqs. (5),

(6) and (8), we can write the fourth equation of Eq. (10) as:

ρM
ρx = (11)
d(B/B0)
 d(ln X) 
Changing the natural logarithm into the common logarithm, we
can obtain:

ρM
ρx = 2.303 × (12)
d(B/B0)
 d(log X) 
This equation is used as an approximate precision profile (see
Fig. 1 B/B0, differentials of B/B0, RSD of dose estimates in the Figs. 1C and 1C′). At LD, ρX = 30% and Eq. (12) becomes equal
competitive ELISA with incubation periods of 20 h (A – D) and 3 h to Eq. (9).
(A′ – D′). A and A′: B/B0; B and B′: differentials of B/B0; C and C′:
RSD of dose estimates (m from Eq. (12): s from experiments (n =
8)); D and D′: RSD of dose estimates (m from Eq. (14) of Ref. 6;
s from experiments (n = 8)). The experimental RSD values (s) in C
Results
and D are the same and so are those in C′ and D′.
Figures 1A – 1D show the results from the competitive ELISA
for 17α-hydroxyprogesterone with 20 h incubation. The line (–)
of Fig. 1D displays the precision profile based on the method of
detection limit. An application to competitive ELISA is given the previous paper.6 We can easily spot the LD (= 0.007 µg/L)
below. In competitive ELISA, the calibration curve is from the precision profile at RSD of 30%.6
expressed as the four-parameter logistic function,2,7–9 and its As shown in Figs. 1A and 1B, the slope of the calibration
standard form is known as B/B0: curve varies depending on concentration. The slope is near zero
at both the edges, but takes the maximum value at the inflection
Y= C0 – C3 + C point. The absolute value is plotted in Fig. 1B.
3 (5)
1 +  X 
C 1
As shown in Fig. 1B and also by the previous paper,6 the
C2 wide-range survey of uncertainty is inevitable to find LD.
However, the differential method based on Eq. (9) can dispense
1 with the survey, while retaining the stochastic aspect (30%
B/B0 = (6)
1 +  X 
C1 RSD). The most significant practical advantage of the
C2 differential method is the minimum demand for the detection
limit: that is, the slope of the log-dose B/B0 curve and RSD of
where C0, C1, C2 and C3 are coefficients. Noticing that dY = (C0 blank responses.
– C3) dB/B0, we can write Eq. (4) as: In the ELISA examined here, the major error source at blank
and low doses around LD was identified as the pipetting of the
dB/B sY/(C0 – C3) viscous antiserum solution (RSD = 0.019).6 Therefore, ρM of
d log X0 = 0.13
(7)
Eq. (9) can be substituted for by the pipetting RSD and the
slope of the semi-log B/B0 curve at LD is calculated as 0.15 (=
Coefficient, C0, denotes the largest response for the blank 0.019 ÷ 0.13). From Fig. 1B, we can see that the concentration
sample and C3 the smallest one at the infinite concentration (see at which |d(B/B0)/d(log X)| = 0.15 is exactly the same as the LD
Eq. (5)). Therefore, sY/(C0 – C3) is approximately equal to obtained from Fig. 1D (= 0.007 µg/L).
sY/C0, meaning the RSD of blank responses. Let ρM be defined The experimental RSD (s of Figs. 1C and 1D) is calculated
as: from the eight dose estimates which are transformed from the
respective absorbance responses by the calibration curve. As
sY sY demonstrated previously,6 the line (m) of Fig. 1D is in excellent
ρM = ——— ≈ — (8)
C0 – C3 C0 agreement with the experimental RSD in a wide concentration
ANALYTICAL SCIENCES FEBRUARY 2005, VOL. 21
range. On the other hand, the profile of Fig. 1C (Eq. (12)) is
satisfactory only at low doses (< 0.1 µg/L). This fact originates
from the assumption taken during the derivation of Eq. (12).
That is, ρM is fixed at the RSD of blank responses (ρM is
constant). We should note that the precision profiles (m) are
not the least squares fittings to the real data (s).
Figures 1A′ – 1D′ show the results from the same experiments
except for the incubation time (3 h). According to Eq. (9), the
slope at LD for the 3 h incubation is the same as that for the 20 h
incubation, since the major error source, ρM, at blank and low
doses is common to both the experiments. However, the
different calibration curves lead to the different LD values. The
contact point of the tangent with the slope (= 0.15) corresponds
to the LD (= 0.028 µg/L) (see Fig. 1B′). The incubation periods
affect the calibration curves (and also B/B0) and consequently
change the precision profiles.
Discussion
This paper describes a general rule for calibration curves at
detection limit and its application to competitive ELISA.
In case of linear calibration (Y = aX + b), the slope is constant
irrespective of analyte concentration, X. Given the response
SD, sY, and slope, a, the detection limit, LD, can be calculated
from the right side of Eq. (1). However, if the calibration curve,
Y = f(X), is non-linear, the detection limit cannot be estimated
simply from Eq. (1). The slope, a (= dY/dX), is variable and its
specific value at X = LD cannot easily be known, unless LD is
determined. That is, the definition (Eq. (1)) involves two
unknown quantities (LD and a). Of course, if the differential
coefficient, dY/dX, is expressed mathematically as a function of
X, Eq. (1) can be solved straightforwardly. In competitive
ELISA, we can imagine that it is not easy to solve the equation,
169
df(X)
X = 3.3sY  dX , for X (see Eq. (5)). Alternatively, this paper
has given a smart answer.
Competitive ELISA is a good example for the general rule
(Eq. (4)). The calibration curve is non-linear and differentiable.
A computer is helpful to calculate the differential coefficients of
the semi-logarithmic calibration curve over a wide range of
concentration, X, and to spot the detection limit at which the
differential coefficient takes the specific value.
Acknowledgements
This work was supported in part by a grant from Japan Health
Sciences Foundation.
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