METHOD FOR DETERMINING THE PRESENCE OF SUBSTANCES OF DIAGNOSTIC RELEVANCE, IN PARTICULAR ANTIBODIES OR ANTIGENS, BY THE ELISA METHOD WITH PHOTOMETRIC EVALUATION

METHODE POUR DETECTER LA PRESENCE DE SUBSTANCES A SIGNIFICATION DIAGNOSTIQUE, PARTICULIEREMENT DES ANTICORPS OU DES ANTIGENES PAR LA METHODE ELISA AVEC EVALUATION PHOTOMETRIQUE

English Abstract

Abstract of the disclosure
A method for the detection and stepless quantification
of substances of diagnostic relevance is indicated, in
which a single sample mixture suffices for examination of
the sample. Assays which can be used are ELISA, RIA,
nucleic acid hybridization, nephelometry and other methods
of determination. Examples of substances of diagnostic
relevance which are detected are antigens, antibodies of
various immunoglobulin classes, nucleic acids or metabolic
products of medical importance. The extinction of a sample
in the selected assay dilution with colored, enzymatically
labeled antigen/antibody complexes is measured, and the
titer is calculated from the resulting signal AOD using
the formula:
Log titer = alpha . <IMG>
and can be set equal, with satisfactory accuracy, to the
final dilution titer obtainable by serial dilutions. The
values for alpha and beta are established experimentally
in separate series of tests.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for determining the presence of antibodies
or antigens, in assay fluids, with photometric
evaluation of the samples, prepared from the assay
fluids, with the aid of an enzymatically labelled
antigen or antibody (labelled species) in which
only one single assay dilution of the sample is
prepared, an antigen/antibody binding reaction is
initiated in this assay dilution, wherein one
reactant is immobilized on a solid surface, and
either the free or bound labelled species is
subjected to a color reaction brought about
enzymatically, in order to prepare a colored
solution, and a titer corresponding to the final
dilution titer is determined from the extinction
measured on this colored solution, using previously
measured reference data, which comprises the
extinction (or optical density) of the colored
solution being measured, and the titer being
calculated from the resulting signal AOD by the
following formula:
log titer = alpha . <IMG> (1)
and being set equal to the final dilution titer
obtainable by serial dilutions, with the "titer" in
the Formula (1) being the reciprocal of the final
dilution at which the signal AOD corresponds to the
limiting signal at the detection limit compared
with negative control samples, and the values for
- 14 -

alpha and beta at a fixed assay dilution being
determined experimentally, by a series of tests on
samples of known final dilution titer of the
analyte, separately for the particular combination
of immunologically reactive surface and enzyme-
labelled immunoglobulin or antigen as detector
under the same reaction and immobilization
conditions.
2. The method as claimed in claim 1, wherein the
signal AOD for the extinction or optical density
which is inserted in Formula (1) is the specific
color signal which has been obtained
a) by substraction of the extinction signal for a
control also measured in the ELISA or of the
extinction signal for a parallel mixture of
the test sample on a surface coated with a
control preparation, or of the extinction
signal as the start of a measurement of the
increase in color, from the extinction signal
of the test sample,
b) by multiplication of the extinction signal for
the test sample by a correction factor
resulting from a test control, which may be a
standard, which is also measured, or
c) after formation of the quotient or index from
the extinction signal for the test sample and
that for a test control, which may be a
negative sample, which is also measured.
-15-

3. The method as claimed in claim 1, wherein the assay
dilution used are sample dilutions in the range
from undiluted to 1:800, preferably about 1:150.
4. The method as claimed in claim 1, wherein the
values used for alpha are in the range from 3.0 to
3.5, and for beta are in the range from 0.10 to
0.27.
5. The method as claimed in claim 1, wherein the
values for alpha and beta are determined by series
of tests on a large number of assay fluids or
samples, by measuring the signal AOD at the desired
assay dilution of each of these samples and,
moreover, determining by endpoint dilution, the
titer, and then iterative fitting is used to find
from the resulting pairs of values (titer, AOD) for
the individual assay fluids or samples the values
of alpha and beta which are suitable for optimizing
the agreement between the titers (Tmeas) measured
by endpoint dilution and the titers (Tcalc)
calculated by insertion of the signal AOD in the
Formula (1).
6. The method as claimed in claim 5, wherein the pair
of values for alpha and beta at which the
particular quotient Tmeas/Tcalc for the individual
assay fluids or samples is most often in the range
0.8 to 1.25 is determined by iteration.
- 16 -

7. The method as claimed in claim 1, for determining
the presence of rubella (IgG; IgM) antibodies,
cytomegalovirus (IgG; IgM) antibodies or hepatitis
B surface antigen (HBsAg) in sera using
microtitration plates on which are immobilized,
respectively, rubella antigen, cytomegalovirus
antigen or antibodies against HBsAg.
8. The method as claimed in claim 7, wherein alkaline
phosphatase or peroxidase is used as indicator
enzyme.
- 17 -

Description

~28~7~3~3
~EHRI~G~ERKE AKTIENGES~LLSCHAFT 86/~ 0 39J ~ Ma 515
Dr. Ha/Li
A method for determining the presence of substances o-f
diagnostic relevance~ in particular antibodies or anti-
gens, by the ELISA method with photometric evaluation
_
The invention relates to a method for determining the
presence of substances of diagnostic relevance, in parti-
cular antibodies or antigens, in assay fluids, preferably
by the ELISA method with photometric evaluation of the
samples, prepared from the assay fluids, with colored,
enzymatically labeled antigen/antibody complexes, in which
only one single assay dilution of the sample is prepared,
an antigen/antibody binding reaction is initiated in this
assay dilution, with preferably one reactant beiny immobi-
lized on a solid surface, and the antigen/antibody com-
plex is subjected to a color reaction brought about enzy-
matically, in order to prepare a colored solution, and atiter corresponding to the final dilution titer is deter-
mined from the extinction measured on this colored solu-
tion, using prPviously measured reference data.
The invention is used for the detection and stepless quan-
tification of substances of diagnostic relevance, with a
single sample mixture sufficing for examination of the
sample. It is possible to use as assay for this the
enzyme-linked immunosorbent assay (ELISA), the radio-
immunoassay (RIA), nucleic acid hybridization, nephelo-
metry or other methods of determinatior,. Examples of sub-
stances of diagnostic relevance are regarded as being
antigens, antibodies of the various immunoglobulin classes,
nucleic acids or other metabolic products of medical im-
portance.
Two fundamental principles can be regarded as the bases
of the current methods of quantification of substances of
diagnostic relevance. ~n the one hand~ the substance

~8~799
- 2 ~
which is to be determined can be quantified via a refer-
ence curve which has previously been constructed from
samples with a known content of the substance. The alter-
native option comprises titration, using endpoint dilu-
tion of the substance which is to be determined. Thelatter route is always advisable if in the case of samples
which are to be assayed undiLuted or only slightly diluted
the abovementioned reference curve is difficult to con-
struct or can be used only unsatisfactorily. This is the
case in, for example, the determination of assay-specific
antibodies in the ELISA.
For this reason the method according to the invention is
also preferably used in this case, and is explained in
detail in this embodiment hereinafteru Thus, antibodies
are used as an example of substances of diagnostic rele-
vance, and the ELISA is used as an example of the assays
mentioned in the introduction. Accordingly, in the des-
cription and in the claims, a "specific coLor signal"
also means, analogously for other assays, for example
radioactive disintegrations (counts) or the relative scat-
tered light signal.
- The ELISA method is a ~nown enzyme immunoassay which is
used for the detection of antibodies or antigens in para-
sitic, bacterial or viral infections. The invention is
used to improve the evaluation of colored solutions which
are formed by bringing, for example, the serum which is
to be assayed for particular antibodies and is in a speci-
fic assay dilution into contact with antigens immobilized
on microtitration plates, followed by enzymatic detection
of the antibody/antigen complexes which have been produced
where appropriate. In particular, the invention relates
to the photometric evaluation of solutions of this type,
in which the extinction or optical density of the solution
which has been prepared in this way is measured, this
being a measure of the enzymatically labeled color-forming
immune complexes formed in the solution~ As a rule, a

~2~ 9
3 -
serum is assessed as pos;tive if the extinction of the
solution which has been formed as described exceeds a
def;ned limiting or threshold value. Discussiorls of this
type of evaluation are to be found in, for example, Immun.
In-fekt~ 9, 33-39, 1981.
However, ;t is desirable to be able not only to establish
whether the limiting value has been exceeded or not, but
also to gain quantitative information on the presence of
antibodies in the serum which is to be assayed. It is
known to carry out for this purpose what are known as
"serial dilutions", i.e. to carry out the ELISA with vari-
ous dilutions of the sample, and to measure the extinc-
tions or optical densities of the colored solutions resul-
ting in each case. Connection of the individual photo-
metric measurements in a graph results in what is calleda sample diLution curve for each serum sample (Fig. 1).
The sample dilution at ~hich the sample dilution curve
intersects the predefined limiting value indicates the
final dilution for the antibody which is to be determined,
2û this final dilution being the reciprocal of the antibody
titer (final dilution titer). Thus, the antibody is quan-
tified by stating the sample dilution at which the detec-
tion limit is reached.
The measured extinctions or optical densities are often
not used in the form of the directly measured figures
for the points on the sample dilution curve, usually a
correction of the measurements is carried out. The cor-
rection may take the form of subtraction of the measure-
ment for a suitable control (buffer~ negative serum) which
is also measured in the ELISA or for a mixture in paral-
lel to the serum which is to be determined, with a control
antigen immobilized on the microtitration plate, or of an
initial extinction when the kinetics of color formation
are being observed. Other possibilities are multiplica-
3; tion of the directly measured value by a correction fac-
tor which results in a suitable manner from a standard

9~3
-- 4
which is also measured, or the statement of a quotient or
;ndex ~h;ch results from division of the d;rectly measured
value by that for a negative sample wh;ch is also mea-
sured. Th;s corrected measurement is called the specific
color signal hereinafter and forms the basis for the state-
ment of the final d;lut;on titer.
The essential disadvantage of a quantitative evaluation
in the manner described is that the procedure is costly
in time and reagents. For this reason attempts have been
made to draw conclusions about the final dilution titer
of the serum from the extinction measured on a single
assay dilution in the ELISA. Thus~ for example, van Loon
and van der Veen have described in J. Clin. Pathol. 33,
635-639, 1980, the detection of toxoplasma antibodies
with the ELISA method using a single serum dilution in
conjunction with a reference curve. This entails the
extinction measured in the ELISA at a serum dilution of
1:800 being compared with the same extinction in the
reference curve which indicates the extinct;on found on a
series of sera in the assay dilution 1:800 as a function
of the final dilution titer. This re~erence curve was
constructed by plotting the means o-f the measurements on
a large number of sera. An improvement of this method is
described by van Loon et al. in ~. Clin. Pathol. 34, 198~,
665-669, in which the extinctions are replaced by the
specific color signals which are obtained by subtracting
the extinction of a negative control serum which has been
diluted 1:800 from the extinction of the assay serum. It
was possible in this ~ay to improve, at least in some
cases, the deviations in experimentally measured sera
from the reference curve. However, in other cases the
improvement was so slight that comparison with a negative
control serum was unnecessary.
Although a simplification for practical use had already
been achieved by this known method, namely that it is now
necessary to prepare only a single assay dilution,

~l28'7~9~
nevertheless this method has considerable disadvantages.
The assay dilution of l:800 which is suitable and recom-
mended for this method is very high, so that both the
sensitivity of the method becomes too lo~
and ~here is multiplicat;on of
pipetting errors~ In addition, in practice it is tiresome
to use a reference curve, or a computer is needed for auto-
matic evaluation of the measured data.
The object of the invention is to provide a method for
the quantitative determinat;on of antibodies or antigens,
for example in the ELISA, in ~hich it is possible to use
the smaLLest possible diLution of the sample ~hich is to
be investigated, and the titer can be determined in a
straightforward manner, for exampLe using a pocket calcu-
lator.
In addition, the intention ;s to ;nd;cate areas of use ofthe method according to the invention.
This object is achieved by a method of the type indicated
in the introduction in such a ~ay that She ext;nction (or
optical density) of the colored solution is measured~ and
the titer is caLcuLated from the resulting signal AoD by
the foLLo~;ng formula:
Log titer = alpha . AoDta ~1)
and is set equal to the final dilution titer obtainable
ZS by serial dilutions, with the "titer" in the Formula (1
being the reciprocal of the final dilution at which the
signal AoD corresponds to the limiting signal at the
detection limit compared with negative control samples,
and the values for alpha and beta at a fixed assay dilu-
tion being determined experimentally, by a series of testson samples of known final dilution titer of the analyte,
separately for the particular combination of immunologic-
ally reactive surface and enzyme-labeled immunoglobulin

~l2~ 39
-- 6
or antigen as detector under the same reaction and immo-
bilization conditions~
The signal AoD for the extinction which is preferably
inserted in the Formula (1) in the method ;s the specific
color signal defined above A
The assay dilutions which can be used are sample dilutions
in the range from undiluted to 1:800, preference being
given to an assay dilution of only 1:150. In the latter
case the assay dilution is stiLl sufficientLy low to
guarantee high sensitivity of the immunoassay method, and
on the other hand experiments have shown that it is pos-
sible with an assay dilution of 1:15û to obtain results
which can be reproduced relatively readily.
Lower assay dilutions are possible but require a high
degree of purity associated with high reactivity both of
the immunologically reactive surface, for example the
antigen-coated microtitration plate, and of the conjugate.
It has emerged that utilizable values for alpha are in
the range from 3.0 to 3.6 and for beta are in the range
ZO from 0.10 to 0.27~
Correct determination of these values alpha and beta forms
the basis for the advantageous use of the method according
to the invention in practice. Once optimal values for
alpha and beta have been found the evaluation~ i.e~ the
determination of the titer of the assay sample, involves
very simple calculation. The values for alpha and beta
can be determined, for example, by the reagent manufactu-
rer on the basis of a series of tests on a large number of
trial samples, entailing measurement of the specific color
signal at the desired assay dilution of these sera and,
in adcdition, determination of the titer in a manner known
per se by endpoint dilution. Then the pair of values
obtained for each sample is subjected to iterative fitting

377~
to find suitable values for alpha and beta -for which the
agreement between the titer Tmeas, measured by endpoint
dilution, and the t;ter TCalcr calculated From th~ formula
(1) after insert;on of the spec;f;c color signal, reaches
an optimum~ In an iterative procedure of th;s type, ~or
example arb;trarily assumed f;gures for alpha and beta
are used in;t;ally to calculate the relevant t;ter TCalc
from the specific color signal for each sample using For-
mula (1). In add;t;on, the t;ter TmeaS is determined by
endpoint dilution~
If the quotient TmeaS/Tcalc = 1.0, this would mean that
the correct titer has been der;ved from the measurement
of extinction at the assay dilution. Under realistic con-
ditions, a quotient between 0~8 and 1.25 must be regarded
as an "accurate hit on the titer". ~uotients below 0.5
and above 2.0 mean that the reproducibility limits per-
missible for a classical titration have been exceeded.
After the first test, which normally provides a calculated
titer prediction which is still poor, alpha and beta are
subsequently changed stepwise, and then the titer calcu-
lated for each serum sample is again compared with the
measured titer This iteration procedure is continued
until the agreement is satisfactory.
The tests have shown that it can be assumed that the opti-
mum has been reached when the following applies to about
50 % of the sera in a series of tests
0.~ < Tmeas/Tcalc < 1.25. (2)
This is based on experience with series of assays with
alkaline phosphatase as the indicator enzyme. If it is
possible to use peroxidase as the indicator enzyme, the
"proportion of hits" can be increased to about 65 %. How-
ever, these limits are within the range of variation of
the ELISA method itself~ and thus cannot be exceeded-by

~2~77~
-- 8 --
the procedure according to the invention for the quanti-
tat;ve evaluation of the results of measurement~
It has also emerged that the values of alpha and beta
depend not only on the nature of the diagnostic assay but
also on the production batches of the immunologicaLly re-
active solid phase and of the conjugate wh;ch are used.
Accordingly, for practical use of the method, the two con-
stants must be determined anew, for example, for each com-
bination of batches of assay plates and conjugate, but
they then allow investigation of samPles by serial measure-
ments with satisfactory reproducibility and very satis-
factory accuracy for practical use.
The invention is illustrated in detail by means of examples
and results of measurements hereinafter. This also en-
tails reference being made to the drawings which are
attached.
In the drawings,
Figure 1 shows serial dilutions carried out according to
the state of the art, in the form of sample dilu-
tion curves which show the specific color signal
as a function of the sample dilutions,
Figure 2 shows the dependence of the specific color sig-
nal at a sample dilution of 1:150 on the actual
titer determined by final dilution (TmeaS)~
Figure 3 shows the dependence of the titer calculated by
Formula (1) tTCalc) on the specific color signal
at a sample dilution of 1:150 on the same assay
reagents as in Figure 2, and
Figure 4 shows the measured titer TmeaS from Figure 2
compared with the calculated titer TCalc from
Figure 3 at the same specific color signal of

3 28~7799
the test samples diluted 1:150.
The method according to the invent;on was examined by
carrying out tests to determine antibodies aga;nst cyto-
megalov;rus ;n sera us;ng microt;trat;on plates on whose
walls cytomegalovirus antigen was ;mmob;l;zed, and to
determ;ne antibod;es aga;nst rubella ;n sera us;ng m;cro-
titration plates on whose wall rubella antigen was immo-
bili~ed. The test samples in the assay dilution 1:150
were reacted with the immobilized antigens9 the unbound
antibodies were washed out, 3 conjugate solution was used
to bind an enzyme to the anti~ody/antigen complexes which
had formed, the excess conjugate solution was washed out,
a chromogenic substrate solution was used to effect the
coloring via the enzyme, and then the react;on was termi-
nated with a stop solution. The samples which had beenprepared in this way, whose color depended on the content
of virus-specific antibodies~ were then evaluated by
determination of their optical density or extinction AOD
by photometry (at a defined wavelength).
In ser;al dilutions, the final dilution titer TmeaS for
the same samples was determined in a manner known per se
and was then compared with the titer TC3lC calculated
using the Formula (1) according to the invention.
To improve the reproducibility and accuracy of the method,
in alL cases the specific extinction or the "specific
color signal AoD" was used.
Figure 1 shows a graph of the specific color signal AoD
as a function of various samPle dilutions (sample dilu-
tions 1) for three positive sera A, B and C and for a
3û negative serum D. Parallel lines can be obtained for the
plots of the examples only in the higher assay ~;lution
ranges, for example 1:~00. This is the basis for the
construction of a suitable reference curve as specified
by van Loon.

~l2~77~9
- 10 --
Whereas the deviation downwards of the sampLe dilution
curve for a serum with a high antibody titer (Example A)
represents the prozone effect known from the literature,
it is not entirely clear why the dilution curves are
flattened in the lo~er region, in the neighborhood of the
final dilution titer.
It has now been found that the final dilution titer deter-
mined by seriaL dilution of a wide variety of samples is
related to the extinction measured at an assay dilution
lD of, for exampLe, 1:150 as follows:
log titer = alpha . AoGeta (1)
with the constants alpha and beta allowing adjustment
for the sample and assay conditions.
This fact is evident from Figures 2 to 4. Figure 2 is a
plot of the dependence of the specific color signal AOD
of 58 sera in a dilution of 1:150 as a function of the
titer Tmeas determined by final dilution. On the other
hand, Figure 3 shows the titer calculated using Formula
(1) from the extinction AoD at the optimal values for
alpha and beta. The lo~ degree of scatter of this curve
results from the mathematical construct;on.
Figure 4 now shows clearly that the curve o-f the titers
calculated by Formula (1) is an excellent average of
measurements. Thus, it is ev;dent from this that an opti-
mal prediction of the final dilution titer can be obtainedwith the method of the invention.
This good agreement between Tcalc and Tmeas is~ however~
only obtained when care is taken about the determination
of the values for alpha and beta.
The procedure for determination and optimization of alpha
and beta is now explained by the use of the following

~3779~
table of measured and calculated data from a serles of
tests on 60 sera as an example.
TABLE 1
specific T / T
co,lor meas ca~c
slgrlal neas calc ~o~ler in the rancJe greater
at, 1:150 than 0.8 0,8-1.25 ,than 1.25
0.3611: 466 1: 610 0.763
0.6061: 1372 1: 1285 1.068
0.6601: 1247 1: 1464 0.852
0.7681: 1862 1: 1857 1.003
0.8051: 1225 1: 2002 0.612
0.8381: 1985 1: 2137 0.929
0.8431: 1446 1: 2157 0.67
0.8651: 2058 1: 2250 0.915
0.8791: 1348 1: 2310 0.584
0.8861: 2298 1: 2340 0.982
0.9281: 1730 1: 2526 0.685
0.9581: 2264 1: 2664 0.85
0.96~1: 2166 1: 2710 0.799
, 0.9991: 2622 1: 285~ 0.918
1.0411: 3185 1: 3064 1.039
1.0811: 1911 1: 3268 0.585
- 1.1661: 2230 1: 3725 0.599
1.1841: 4410 1: 3825 1.153
1.1851: 4165 1: 3831 1.087
1.2021: 1127 1: 3928 0.287
1.2331: 6096 1: 4108 1.484
1.2551: 6321 1: 4238 1.492
1.2661: 4459 1: ~304 1.036
1.2701: 6591 1: 4328 1.523
1.2811: 5317 1: 4395 1.21
1.3051: 2842 1: 4543 0.626
1.3121: 4802 1: 4586 1.047
1.3211: 5390 1: 4643 1.161
1.3481: 3700 1: qB14 0.769

77~9
- 12 -
specific Tmeas / TCalC~
signal Tmea~ calc lower ;n the range greater
a~ 1:150 than 0.8 0.8-1,25 thanl. 25
1.349 1: 3112 1: 4820 0.646
1.354 1: ~753 1: 4853 0.979
1.363 1: 5537 1: 4911 1.128
1.364 1: 6223 1: 4917 1.266
1.429 1: ~076 1: 5349 1.136
1.470 1: 4116 1: 5633 0.731
1.471 1:106B2 1: 5640 1.894
1.476 1: 7473 1: 5675 1.317
1.477 1: 11172 1: 5682 1.966
1.486 1: 5341 1: 5746 0.93
1.496 1: 7718 1: 5817 1.327
1.496 1: 8232 1: 5817 1.415
1.515 1: ~983 1: 5953 1.173
1.537 1:10633 1: 6114 1.739
1.586 1: 9212 1: 6480 1.422
1.616 1: 7473 1: 6710 1.114
1.632 1:10266 1: 6835 1.502
1.674 1: 8134 1: 7168 1.135
1.684 1: 6689 1: 7249 0.923
1.727 1: 6101 1: 7603 0.802
1.747 1:20355 1: 77~0 2.62
1.780 1: 5635 1: B052 0.7
1.793 1: 10633 1: 8164 1.302
1.846 1: 7032 1: 8633 0.815
~0 1.898 1: 9408 1: 9107 1.033
1.901 1: 11074 1: 9135 1.212
1.937 1:14945 1: 9473 1.578
1.980 1: 18718 1: 9886 1.893
2.012 1: 21805 1: 10200 2.138
2.211 1: 25725 1: 12289 2.093
2.224 1: 18375 1: 12433 1.478

~7~99
- 13 -
Column 1 of the table shows measurements of the specific
color signal on samples o-f 60 sera ;n an assay dilution
of 1:150. Column 2 shows the titer Tmeas measured by
final dilution series for the same sera. CollJmn 3 shows,
S for the same sera ;n each case, the t;ter TCa~c calculated
from Formula (1) from the color signal in column 1 using
the values alpha = 3.4514 and beta = 0.2106 found after
optimi~ation.
Using the modern calculators now available it is no great
effort for an expert to draw up a program for the itera-
tive procedure used to optimize alpha and beta for parti-
cular reagent combinations.
The quotient Tmeas/Tcalc is then shown, being divided
into "too lo~" tlower than 0.8), 'lapproximately equal to 1"
(in the range 0.8-1.25) and "too high" (greater than 1.25~.
It is evident in the case which is shown that a good
approximation has been achieved for 45 % of the sera.
Taking into account the range of variation of the final
dilution titers, which is evident from Figure 2, the
choice of the values for alpha and beta can thus be
regarded as very satisfactory for practical use.