FI81448B - PHOTOMETRIC FOLLOWING FOR THE PURPOSE OF CONTAINERS AND REACTORS, FOLLOWING AND UNDER IMPLEMENTATION OF SPRIDNING SCENTRALERS. - Google Patents

Abstract

1. A method for the photometric determination of the concentration of a reactant in a reaction in which scattering centers are formed or consumed in the sens of a transition from Rayleigh to Mie scattering where the peak rate of reaction Vmax and the time from the start of the reaction to the appearance of the peak rate of reaction tmax are determined and to obtain a single value for the concentration the functional relationship between concentration, Vmax and tmax is found empirically using a standard preparation, and Vmax and tmax are measured on the sample, which comprises choosing between the two possible concentration values on the basis of the 2nd reaction parameter, namely Vmax or tmax .

Description

1 81448

The invention relates to a method for the photometric determination of the concentration of a single reactant in a reaction proceeding to form or consume scattering centers to effect a transition from Rayleigh scattering to Miesiron, measuring the maximum reaction rate. ^ max ΓΘ3 ^ ϋοη from the beginning to the occurrence of the highest reaction rate, for the unambiguous determination of the concentration, the functional dependence between the concentration, the V-value and the t-value is determined empirically with the standard preparation avul-max max la, and the V and t values of the sample are measured.

1 max J max 15 Light scattering phenomena on particles in a homogeneous medium are used to determine the concentration by measuring both the intensity of the scattered light (nephelometry) and the loss of intensity of the light beam entering the medium (coexistence measurement).

20 Many chemical reactions lead in several stages to molecular aggregates or macromolecules whose light scattering behavior differs greatly from that of the starting materials and the concentrations of which can be determined on the basis of this property.

For example, when the solubility input is exceeded, a very large number of ions accumulate into crystals, which first appear in the precipitate and finally grow so large that they separate by precipitation from the liquid phase.

Another example is the immunochemical reaction between a Liu-30 antigen and a bivalent antibody, which can form large and highly light-degrading molecular clumps.

The time course of these reactions very often corresponds to the general kinetic course of 1st order sequential reactions, this means that in the concentration curve 2 81448 there is a turning point for the formation of the final product and thus the highest reaction rate occurs only during the reaction. As an example of an immunochemical reaction, this procedure is shown in Figure 1 for two antigen concentrations.

5 From the signal / time curves in Figure 1, the concentration-dependent measurement signal can be obtained in different ways (Table 1).

Table 1 Time boundary conditions for determining concentration-dependent measurement quadrants (S = signal, t = 10 time).

Name Principle 1) Endpoint- The measurement signal is determined at such a late method at the time that experience has shown that it no longer changes, but the precipitate does not yet appear ny.

2) Kinetic The actual measurement signal is the difference between two signaling methods, which are determined by different but "fixed-time fixed pre-set times". 3) Kinetic The maximum reaction rate, i.e. signal method Iin maximum change in time unit: "rate eter- (a) by measuring AS

FLAT

(b) form an electronically differentiated form below.

FLAT

by setting a maximum value;

At J

c) forming a tangent to the signal / time curve and determining the maximum slope.

30

A large number of analytes can currently be quantified according to the methods shown in Table 1 using direct or indirect light scattering measurement.

35 Considering the dependence of a suitable measurement signal (Table 3 81 448 1) on the concentration of one component involved in the reaction, for example the concentration of antigen, and the other component in the reaction being added at a constant concentration, the dependence shown in Figure 2 can be observed. The surprising fact that the same measurement signal can be used for both low and high analyte concentrations leads to ambiguity in the signal / concentration equation, which is known to those skilled in the art as the antigen excess phenomenon or Heidelberg curve.

This ambiguity can in principle always be observed where complexes of different stoichiometry are possible according to the excess of the first or second component involved in the reaction and these complexes do not differ in their signaling properties, for example in light scattering.

Mon CoMon and Mon COMon where Mon = monomer, x x + m J x + m x J '

CoMon = comonomer,

Ag £ Ak and AgAk2 wherein Ag = antigen, 20 Ak homologous antibody;

Lec2 GP and Lee GP2 where Lee = lectin, GP = glycoprotein; (AgxClx + 1) - and (Agx + 1Clx) + where Ag = silver ion.

Cl = chloride ions.

25

If these reactions are performed for concentration determinations, the method for a given excess situation is defined and optimized. In practice, this leads to difficulties where the analyte can be in a very large concentration range and an excess concentration of one of the substances involved in the reaction cannot be ensured.

This situation is particularly significant in the immunochemical assay of immunoglobulin, where the concentration may vary depending on factor 1000. The test conditions and 35 economically optimal antibody concentration to determine the normal 4,81448 and subnoral concentration do not allow for an increased concentration of 1000. Thus, the following presentation only considers immunochemical reactions, although the method can be applied to all reactions that exhibit the above-mentioned properties and whose behavior is analogous to kinetic.

With respect to immunochemical reactions, several methods and embodiments have been proposed to date to determine on which part of the Heidelberger curve the assay sample is located.

The following are some examples: 1. The oldest and safest method for detecting antigen excess is to perform a duplicate assay with two different sample dilutions. In the presence of an excess of antigen, a higher dilution gives a higher signal and a seemingly higher protein content is observed than with a more concentrated sample. (H. E. Schultze and G. Schwick; Prot. Biol. Fluids 5, 15-25 (1958).

In improved embodiments of this method, further addition of antibodies to the test sample is used when the reaction has reached a certain equilibrium. In the presence of an excess of antigen, an increase in the signal then occurs (T. O. Tiffany et al., Clin. Chem. 20, 1055-1061 (1974)). Excess antigen can also be detected by post-administration of antigenic material of known concentration (j.C.

Sternberg; Clin, Chem. 23, 1456-1464 (1977)).

2. In a special continuous flow technique, the presence of an excess of antigen is observed by means of a double peak (R.F. Ritchie; Protides Biol. Fluids 21, 30 569 (1974)).

3. By looking at the reaction kinetics using a graphical representation of the course of the reaction, a turbidimetric measurement can be used to distinguish between antigen excess and "weak" antigen excess (I. Deverill; Protides Biol.

35 Fluids 26, 697 (1979)). (P. J.J. Van Munster et al., Clin.

5,81448

Chlm. Acta 76, 377-388 (1977)).

4. By determining the reaction time until the occurrence of the maximum reaction rate by means of nephelometric measurement, a quantity can be calculated which allows a distinction to be made between 5 excess antigen and antibody (DE-A 27 24 722 C2).

The methods mentioned in items 2 and 4 preferably differ from other methods in that it is not necessary to examine all samples for the presence of excess antigen, but to obtain information during the measurement event as to whether an antigen excess determination is required.

Method 2 is subject to a specific technique and cannot be used in general as a nephelometric or turbidimetric sample assay.

According to the state of the art, DE-A-27 24 722 C2 assumes that there is a function of the maximum reaction rate which allows a distinction between the excess antigen and the excess antibody by means of a threshold value for this function (claim 1). In paragraph 12 of paragraph 12 of this patent, lines 8-21 state that the time (T) extending from the onset of the reaction to the occurrence of the maximum value of the reaction rate (H) can be used as the separation function. However, these implementations are severely limited in paragraph 21, lines 55-68, where it is stated that there is no single time value for the antibody excess of the curve to be on one side of this curve and the antigen excess of the curve to be on the other side. The invention solves the difficulty by means of coordinate transformation and the use of new variables.

The method is further characterized in that, in order to detect an excess antigen state, the measurement is repeated according to the appropriate dilution of the sample to achieve an antibody excess state. This additional measurement of the sample to detect an excess of antigen is material and time consuming.

6 81 448

The object of the invention is therefore to develop a method which allows the antigen or antibody content of a sample to be determined without further measurement, even if this excess antigen situation prevails in the respective 5 test samples.

Surprisingly, it has now been found that by measuring the time from the onset of the reaction to the occurrence of the maximum value of the reaction rate, the kinetic method, i. by determining the maximum reaction rate as a function of concentration, perform a quantitative measurement on both sides of the hei-delberger curve. The distinction between antibody excess and antigen excess is no longer necessary.

The invention thus relates to a process of the type 15 described in the introduction, which is characterized in that the correct concentration is selected from the two possible concentrations by means of a second reaction parameter, V or t.

^ max max

The dependence between the concentration and the V-value and t -ar-max max von can be represented, for example, by means of a table, a matrix-20 sin or one or more mathematical functions.

It is known that the reaction rate and signal strength in antigen-antibody reactions can be affected over a wide range by the ionic strength, ionic quality and polymer addition of the reaction medium. Thus, the following presentation is not limited to the conditions set forth in the Examples, but is directed to precipitation reactions that can be affected in the above sense.

If the maximum reaction rate (vmax) is determined as a function of concentration, the dependence shown in Fig. 2 is obtained. If the max time taken from the beginning of the reaction to reach the V value ΠΙαΧ (tjnax) 'is determined simultaneously with the V value, the dependence shown in Fig. 3 is obtained. Both signal / concentration curves show the case of antigen excess; the measured signals can be assigned to two pitches.

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If both functions are projected into a single image (Figure 4), it is observed that there is no security (vmax / corresponding to two different concentrations. Maximum reaction rate (V) and time of maximum reaction

ITldX

The 5 speed (s) to achieve are apparently apart

IuqX

independent concentration functions and the Heidelberger curve is a three-dimensional curve (Figure 5) in which, when the reaction is performed appropriately, there is no ambiguity in concentration.

10 In measurement methods that use only a two-dimensional projection of this curve, for example the measurement of vmax as a function of concentration (curve 6), there is always ambiguity with respect to concentration.

For a practical evaluation of the observed dependencies, it should be noted that the concentration of the signal / concentration curve in the vicinity of the minimum value can only be measured with reduced accuracy, because relatively small signal differences correspond to large concentration differences.

In the new method, when measuring two independent measurement signals, the accuracy improves especially when the minimum value of the concentration function f (t) is as far as possible from the concentration function f (VmaxMaximum value).

The working method and reaction conditions of the new process are illustrated in the following examples.

25 Example 1

Determination of the maximum range of the calibration curve for IgG.

Photometer: DU-8 with accessory for nephelometric measurements (Beckman Instruments).

Reaction medium: 0.02 mol / l potassium phosphate buffer, pH 7.3 with 40 g / l polyethylene glycol 6000 (Serva), Heidelberg, Federal Republic of Germany and 64 g / l sodium bromide.

Sample diluent: physiological NaCl solution.

35 Antiserum: LN antiserum against human IgG / kappa ket- β 81448 (from rabbits) (Behringwerke AG, PART 14), diluted 1:31 with reaction medium.

Standard: Standard T-protein serum (human), (Behringwerke AG, OSKT 06).

Test sample: 20 microliters of sample / standard dilution is mixed with 500 microliters of antiserum dilution and measured immediately in a photometer using a Kinetik II program for up to 3 minutes at 224 nm.

10 9 81448 a) turbidimetric measurement

Standard dilution - Maximum reaction - Time to reaction medium concentration mg / dl rate (V_a mE / ta maximum up to max 5 IgG min (tra5i .s.)

IuoX

110.4 425 38 92 466 30 10 78.9 542 24 73.6 569 20 69 583 18 64.9 576 16 61.3 567 16 15 55.2 548 14 22.1 219 20 11.0 75 30 b) nephelometric measurement 20

Standard dilution - Maximum reaction - Time to reaction medium concentration mg / dl rate (vmaxE / ta for maximum IgG evolution) 25 110.4 1410 36 92 1490 30 78.9 1710 22 73.6 1730 22 30 69 1780 20 64.9 1800 20 61.3 1810 20 55.2 1720 18 22.1 706 24 35 11.0 325 42 10 81 448

Example 1 shows that in both turbidimetric and nephelometric measurements, concentrations can be unambiguously determined at and at the maximum point of the Heidelberger curve. Unknown sample

5 is determined by interpolating V - la T

max After measuring the max max values between the standard values tabulated. The measuring range is determined by appropriate dilution of the samples, in Example 1 a sample dilution of 1:51 is 560-5600 mg / dl IgG and can be selected so that the 10 sub-ranges with lower accuracy do not fall within the clinically relevant decision range of the corresponding parameter.

Example 2

Measurement of the calibration curve for IgM (turbidimetrically) 15 Photometer: DU-8 (Beckman Instruments) Reaction medium: 0.15 m NaCl phosphate buffer, pH 7.2 together with 39 g / l PEG 6000 (Serva) and 8 g / 1 NaCl Sample dilution medium: physiological NaCl solution

Antiserum: LN antiserum against human IgM / β chain 20 (from rabbits) (Begringwerke AG, PARTS 14), diluted 1:11 with reaction medium

Standard: T-protein standard serum (human), (Behrinwerke AG, OSKT 06)

Test sample: 200 microliters of sample / standard diluent is mixed with 500 microliters of antiserum dilution and measured immediately in a photometer using a Kinetik II program for up to 3 minutes at 312 nm.

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Standard law- Sample pi- Maximum reac- Time reaction concentration concentration plant rate from the beginning maximum concentration, mg / dl with reduction (V e »_, min (t, s) igM 1:21 mg / dl mi8ax) 'mE / max

5 IgM

110 2310 371 34 74 1540 508 28 10 55 1155 591 18 44 924 639 14 37 770 639 12 31 660 620 12 28 578 611 10 15 24 513 598 10 22 462 556 10 20 420 508 10 18 385 488 10 17 355 467 10 20 16 330 460 10 15 308 419 10 14 290 405 10 13 272 378 10 12 257 368 10 25 11 231 330 10 5.5 116 158 10 2.8 58 69 10 1.4 29 27 12 30 From Example 2 it can be seen that that the selected sample dilution can measure a concentration between 30-600 mg / dl IgM with greater accuracy than between 600-900 mg / dl. However, in the normal range of 70-300 mg / dl IgM in serum, values greater than 600 mg / dl are always 35 suspected to be close to monoclonal gammopathy and thus require further studies regardless of the accuracy of the values.

Claims (5)

A method for photometric determination of a single reactant concentration in a reaction proceeding to form 5 or consume scattering centers to effect a transition from Rayleigh scattering to Mie scattering by measuring the maximum reaction rate V and time t from the onset of the reaction to the maximum max max reaction rate for unambiguous concentration determination 10 dependence between the concentration, the V-value and the t-value using the max max standard preparation, and measuring the V max and t values of the sample, characterized in that the correct concentration is selected from the two possible max-J max concentrations by means of another reaction parameter, or t. * max max ' Process according to Claim 1, characterized in that the reaction is an immunochemical reaction. Method according to Claim 1, characterized in that the photometric determination is carried out by means of a light transmission method. Method according to Claim 1, characterized in that the photometric determination is carried out using radiation having a wavelength of 240 to 700 nm, but preferably between 300 and 380 nm. Method according to Claim 1, characterized in that the photometric determination is carried out by means of a measurement of scattered light (nephelometric measurement). 13 81 448