AU602170B2 - A method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the ELISA method with photometric evaluation - Google Patents

Description

COMMONWVEALTH OF AUSTRALIA PATENTS ACT 195269 COMPLETE SPECIFICATION (OR IGINAL) Class Application Number: Lodged: Complete Specification Lodged: Accepted: o Published: PrioFity: I nt. Class $I Rcla'ted Art: This document contaiins tli.d amendments imade vader Section 49 arid is coricct for priiiting.I 4% rimme of Applicant: Address of Applicant: 4C f C Actual Inventor: Address for Service: BEHRINGWERKE AKTIENGESELLSCH-AFTI D-3550 Marburg, Federal Republic of Germany HANS-DETLEF DOPATKA EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Coi~lete Specification for the invention entitled: A METHOD FOR DETERMINING THE PRESENCE OF SUBSTANCES OF DIAGNOSTIC RELEVANCE, IN PARTICULAR ANTIBODIES OR ANTIGENS, BY THE ELISA METHOD WITH PHOTOMETRIC EVALUATION Thie following statement Is a full description of this Invention, Includ~ng the best method of performing It known to Us 1.

BEHRINGWERKE AKTIENGESELLSCHAFT 86/9 0 39J Ma 515 Dr. Ha/Li A method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the ELISA method with photometric evaluation The invention relates to a method for determining the presence of substances of diagnostic relevance, in particular 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 being immobilized on a solid surface, and the antigen/antibody complex is subjected to a color reaction brought about enzymatically, in order to prepare a colored solution, and a "c titer corresponding to the final dilution titer is determined from the extinction measured on this colored solution, using previously measured reference data.

tc The invention is used for the detection and stepless quantificatio.; of substances of diagnost c relevance, with a single sample mixture sufficing for examination of the sample. It is possible to use as assay for this the enzyme-'inked immunosorbent assay (ELISA), the radioimmunoassay (RIA), nucleic acid hybridization, nephelometry or other methods of determination. Examples of substances of diagnostic relevance are regarded as being antigens, antibodies of the various immunoglobulin classes, nucleic acids or other metabolic products of medical importance.

Two fundamental principles can be regarded as the bases of the current methods of quantification of substances of diagnostic relevance. On the one hand, the substance m.L I I 2 which is to be determined can be quantified via a reference curve which has previously been constructed from samples with a known content of the substance. The alternative option comprises titration, using endpoint dilution of the substance which is to be determined. The Latter 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 construct or can be used only unsatisfactorily. This is the case in, for ,xample, 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 hereinafter. Thus, antibodies are used as an example of substances of diagnostic relevance, and the ELISA is used as an example of the assays mentioned in the introduction. Accordingly, in the description and in the claims, a "specific color signal" also means, analogously for other assays, for example radioactive disintegrations (counts) or the relative scattered light signal.

The ELISA method is a known enzyme immunoassay which is used for the detection of antibodies or antigens in parasitic, 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 specific 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 ~~~rnnn A 3 serum is assessed as positive if the extinction of the solution which has been formed as described exceeds a defined limiting or threshold value. Discussions of this j type of evaluation are to be found in, for example, Immun.

Infekt. 9, 33-39, 1981.

However, it 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 i '"serial dilutions", i.e. to carry out the ELISA with vari- S t ous dilutions of the sample, ard to measure the extinctions or optical densities of the colored solutions resul- I o ting in each case. Connection of the individual photoeo2c 15 metric measurements in a graph results in what is called a a sample dilution curve for each serum sample (Fig. 13.

The sample dilution at which the sample dilution curve intersects the predefined limiting value indicates the e final dilution for the antibody which is to be determined, o o 20 this final dilution being the reciprocal of the antibody titer (final dilution titer). Thus, the antibody is quan- Soo tified by stating the sample dilution at which the detec- 0 tion limit is reached.

i 0 0 o 0 The measured extinctions or optical densities are often 00 0 a 25 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 correction may take the form of subtraction of the measure- Sment for a suitable control (buffer, negative serum) which is also measured in the ELISA or for a mixture in parallel to the serum which is to be determined, with a control antigen immobilized on the microtitration p{ ate, or of an inita- l extinction when the kinetics of color formation are being observed. Other possibilities are multiplication of the directly measured value by a correction factor which results in a suitable manner from a s;ndard I which is aLso meas index which result value by that for sured. This corre 4 ured, or the statement of a quotient or s from division of the directly measured a negative sample which is also meacted measurement is called the specific nafter and forms the basis for the statedilution titer.

color ment o signal hereii ,f the final 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 extinction found on a series of sera in the assay dilution 1:800 as a function of the final dilution titer. This reference curve was constructed by plotting the means of the measurements on a large number of sera. An improvement of this method is described by van Loon et al. in J. Clin. Pathol. 34, 1981, 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 way 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, nevertheless this method has considerable disadantages.

The assay dilution of 1:800 which is suitable and recommended for this method is very high, so that both the sensitivity of the method becomes too Low (see the weakly positive serum in Figure 1) and there is multiplication of Spipetting errors. In addition, in practice it is tiresome to use a reference curve, or a computer is needed for automatic evaluation of the measured data.

The object of the invention is to provide a method for the quantitative determination of antibodies or antigens, for example in the ELISA, in which it is possible to use o the smallest possible dilution of the sample which is to be investigated, and the titer can be determined in a straightforward manner, for example using a pocket calcuti 15 lator.

In addition, the intention is to indicate areas of use of the method according to the invention.

This object is achieved by a method of the type indicated in the introduction in such a way that the extinction (or optical density) of the colored solution is measured, and the titer is calculated from the resulting signal AOD by the following formula: beta log titer alpha A (1) and is set equal to the final dilution titer obtainable 25 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 dilution being determined experimentally, by a series of tests on samples of known final dilution titer of the analyte, separately for the particular mbination of immunologically reactive surface and enzyme labeled immunoglobulin 1cL 6 or antigen as detector under the same reaction and immobilizationr conditions.

The signal AOD for the extinction which is preferably inserteed in the Formula in the method is the specific color iignal defined above.

The as;say 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 possible with an assay dilution of 1:150 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 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 manufacturer 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 addition, 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 7 to find suitable values for alpha and beta for which the agreement between the titer Tmeas, measured by endpoint dilution, and the titer Tcalc, calculated from the FormuLa after insertion of the specific color signal, reaches an optimum. In an iterative procedure of this type, -or example arbitrarily assumed figures for alpha and beta Sare used initially to calculate the relevant titer Tcalc Sfrom the specific color signal for each sample using Formula In addition, the titer Tmeas is determined by endpoint dilution.

If the quotient Tmea/Tcalc 1.0, this would mean that 0000 the correct titer has been derived from the measurement 0°0 e o of extinction at the assay dilution. Under realistic con- 00oo00 o° o ditions, a quotient between 0.8 and 1.25 must be regarded 0o 15 as an "accurate hit on the titer". Quotients below 000 o o and above 2.0 mean that the reproducibility limits permissible for a classical titration have been exceeded.

0 o After the first test, which normally provides a calculated titer prediction which is still poor, alpha and beta are 0°°O° subsequently changed stepwise, and then the titer calcul o' lated for each serum sample is again compared with the I measured titer. This iteration procedure is continued until the agreement is satisfactory.

The tests have shown that it can be assumed that the optimum has been reached when the following applies to about of the sera in a series of tests 0.8 Tmeas/Tcalc I 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 However, these limits are within the range of variation of the ELISA method itself, and thus cannot be exceeded by

I

8 the procedure according to the invention for the quantitative 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 reactive solid phase and of the conjugate which are used.

Accordingly, for practical use of the method, the two constants must be determined anew, for example, for each combination of batches of assay plates and conjugate, but they then allow investigation of samples by serial measurements with satisfactory reproducibility and very satisfactory accuracy for practical use.

The invention is illustrated in detail by means of examples and results of measurements hereinafter. This also entails reference being made to the drawings which are attached.

In the drawings, Figure 1 Figure 2 shows serial dilutions carried out according to the state of the art, in the form of sample dilution curves which show the specific color signal as a function of the sample dilutions, shows the dependence of the specific color signal at a sample dilution of 1:150 on the actual titer determined by final dilution (Tmeas), shows the dependence of the titer calculated by Formula (Tcalc) on the specific color signal at a sample dilution of 1:150 on the same assay reagents as in Figure 2, and shows the measured titer Tmeas from Figure 2 compared with the calculated titer Tcalc from Figure 3 at the same specific color signal of Figure 3 Figure 4 9 the test samples diluted 1:150.

The method according to the invention was examined by carrying out tests to determine antibodies against cytomegalovirts in sera using microtitration plates on whose Walls cytomegalovirus antigen was immobilized, and to j determine antibodies against rubella in sera using microi titration plates on whose wall rubella antigen was immobilized. The test samples in the assay dilution 1:150 j were reacted with the immobilized antigens, the unbound antibodies were washed out, a conjugate solution was used to bind an enzyme to the antibody/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 reaction was terminated with a stop solution. The samples which had been prepared 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 serial dilutions, the final dilution titer Tmeas for i the same samples was determined in a manner known per se and was then compared with the titer Tcalc calculated using the Formula according to the invention.

To improve the reproducibility and accuracy of the method, in all cases the specific extinction or the "specific Scolor signal AOD" was used.

IF Figure 1 shows a graph of the specific color signal AOD as a function of various sample dilutions (sample dilutions for three positive sera A, B and C and for a negative serum D. Parallel lines can be obtained for the plots of the examples only in the higher assay dilution ranges, for example 1:800. This is the basis for the construction of a suitable reference curve as specified by van Loon.

I l I 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 Lower region, in the neighborhood of the final dilution titer.

It has now been found that the final dilution titer determined by serial dilution of a wide variety of samples is related to the extinction measured at an assay dilution of, for example, 1:150 as follows: L Log titer alpha beta (1) «e0D f 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 t 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 ;alculated using Formula f ro the extinction AOD at the optimal values for aLpha rnd beta. The low degree of scatter of this curve results from the mathematical construction.

Figure 4 now shows clearly that the curve of the titers calculated by Formula is an excellent average of measurements. Thus, it is evident from this that an optimal prediction of the final dilution titer can be obtained with the method of the invnt ion.

This good agreement between Tcact 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 r 11 table of measured and calculated data from a series of tests on 60 sera as an exampe.

TABLE 1 specif i c color signal at 1:150 T T meas caie T T meas cate Lower in the range greater than 0.8 0.8-1.25 than 1.29

I

I

0,361 0.606 0.660 0.768 0,805 04838 0,843 0, 865 0,879 0,886 0,928 01958 0,968 04999 1.041 20 1.081 1,166 14184 L 185 1.202 1,233 1.255 1,266 3,270 1.281 1.305 1.312 1,321 1348O 466 1372 1247 1862 1225 1985 1446 2058 1348 2298 1730 2264 2166 2622 3185 1911 2200 4410 4165 1127 6096 6321 4459 6591 5317 2842 4802 5390 3700 1: 610 1t 1285 1: 1464 1 1857 1: 2002 11 2137 1i 2157 1 2250 i1 2310 11 2340 11 2526 1: 2664 1; 2710 1; 288 1: 3064 10 3268 i 3725 i; 3825 1: 3831 It 3928 1s 4108 1 4238 1i 4304 it 4a20 ii 4395 Is 4543 it 4586 1i 4643 15 4814 0.632 0.67 0.584 0 685 0.799 0,585 0.599 0.287 0.763 1.068 0.852 1.003 0.929 0.915 0.982 0.85 0,918 1,039 1,13 I..087 1.484 1.492 1.s523 1.036 1,461 1.161

K

Ii Q LaSpecific 12 signal at 1:150 T m T caLc T /IT rneas, caLd.

Lower in the rane groate: than 0 .8 0.8-1,25 than 1.-25 1.349 1.354 1.363 1.364 1.429 1.470 1.471 1.476 1.477 1.486 1.496 1.496 1.515 1.537 1.586 1.616 1.632 1 .6 74 1.684 1.727 1.747 1.780 *1 .9 3 1.846 1.898 1.19 01 1.937 1.980 2, 012 2.211 2. 24 1 3112 1 4753 5537 1 6223 1 6076 1: 4116 1:10 68 2 1; 7473 1:11117 2 1: 53 43, 1: 7 718 1: 8 2 32 1: 6 983 1 10 633 1 92,12 1 74 73 1:10 26 6 1: 8 134 1: 6689 1: 610 1 1 :2 0355 1: 563 5 1 :10 633 1: 7 032 1: 9 40 8 1 :11074 1 14 94 5 1 18 718 1 :218 05 1 257 25 1 :18 375 1: 4820 1: 4853 1: 4911 1: 4917 1: 5349 1: 5633 1: 5640 1: 5675 1 5682 1 5746 1 5817 1 5817 1 5953 1: 6114 1: 6480 1: 6710 1: 6835 1: 7168 1: 7249 1: 7603 1: 7770 1: 8052 1: 8164 1: 8633 1: 9107 1: 9135 1 9473 1 9886 1 10 200 1 :12 28 9 1 :12433 0.646 0.979 1.128 1.136 1.266 0.731 0.93 1.894 1.317 1.966 1.327 1.415 1.739 1.422 1.502 1.173 1.114 1. 1,35 0.923 0.802 0.7 2.62 1.302 0.815 1.*033 1.212 1.578 1.893 2.13 8 2.093 1,478 U 13 Columr 1 of the table shows measurements of the specific color signal on samples of 60 sera in an assay dilution of 1:150. Column 2 shows the titer Tmeas measured by final dilution series for the same sera. Column 3 shows, for the same sera in each case, the titer Tcalc calculated from Formula from the color signal in column 1 using the values alpha 3.4514 and beta 0.2106 found after optimization.

Using the modern calculators now available it is no great effort for an expert to draw up a program for the iterative procedure used to optimize alpha and beta for particular reagent combinations.

The quotient Tmeas/Tcalc is then shown, being dividec into "too low" (lower than "approximately 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 vrriation 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.

sT"

Claims (4)

1. A method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, in assay fluids, Ji by the ELISA a method with photometric evaluation of the saiples, prepared from the assay fluids, with colored, enzy- matically labeled antigen/antibody complexes, in which only one single assay dilution of the sample is pre- pared, an antigen/intibody binding reaction is initi- ated in this assay dilution, with preferably one reac- tant being immobilized on a solid surface, and the r tC antigen/antibody complex is subjected to a color reac- tion brought about enzymatically, in order to prepare b j ta colored solution, and a titer corresponding to thej 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 i l c measured, and the titer being calculated from the re- suiting signal AOD by the following formula: log titer alpha Ata I

3. T and being set equal to the final dilution titer a obtainable by serial dilutions, with the "titer" in S' the Formula being the reciprocal of the final dilution at which the signal A0D corresponds to the limiting signal at the detection Limit compared with 4 negative control samples, and the values for alpha and beta at a fixed assay dilution being determined 3 experimentally, by a series of tests on samples of Ti known final dilution titer of the analyte, separately v. for the particular combination of immunologically t reactive surface and enzyme-labeled immunoglobulin b or antigen as detector under the same reaction and imiobiI izat ion conditions. d d immiqbiL izat ion conditions. 2. The method as claimed in claim 1, wherein the signal Pt 15 AOD for the extinction or optical density which is inserted in Formula is the specific color signal which has been obtained a) by subtraction of the extinction signal for a con- trol (for example buffer or negative sample) 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 Ithe extinction signal as the start of a measurement of the increase in color, from the extinction sig- jil D nal of the test sample, S b) by multiplication of the extinction signal for the Li I test sample by a correction factor resulting from i a test control (for example a standard) which is i also measured, or c) after formation of the quotient or index from the o I Sextinction signal for the test sample and that for i a test control (for example negative sample) which is also measured. 3. The method as claimed in claim 1 or 2, wherein the assay dilutions used are sample dilutions in the range from undiluted to 1:800, preferably about 1:150.

4. The method as claimed in claim 1, 2 or 3, wherein the values used for alpha are in the range from 3.0 to 3.6, and for beta are in the range from 0.10 to 0.27. The method as claimed in claim 1, 2 or 3, 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, in a manner known per se, the titer, and then iterative fitting is used -16 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 opti- mizing the agreement between the titers (Tmeas) measured by endpoint dilution and the titers (Tcalc) calculated by insertion of the signal AOD in the For- mula

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 ooae or samples is most often in the range 0.8 to 1.25 is oo determined by iteration. O 0 0 o0 "oona 7. The use of the method as claimed in one of claims 1 0000 0a 0 n to 6 for determining the presence of rubella (IgG; IgM) oo o antibodies, cytomegalovirus (IgG; IgM) antibodies or 0o s hepatitis B surface antigen (HBsAg) in sera using microtitration plates on which are immobilized, res- 04 a o00 pectively, rubella antigen, cytomegalovirus antigen o0 0" or antibodies against HBsAg. 0 00 0600 o,oo 8. The use as claimed in claim 7, wherein alkaline phos- 0oooo phatase or peroxidase is used as indicator enzyme. 00 00D 0 0 0 o"o g DATED this 22nd day of October 1987. BEHRINGWERKE AKTIENGESELLSCHAFT EDWD. WATERS SONS PATENT ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3000.