NO170908B - IMMUNO CHEMICAL PROCEDURE FOR DETECTION AND DETERMINATION OF DIAGNOSTIC RELEVANT SUBSTANCES AND APPLICATION OF THE PROCEDURE - Google Patents

Description

The invention relates to a method for detecting and determining the presence of diagnostically relevant substances, especially antibodies or antigens, in sample liquids, preferably according to an ELISA method, by photometric assessment of the samples prepared from the sample liquids, with dyed, enzymatically marked antigen-antibody complexes where only a single sample dilution of the samples is prepared, in this sample dilution an antigen-antibody binding reaction is triggered, preferably a reaction participant is fixed to a solid surface, and the antigen-antibody complex is subjected to an enzymatically produced color reaction, to produce a color solution , and from the spectral absorbance measure measured on this color solution, a titer corresponding to the final dilution titer is determined using previously measured reference data.

The invention also relates to the application of the method by

. detection of substances as indicated above.

By "spectral absorption measure" is meant above and in the following the term that previously went under the term "extinction".

The invention serves for the detection and stepless quantification of diagnostically relevant substances, as a single sample mixture is sufficient for the examination of the sample. The "enzyme-linked" immunosorbent assay (ELISA), the radio-immunoassay (RIA), nucleic acid hybridization, nephelometry or other determination methods can be used as a test. For example, antigens, antibodies of the different immunoglobulin classes, nucleic acids or also metabolic products of medical importance are considered diagnostically relevant substances.

The usual quantification methods of diagnostically relevant substances can be traced back to two principles. On the one hand, the quantification of the substance to be determined can take place over a reference curve that was constructed in advance from samples with known substance content. The alternative possibility consists in a titration of the substance to be determined by means of endpoint dilution. The latter method always pays off when too undiluted or only slightly diluted samples are to be examined, it is difficult to set up the above-mentioned reference curve or it is simply unsatisfactory to use it. This is, for example, the case with

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determination of sample-specific antibodies in ELISA.

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For this reason, the method according to the invention is also preferably used in this case, and is explained below in more detail in this embodiment. As an example for diagnostically relevant substances, antibodies are thus used as examples for the initially mentioned test, ELISA. Consequently i means

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description and requirements a "specific color signal" in the same way for other samples, for example also radioactive decay, (counts), or relative scattered light signal.

The ELISA method is a well-known enzyme-immune test that serves for detection! of antibodies or antigens in parasitic bacterial or viral infections. The invention serves to improve the assessment of color solutions which, for example, are formed by contact between ~the serum to be examined for specific antibodies in a special sample dilution, and ; antigens fixed to microtitration plates, with subsequent enzymatic detection of the possibly formed antibody-antigen complex.

In particular, the invention relates to the photometric assessment of such solutions, where the spectral absorption target for the thus prepared solutions is measured, and which is a target for the formed enzymatically marked colour-forming immune complexes in the solution. Usually, a serum is considered positive when the spectral absorption measure for the above-mentioned formed solution exceeds a certain limit or threshold value. Discussions of this type of assessment can be found, for example, in "Immun.Infekt." 9, 33-39 1981.

However, it is desirable not only to determine whether or not the limit value is exceeded, but also to be able to make quantitative statements about the presence of antibodies in the serum to be examined. It is known for this purpose to carry out so-called "dilution series", that is to say at different sample dilutions to carry out ELISA and to measure the spectral absorption target for the respective resulting color solutions. By combining the individual photometric measurement values in a graphic, a so-called sample dilution curve appears for each serum sample (Fig. 1). The sample dilutions where the sample dilution curve intersects the previously given limit value indicate the final dilution for the antibody to be determined the final dilution, this final dilution being the reciprocal value of the antibody titer (final dilution time). The quantification of the antibody thus takes place above the specified sample dilution, whereby the detection limit is achieved. For the measurement points of the sample dilution curve, the measured spectral absorption values are often used, not in the directly measured numerical value, but a measurement value correction is mostly carried out. The type of correction can consist of subtracting the measured value from a suitable control included in the ELISA (buffer, negative serum), or from a parallel mixture of the serum to be determined with control antigen fixed to microtiter plates or a starting extinction by observing a color development kinetics. Furthermore, it is possible to multiply the directly measured measured value with a correction factor that appears in a suitable way from an included standard or also the indication of a coefficient or index that appears by dividing the directly measured measured value by that of an included negative sample. This corrected measurement value is referred to in the following as specific color signal, and is the basis for specifying the final dilution titer.

The significant disadvantage of a quantitative assessment in the manner mentioned is that the implementation is time- and reagent-consuming. An attempt was therefore made, from the spectral absorption measure measured on a single sample dilution in ELISA, to deduce the final dilution titer of the serum. Thus, for example, van Loon and van der Veen mention in "J.Clin. Pathol." l 33, 635-639 (1980), detection of Toxoplasma antibodies by the ELISA method using a single serum dilution in conjunction with a reference curve. Thereby, the spectral absorbance measure that was measured by ELISA at a serum dilution of 1:800 is compared with the same spectral absorbance measure in the reference curve which indicates the spectral absorbance measures found on a number of sera in a sample dilution of 1:800 as a function of the final dilution titer. Thereby, this reference curve was constructed graphically as the mean value of the measured values for the majority of sera. An improvement of this method is described by van Loon et al., in "J. Clin. Pathol." 34, 1981, 665-669, where the values for the spectral absorbance measures are replaced by the specific color signals obtained by subtracting the spectral absorbance measure for a negative control serum diluted to 1:800 from the spectral absorbance measure for the sample serum. In this way, the deviations of experimentally measured sera from the reference curve could be improved, at least in some cases. In other cases, however, the improvement was so small that comparison with negative control serum was unnecessary.

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Although Iman by this known method for practice had already achieved a simplification in that only a single test dilution had to be prepared, this method does have considerable disadvantages. The suitable and recommended sample dilution of 1:800 for this method is very high, so that, firstly, the sensitivity of the method is too low (see weak positive serum in Fig. 1), and secondly, pipetting errors become potentiated. Moreover, in practice, working with a reference curve is cumbersome and requires a computer for automatic measurement data assessment.

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OppfinnéIsen's task is to provide, a method for quantitative determination of antibodies or antigens, for example by ELISA, where the lowest possible dilution of the sample to be examined can be used, and the titer determination can be carried out in a simple way, for example with the help of a pocket calculator.

In addition, the areas of application for the method according to the invention must be stated.

According to this, the present invention relates to an immunochemical method for the detection and determination of diagnostically relevant substances, in particular an ELISA method, and the method is characterized by the fact that, per sample only a single sample dilution of approx. 1:150 and that the spectral absorbance measure (Aqd) for the color solution is measured and that the final dilution titer is determined from the obtained signal according to the formula:

whereby in formula (1), the final dilution titer is the reciprocal value of the final dilution which, at the signal Aqd, corresponds to the limit value signal at the detection limit for negative control samples, and the value for a and p is determined separately, experimentally by series of tests on samples with a known final dilution titer for the analytes at a fixed sample dilution dilution for the relevant combination of Immunologically reactive surface and enzyme-labelled immunoglobulin, respectively antigen as detector under equal turnover and fixation conditions, whereby values in the range 3.0 to 3.6 are used for a and for e values in the range 0, 10 to 0.27 and the value pair for a and p is determined where the relevant quotient <T>measured/Tcalculated for the individual samples lies in a range of 0.8 - 1.25.

Preferably, in this method, the specific color signal defined above in formula 1 is used as signal Aqd for the spectral absorption measure.

As indicated above, the invention also relates to the use of the described method for determining the presence of rubella antibodies, cytomegalovirus antibodies or hepatitis B surface antigen (HBsAg) in sera by means of microtiter plates on which corresponding rubella antigens, cytomegalovirus antigens or antibodies against HBsAg are fixed.

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Furthermore, the invention relates to the use of this method

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where alkaline phosphatase or peroxidase is used as an indicator system.

As test dilutions, test dilutions in the range from undiluted to 1:800 can be used, with a test dilution of only 1:150 being preferable. In the latter case, the sample dilution is still so small that a high sensitivity of the immunoassay method is ensured, secondly, has

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experiments have shown that with a sample dilution of 1:150 relatively well reproducible results can be obtained.

Lower sample dilutions are possible, however both the immunologically reactive surface, for example of the antigen-coated microtitration plates and also the conjugate, require a high purity with high reactivity.

It has been found that usable values for a lie in the range of 3.0 to 3.6, and for p in the range of 0.10 to 0.27.

The correct determination of these values a and P forms the basis for the advantageous application of the method according to the invention in practice. Once optimal values for a and p have been found, the assessment, i.e. the titer determination of the sample, can be carried out mathematically very

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single. The values for a and 3 can, for example, be determined by the reagent manufacturer using a series of tests on a majority of research samples, with the specific color signal measured on these sera respectively at the desired

test dilution and additionally determine the titer in a manner known per se by endpoint dilution. The pair of values formed for each sample is then sought by iterative adaptation of suitable values for ot and p, for which an optimum is achieved for the agreement between the titer Tm^^t- measured by endpoint dilution and that from formula (I) after applying the specific color signal calculated titers TDs. In such an iterative method, one calculates, for example, the associated titer TDer with primarily arbitrarily assumed numerical values for a and P via formula (I) from the specific color signal of each sample. Also, the titer T^ it is determined by endpoint dilution.

When the quotient ^ measure/^ is <=> 1>0", this would mean that the correct titer of the spectral absorption measure at the sample dilution has been achieved. Under realistic conditions, a coefficient between 0.8 and 1.25 must be considered "hit exactly right". Coefficients below 0.5 and above 2.0 mean that the

for a classic titration, the permissible reproducibility limit was exceeded.

After the first attempt, which usually gives an even worse mathematical titer prediction, a and p are then changed step by step, and then again compared for each serum sample of the calculated titer with the measured titer. This interaction procedure is continued until the agreement is satisfactory.

The experiments show that it can be assumed that the optimum is achieved when, in a series of experiments, the equation

applies to approx. 5OSé of the tested sera.

These experiences apply to test series with alkaline phosphatase as indicator enzyme. When peroxidase can be used as an indicator enzyme, the "hit rate" can be increased to approx. 65%. These limits, however, lie within the range of the spread in the 1ELISA method itself, and thus cannot be overcome by the method according to the invention for quantitative assessment of the measurement results.

Furthermore, it has been shown that the values for a and p do not depend on the type of diagnostic sample, but also on the production batches of the immunologically reactive solid phases used and the conjugate. Consequently, for the practical application of the method, the two constants must, for example, be determined anew for the combination of a sample plate and a conjugate charge, which, however, enables series measurements on samples to be examined, with good reproducibility, and in practice very good accuracy.

In the following, the invention is explained in more detail using examples and measurement results. Thereby, reference is also made to the drawings.

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Fig. 1, according to the state of the art, carried out dilution series in the form of sample dilution curves showing the specific color signal as a function of the sample dilutions; Fig. 2 the dependence of the specific color signal at a sample dilution of 1:150 on the actual titer determined at the final dilution (Tm^n)5 Fig. 3 the dependence of the titer (Ttøer) calculated according to formula (1) on the specific color signal at a sample dilution of 1:150 on the same sample reagents as in fig. 2; and Fig. 4 the measured titer T m^^ from fig. 2, compared to the calculated titer T^er from fig. 3, by identical specific color signal of the 1:150 diluted examination samples.

To test the method according to the invention, tests were carried out for the determination of antibodies against cytomegalovirus in sera using microtiter plates to whose walls cytomelagovirus antigen was fixed and for the determination of antibodies against rubella in sera using microtiter plates on whose walls was fixed rubella antigen. The test samples in the sample dilution 1:150 were reacted with the fixed antigens, the unbound antibodies were washed out, an enzyme bound to the formed antibody-antigen complexes over a conjugate solution, and the excess conjugate solution was washed out, a staining caused by means of a chromogenic substrate solution, via the enzyme, after which the reaction is terminated with a stop solution. The thus prepared, depending on the content of the virus-specific antibodies colored samples, were then assessed, their spectral absorption measure Aqd being determined photometrically (at a defined wavelength).

In dilution series, for the same samples, the final dilution titer Tm^^ was first determined in a manner known per se, and then compared with the titer TDer calculated via formula (1) according to the invention.

To improve the reproducibility and accuracy of the method, in all cases the specific absorption measure or the "specific color signal Aqd" was used, the specific extinction or the "specific

Fig. 1 shows a curve representation of the specific color signal Aqd as a function of different sample dilutions (sample dilutions -<1>) for three positive sera A, B and C, for a negative serum D. This example shows curve progression that only in the range of higher sample dilutions , for example 1:800, can be parallelised. This is the basis for a suitable reference curve according to van Loon.

While the break in the sample dilution curve for a serum with a high antibody titer (example A) shows the literature-known prozone effect, it is not entirely clear why the dilution curves flatten out in the lower area near the final dilution titer.

It had now been found that the final dilution titer determined by a series of dilutions for a wide majority of samples consists in relation to the spectral absorption measure measured by a sample dilution of, for example, 1:150:

as the constants a and p enable an adaptation to the test and examination conditions.

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This fact appears from fig. 2-4. In fig. 2, the dependence of the specific color signal Aqd on 58 sera in a dilution of 1:150 is listed as a function of the titer Tm^i determined at the final dilution. On the other hand, fig. 3 the titer calculated according to formula 1 from the spectral absorption measure Aqd at optimal values for a and P. The small dispersion area of this curve appears from the mathematical arrangement.

Fig. 4 now clearly shows that the curve calculated according to formula I and; the titer values mean the measured values excellent. It thus appears from this that with the method according to the invention an optimal prediction of the final dilution titer can be achieved.

This good agreement between T^er and T m^^ is indeed achieved; only when care is taken in determining the values for a and p.

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With the help of the following table of measured and calculated data from an experimental series of 60 sera, the procedure for determining optimization of a and p is now explained as an example.

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In column 1 1 of the table, the measured values of specific color signal on samples in a sample dilution of 1:150 are given for 60 sera. In column 2, the titer Tm4i measured at the final dilution series is indicated for the same sera. Column 3 shows for the same sera the calculated titer TDs according to formula (1) from the color signal to column 1, using the values a = 3.4514 and p = 0.2106 found after optimization.

With the help of modern disposable calculators, it is not a big task today to program the used interactive method for optimizing a and 3 for specific reagent combinations.

The coefficient ^ measure/^ ber is then specified, namely assessed according to "too small" (less than 0.8) "about equal to 1" (in the range 0.8-1.25), and "too large" (greater than 1.25).

In the case shown, it can be seen that for 4556 of the tested sera a good approximation has been achieved. Taking into account the spread of the final dilution titer, see fig. 2, the choice of the values for a and p can therefore be considered very good for practice.

Claims (5)

1. Immunochemical method for the detection and determination of diagnostic ; relevant substances, especially an ELISA method, characterized by the fact that only a single sample dilution of approx. 1:150 and that the spectral absorption measure (Aqd) for the color solution is measured and that from the obtained signal the final dilution titer is determined according to the formula: i whereby in formula (1), the final dilution titer is the reciprocal value of the final dilution which at the signal Aqd corresponds to the limit value signal at the detection limit opposite in negative control samples, and the value for a and p is determined separately, experimentally in a series of experiments on samples with known final dilution titres for the analytes at a fixed sample dilution for the relevant combination of immunologically reactive surface and enzyme-labeled immunoglobulin respectively antigen as detector under equal turnover and fixation conditions ', whereby values in the range 3.0 to 3.6 are used for a and, for p-values in the range 0.10 to 0.27 and the value pair for ' a and p is determined where the relevant quotient Tmeasured/Tcalculated f °r the individual samples lie in a range of 0.8 - 1.25.i 2. Method according to claim 1, characterized in that1 as the signal Aqd for the spectral absorption target, the specific color signal in formula (1) is used which is obtained a) by subtracting the signal for the spectral absorption target for a control included in the ELISA (e.g. buffer or negative sample), or the signals for the spectral absorption measurements of a parallel approach of the sample i which is to be examined on a surface coated with a control preparation or the signal for the spectral absorption measure as the beginning of a color increase measurement, from the signal for the spectral absorption measure in the sample to be examined, b) by multiplying the signal for the spectral absorption measure in the sample to be examined, with a correction factor that is obtained from an included experimental control (for example standard), or c) after coefficient or index formation, of the signal for the spectral absorption measure of the sample to be examined, and d) to an included experimental control (for example negative sample). 3. Method according to claim 1 or 2, characterized in that the values for a and p are determined on a number of sample liquids or samples, the signal Aqd being measured on these samples at the desired sample dilution and the titer in addition, in a manner known per se, determined by end-point dilution , and then, from the obtained value pairs (titer Aqd). for the individual test liquids or samples by iterative adaptation, suitable values for a and P are sought, for which the agreement between the titer measured at the endpoint dilution (<T>måit) and the titer calculated from formula (1) by inserting the signal Aqd (TDeregne-t ) reaches an optimum. 4. Use of the method according to one of claims 1-3, for determining the presence of rubella antibodies, cytomegalovirus antibodies or hepatitis B surface antigen (HBsAg) in sera by means of microtiter plates on which corresponding rubella antigens, cytomegalovirus antigens or antibodies against HBsAg are fixed. 5. Use of the method according to claim 4, where alkaline phosphatase or peroxidase is used as an indicator system. in