CA1287799C - Method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the elisa method with photometric evaluation - Google Patents
Method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the elisa method with photometric evaluationInfo
- Publication number
- CA1287799C CA1287799C CA 550085 CA550085A CA1287799C CA 1287799 C CA1287799 C CA 1287799C CA 550085 CA550085 CA 550085 CA 550085 A CA550085 A CA 550085A CA 1287799 C CA1287799 C CA 1287799C
- Authority
- CA
- Canada
- Prior art keywords
- titer
- dilution
- signal
- assay
- extinction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 .
Log titer = alpha .
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 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 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 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~
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 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.
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 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 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.
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 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 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 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~
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 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.
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 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 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.
Claims (8)
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 . (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 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.
log titer = alpha . (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 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.
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.
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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3636271.9 | 1986-10-24 | ||
| DEP3639279.0 | 1986-11-17 | ||
| DE3639279A DE3639279C3 (en) | 1986-10-24 | 1986-11-17 | Method for the quantitative determination of antibodies or antigens according to the ELISA method by photometric evaluation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1287799C true CA1287799C (en) | 1991-08-20 |
Family
ID=6314155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 550085 Expired CA1287799C (en) | 1986-10-24 | 1987-10-23 | Method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the elisa method with photometric evaluation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1287799C (en) |
-
1987
- 1987-10-23 CA CA 550085 patent/CA1287799C/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10809258B2 (en) | Method for the detection of the prozone effect of photometric assays | |
| US4401387A (en) | Nephelometric immunoassay and nephelometer | |
| Fielding et al. | Enzyme immunoassay for urinary albumin. | |
| Forest et al. | Evaluation of the analytical performance of the Boehringer Mannheim Elecsys® 2010 immunoanalyzer | |
| Sarkar | TSH comparison between chemiluminescence (Architect) and electrochemiluminescence (Cobas) immunoassays: an Indian population perspective | |
| Tiffany et al. | Specific protein analysis by light-scatter measurement with a miniature centrifugal fast analyzer | |
| US6277584B1 (en) | Method for calibrating a chemical analyzer with improved accuracy at low signal levels | |
| Lucertini et al. | Enzyme-linked immunosorbent assay of retinol-binding protein in serum and urine. | |
| EP0254430B1 (en) | Light-scattering immunoassay system | |
| Blom et al. | Profile analysis of blood proteins with a centrifugal analyzer. | |
| JPS605899B2 (en) | Immunochemical quantitative method | |
| Galen et al. | Enzyme immunoassay of serum thyroxine with the" Autochemist" multichannel analyzer. | |
| CA1287799C (en) | Method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the elisa method with photometric evaluation | |
| AU602170B2 (en) | A method for determining the presence of substances of diagnostic relevance, in particular antibodies or antigens, by the ELISA method with photometric evaluation | |
| Whicher et al. | Formulation of optimal conditions for an immunonephelometric assay | |
| AU631132B2 (en) | Immunoanalysis method for discriminating between antigen excess and antibody excess conditions | |
| JPH05126832A (en) | Immune analyzer and immune analysis method | |
| JPS61280568A (en) | Method for measuring components in bodily fluids | |
| JPH0875740A (en) | Immunological nephelometry | |
| Winkles et al. | Automated enhanced latex agglutination assay for rheumatoid factors in serum. | |
| D'orazio et al. | Potentiometric electrode measurement of serum antibodies based on the complement fixation test | |
| Desjarlais et al. | Basic characteristics and evaluation of a partially automated Behring laser nephelometer for the measurement of IgG, IgA, IgM and C3c in serum | |
| McLachlan | Monoclonal immunoglobulins: affinity blotting for low concentrations in serum. | |
| Spencer et al. | Kinetic immunoturbidimetry: The measurement of pregnancy specific β1 glycoprotein | |
| Wang et al. | A simplified solid-phase immunofluorescence assay for measurement of serum immunoglobulins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |