FI104585B - Method for extending the dynamic of a bioaffinity determination - Google Patents

Description

104585

METHOD FOR EXTENDING THE BIOAFFINANCIAL DETERMINATION OF DYNAMICS

Immunoassay is an established biospecific assay and its various applications are widely used in routine diagnostics and research laboratories. Another biospecific assay is the DNA and RNA Hybridization Assay. The improvement of the biospecific assay method of the present invention relates in particular to assay methods for analyte molecules having at least two specific determinants. These methods use two biospecific reagents, 10 primary reagents and secondary reagents. The primary and secondary reagents may each be, for example, antibodies or DNA or RNA probes, which each bind to specific determinants of the analyte molecule to form a reaction product which is a complex of three molecules (sandwich structure). Depending on the application, either or both of these reagents are labeled. The labels used today are radioisotopes, enzymes, luminescent labels and fluorescent labels.

The most common sample material to be analyzed is 20 blood serum.

Most commonly, methods are used in which the Ag analyte reacts with the specific primary reagent Ab attached to a solid reaction medium and, on the other hand, with the labeled reagent Ab * in solution to form complexes AbAgAb *. The solid reaction medium used is, for example, the inside surface of a reaction cuvette or latex microparticles. The response of the assay is linear when. these assays use excess primary and secondary reagent relative to Ag. In immunology, such assays are referred to as immunometric assays. DNA and RNA assays can also be performed according to the same principle.

2 104585 The method of the present invention relates in particular to the improvement of the methods disclosed in International Patent Applications WO 96/22531 and WO 96/27798.

Each publication discloses a biospecific assay method that provides a signal for a very small and diffraction-limited laser beam targeting volume when this targeting volume contains a fluorescent dye-labeled molecule or microparticle to which the aforementioned 10 molecules are attached. Both methods are suitable for the kinetic monitoring of the bioaffinity reaction, since a special feature of these methods is that they are able to distinguish between the fluorescence signal provided by the AbAgAb * complexes and the free fluorescence signal in solution, and no separation of free and bound label is required.

The application disclosed in WO 96/22531 is based on the use of latex microparticles as a solid reaction medium. Herein, the selective measurement of the fluorescence signal from the AbAgAb * complexes is based, for example, on detecting the microparticle trapped in the targeting volume based on the light scattering or internal fluorescence it emits, while simultaneously measuring the Ab * 25 fluorescent labels associated therewith.

Application In the biospecific assay disclosed in WO 96/27798, the primary reagent and the secondary reagent are labeled with various fluorescent dyes D and F. In the bioaffinity reaction complexes AbDAgAbr are formed. In this method, the selective measurement of the fluorescence signal of% individual complexes is based on detecting a molecule drifting to the targeting volume based on the temporal correlation of the fluorescence signals from labels D and F. 1 In these aforementioned methods, the signals obtained from the targeting volume of 104585 are directly proportional to the concentration of AbAgAb1 or AbDAgAbF 5 complexes formed on the surface of the microparticles or in the reaction solution from the beginning of the reaction. These reaction products are hereinafter also referred to, for simplicity, as the symbol AbAgAb, wherein the first Ab represents the primary reagent and the second Ab the secondary reagent. Either or only one of these reagents may be labeled. The symbols Ag, Ab, AbD, AbF, and Ab1 used in this text generally refer to biospecific molecules and are not limited to immunological antibodies and antigens.

The signal response R of the assay is the value of the signal intensity I obtained from the label as a function of 15 P of analyte. Signal intensity I means the value of the intensity of the signal obtained from the stamp at a given time t. The peak of the signal response is the point at which the signal response of the assay, i.e. the standard curve, reaches its highest value (point H in Figure 5).

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The problem with conventional assay methods is their complexity and the limitation of dynamics to values below the reagents concentration. For example, the determination of a hormone in the blood by fluorometric immunometric assay requires many steps: cell separation from serum, dosing and dilution of serum sample, incubation with solid phase, cuvette removal, incubation with labeled reagent, addition of free label, washing, measuring .

In the previous example, the reagents must be added to the reaction cuvette in two separate steps and occasionally washing the cuvette. The purpose of biphasic and first washing is primarily to limit the signal response of the assay 35 to a value corresponding to a solid phase binding capacity of 4 104585. This is necessary because, in a one-step assay, the signal response R would reverse when analyte concentration P exceeded the binding capacity of the primary reagent. When the Ag concentration of the analyte 5 to be measured exceeds the binding capacity of the reagents, the reaction product is formed not only of AbAgAb complex but also of AgAb relative to the concentration of secondary reagent Ab and excess Ag. The greater the excess of analyte Ag compared to the binding capacity of the primary reagent 10, the smaller the assay signal response R will be. This adverse effect, which is only possible when all the reaction components are added at the same time, is referred to in the literature as "hook effect". Due to the hook effect, the system response is not unambiguous and it has therefore been customary to limit the system's range (dynamics) to the capacity of the primary and secondary reagents and to ensure possible false results by separating the 20 excess if the analyte has reacted with the primary reagent. reaction solution by any suitable means. When one of the reagents is coupled to a solid phase such as a cuvette surface, the most common separation method is cuvette washing.

The possibility of false results due to the Hook effect has prevented the widespread use of single-step assays, although there is a need for simpler and less costly analytics in routine diagnostics.

The novel method of the present invention provides a one-step assay which, in addition to the one-step assay, has a much wider measuring range than conventional multi-step assays without the risk of false results due to the hook effect.

The invention is characterized in that the measurement method used is capable of optically separating the signal given by the AbAgAb complexes formed in the reaction from the free labeled reagent. In addition, 5,104,585 to the present invention are characterized in that the sample and the primary and secondary reagents are simultaneously added to the reaction cuvette and the signal intensity provided by the reaction product is monitored as a function of time from the beginning of the reaction. This is hereinafter referred to as kinetic measurement. Kinetic measurement can provide information on the concentration of the analyte before the reaction proceeds to near equilibrium. The rate of increase in signal intensity dl / dt is proportional to the analyte concentration of 10 P and can be used to predict approximately the concentration of the analyte early in the reaction, and in particular whether the analyte concentration is higher than the reagent binding capacity. The kinetic measurement thus indicates whether the reaction equilibrium is formed to the left 15 or to the right of the peak H of the standard curve (Figure 5). When this information is obtained from reaction kinetics, it is also possible to permit concentrations of the analyte higher than the solid phase binding capacity and to measure higher concentrations of the analyte than conventional assays without the possibility of false results. 20 The scale of the system can thus be significantly increased. For example, normal 3-dimensional dynamics can be grown to 5-6 orders of magnitude by the method of the invention. If there is no need to extend the dynamics, this method provides. 25 optimizing the assay range as desired.

The invention is characterized in that the method according to the invention is characterized by the following steps: - v 30 - Two standard curves are determined using known standard samples to calibrate the assay.

<The first standard curve g = g (P) expresses the growth rate dl / dt of signal intensity I of the assay over a given time interval ti + At as a function of analyte concentration P. Another standard curve 35 indicates the value of the assay signal response R (t2, P) at the end of a given reaction time t2 as a function of analyte concentration 6104585P.

- When measuring unknown samples, the signal intensity I of the assay is recorded as a function of time and 5 parameters g representing the rate of growth of the signal intensity I are determined. In addition, the signal response R (P) is recorded after a certain reaction time.

When the reaction is nearing equilibrium, determine from the inverse function P = P (g) of the standard curve g = g (P) whether the unknown sample has a concentration P less than, about 10, or greater than the solid phase concentration q.

If P <q, read the sample concentration P from the standard curve P = P (R), if P = q read the sample concentration P from the standard curve P = P (g) and if P> q read the sample concentration P from either of the standard curves.

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Example 1

The invention will now be described in more detail by the application example for which the reaction schemes, reaction equations, standard curves and signal intensity curves shown in Figures 1-6 have been produced by mathematical modeling.

As an example of the practice of the invention, we provide an assay using a solid phase-bound biospecific reagent. Figure 1 shows a reaction scheme for the assay. The starting components of the reaction are the solid-phase biospecific reagent Ab, analyte Ag, and labeled reagent Ab *. The components Ab, Ag and Ab * are added simultaneously. Figure 2 shows the reaction equations / · 30 (formulas 1-4), showing all possible reactions and products. The letters k denote the association and dissociation rate constants of the reactions. Naturally, the reaction rates of these reactions are very different. Reactions in the liquid phase are fast 35 and reactions in the solid phase are slower. For this example, the values of the reaction constants typical of these assays have been selected.

7 104585

The equilibrium constant (affinity) of the biospecific reagents Ab and Ab * is chosen to be K = 1-1010 L / mol.

The association rates for the various reactions have been chosen as kj = 1 * 107 L mol'1 s'1 5 k3 = 1 * 109 L mol "1 s'1 k3 * 1 · 107 L mol" 1 s "1 k7 = 1 * 107 L mol ' 1 s'1

Since kdi „= cat / K, the following values for dissociation constants 10 k2 = 1 * 10'3 mol" 1 s "1 k4 = 1Ί0'1 mol" 1 s'1 k6 = 1 * 1 O'3 mol "1 s, respectively, are obtained. '1 k8 = 1 * 10 "3 mol" 1 s'1

It is further assumed that the concentration of the reagent Ab in solid phase A is q = 1 · 10 * 9 mol / L and the concentration of the labeled reagent Ab * is q * = 1 · 10 · 9 mol / L.

For the sake of simplicity, these reaction equations are written to correspond to reactions in solution and not to those required for reaction with the solid phase. The 20 diffusion times have not been specifically considered. The effect of the concentration gradients due to diffusion on the final result of the model shown is not significant because the solid phase is in suspension in the form of uniformly distributed microparticles and the average diffusion distances are short. For the sake of simplification, it is merely 'assumed that the rates of association and dissociation of reactions with the solid phase are two orders of magnitude lower than those of reactions in solution. This assumption includes 30 diffusion slowness in solid phase reactions.

Figure 2 shows the differential equations derived from the reaction equations.

• I

8 104585 Thial equations (formulas 5 to 10) whose solution describes reaction kinetics. The meaning of the variables xL is given in formula group 11 and the units of terms in step 12. The solution of these differential equations is only 5 numerically, and we have used Mathcad 6.0+ mathematics (Matsoft Inc., Cambridge,

Mass, USA). With this mathematical model, the reaction kinetics and the wear and formation of the various reaction components can be modeled with all possible starting values affecting the reaction.

Figures 3 and 4 show the reaction kinetics of the assay, i.e., the value of the signal intensities as a function of time. Since the reaction rate constants introduced in this model are exemplary in nature, the time axis must also be understood as a relative time axis. The signal intensity is directly proportional to the concentration of AbAgAb * produced in the reaction. Figure 3 shows the instantaneous signal intensity as a function of time t. Figure 4 shows the signal intensity integrated over time interval 0 to t as a function of time t.

20 Using the integral improves the signal-to-noise ratio of the measurement. Figures 3 and 4 show signal intensity curves for various analyte concentrations: a) 10 "to 11 mol / L, b) 10 to 10 mol / L, c) 10 to 9 mol / L, d) 10 to 8 mol / L, and: e) 10'7 mol / L Figure 5 shows a signal response curve, or standard curve, of the assay, as a function of the concentration of AbAgAb * in the reaction product, as a function of analyte Ag, peak P of the assay is marked H.

* w

Figure 3 shows that the signal intensity rise rate 30 is directly proportional to analyte Ag concentration P, i.e., higher analyte concentrations achieve a higher signal intensity over a given time. As the reaction approaches equilibrium, the signal intensity curves asymptotically curve toward equilibrium value 35 (curves a, b, and c). When the analyte concentration P exceeds 104585.

With the solid phase binding capacity q, i.e. 10'9 mol / L in this example, the signal intensity increases very rapidly at first (curves d and e) but then reaches a steady-state value corresponding to 5 signal intensities at a lower analyte concentration.

The curve lobe in Fig. 4 shows the integrated signal intensity integrated at time intervals 0 to t as a function of time at different analyte concentrations. In this case, the curves a, b, c, d and e are more gentle.

10

The curves shown in Figures 3 and 4 show the aforementioned hook effect such that, at concentrations of analyte higher than solid phase binding capacity, the signal intensity remains at a specified level, the lower the higher the concentration of analyte. In addition, the reaction equilibrium is rapidly achieved.

Signal intensity increase and potential hook effect can be predicted by recording the signal intensity increase rate dl / dt at different times t. When the reaction 20 reaches equilibrium, the signal intensity growth rate decreases to zero. At higher concentrations of the analyte, this state of reaction equilibrium is achieved earlier than at lower concentrations of the analyte. By fitting the growth rate curve to a first order or higher order polynomial function or some other function over a given time interval, the concentration range of the analyte can be determined from the fitting parameters. Considering the rate of increase in signal intensity is a simple and noise-tolerant method. Naturally, fitting can be done on either a linear or logarithmic scale.

From the linear scale of signal intensity increase dl / dt, the concentration of analyte can be directly deduced.

3 and 4 are a and d ·; 35 fitted to a logarithmic scale and a time interval t = 0 to 300 s for first order functions, i.e. lines denoted by 10 104585, respectively, aa and dd. These regression lines are mathematically denoted by log [R (t)] = g log (t) + c, where R (t) is the time dependent signal response, g is the slope of the regression line representing the reaction rate and c is the constant representing the value of the signal response at t * 1 s. The constant c thus also describes the concentration of the analyte.

When standardizing a measuring device, known standard samples can be used to determine the kinetic signal responses described above for various concentrations of analyte Ag and then the regression lines defined above for these signal responses. The result is a straight flock whose slope changes as a function of analyte Ag concentration, the analyte concentration exceeding the peak concentration. A function can be fitted to the slopes of this straight flock. An example of such a function fitted to the integral curves of Figure 4 is shown in Figure 6. From this curve it is noted that the slope of the assay up to the peak has the same value, but when the Ag concentration exceeds the peak, the value of slope g drastically decreases.

When an unknown sample enters the measurement, its signal response or integral of the signal response is monitored as the reaction progresses near equilibrium as described above, and finally the value of the slope of the regression line is determined. By reading from the curve in Figure 6 the concentration of the sample corresponding to this slope, it can first be determined whether the concentration of the unknown sample is less or greater than the concentration of the analyte corresponding to the peak. If the concentration is found to be lower, the concentration of the unknown 30 samples after the selected reaction time is read normally from the standard curve P = P (R) shown in Figure 5.

If the concentration is found to be in the peak area, read the concentration of the unknown sample from the graph P = P (g) in Figure 6. If the concentration is found to be above ^ · 35 peaks, the concentration of the unknown sample can be read from the 11 104585 curves of either Figure 5 or Figure 6.

The first order linear fit to the intensity growth curves used in the above example is exemplary. It is clear that other mathematical methods of interpretation can be used to determine the standard curve P = 5 P (g). For example, when fitting higher order polynomials to kinetic measurement curves, the coefficients of the polynomials describe the progress of the reaction. By considering the coefficients, the required response curves for the system 10 can be generated.

Example 2

It has already been stated above that the method of the present invention is particularly applicable to assays which can directly monitor the formation of complexes by a signal from a label attached thereto and in which the free label in the reaction solution does not give a significant signal. An example of such an assay is described below.

The assay utilizes a fluorescent molecule labeled biospecific secondary reagent and a primary reagent coated latex microparticles as a solid phase. The sample to be assayed and the secondary biospecific secondary reagent are added to the microparticles, after which complexes formed by the analyte molecule and the labeled biospecific reagent begin to bind to the reagent molecules on the surface of the microparticles.

The microparticle suspension is subjected to a beam of light that is sharply limited in volume and is, for example, limited by diffraction, so that this beam of light can target only one microparticle at a time. Due to the suspension movement, the microparticles drift into and out of the · · targeting volume. The dye on the surface of the microparticle excites and its emission intensity 12 104585 is measured by a photon detector, the intensity of an electrical signal over time being proportional to the rate of reaction and the concentration of the analyte in question.

Such a method may be implemented in particular by employing microparticles as solid phase and excitation of the fluorescent label bound thereto by confocal or double photon excitation, whereby excitation and detection of fluorescence are capable of optically separating light emitted by the microparticle at the point of alignment the stamps produce a significant signal. In this method, the laser beam used for fluorescence excitation 15 can be applied directly through the transparent wall of the reaction cuvette to the reaction suspension. The fluorescence signal can be obtained whenever the microparticle enters the target volume due to diffusion or fluid movement. To accelerate the measurement 20, the suspension in the cuvette may also be otherwise actuated or the cuvette may be moved or rotated at a suitable rate. At the same time, signals are obtained whose amplitude is directly proportional to the number of labeled complexes bound to the microparticle in question at the present time.

In this way, the kinetic progress of the reaction can be followed up to steady state on the basis of fluorescence intensity. The required statistical accuracy is achieved when statistically large numbers of microparticles are observed. The response of the signal response shall be proportional to that of the standard samples used to calculate the concentrations of unknown samples.

It will be apparent to one skilled in the art that various embodiments of the invention may vary within the scope of the following claims.

Claims (4)

A biospecific assay method, wherein an analyte and a labeled secondary reagent are applied simultaneously with a primary reagent to a reaction cuvette, wherein a reaction product is formed of said components, and using such a detection method for the signal delivered by the marker, which distinguishes it from the the reaction product originating from the signal originating from a marker which remains in free form in the solution, and which detection method records the signal intensity of the marker bound to the reaction product as a function of time, characterized in that - at the initial stage of the reaction at one or more time points or time intervals before reaching equilibrium, a parameter g describing the growth rate of the signal intensity of the marker contained in the reaction product is recorded, and - measuring the signal response R of the labeled reaction product after a certain reaction time, and - determine the analyte content P by means of predetermined standard curves P (R) or P (g) depending on whether the analyte content P is lower or higher than the content of the primary reagent. * 2. A method according to claim 1, characterized in that the growth rate of the signal intensity is determined by fitting a polynomial function to a signal intensity curve measured as a function of time, and the analyte content is determined by the constants of the polynomial function. • « Method according to claim 2, characterized in that the function to be fitted is a function of the first degree and that the analyte content of the function is read by the angle coefficient of the function. Method according to claims 1-3, characterized in that a fluorescent marker is used as a marker and 104585. 16 that the measurement method for the signal delivered by the cursor is based either on two-photon excitation or confocal optics. m