FI104586B - Method of monitoring a bioaffinity reaction in realtime - Google Patents

Description

104586

METHOD FOR REAL-MONITORING BIOAFFINITY REACTION - FÖRFARANDE FÖR REALTIDSUPPFÖLJNING AV EN BIO-AFFINITETSREAKTION

Immunoassay is an established biospecific assay and its various applications are widely used in routine diagnostics and research laboratories. Another group of biospecific assays, although still under development, is the DNA and RNA hybridization assay. Biospecific assays generally use two biospecific reagents, a primary reagent and a secondary reagent (e.g., antibody, DNA or RNA probe), each of which binds to the specific determinants of the analyte molecule, forming the three molecule complex (layer structure) and normally stamped. The labels used today are radioisotopes, enzymes, luminescent labels and fluorescent labels. The most common sample material to be analyzed is blood serum.

The method of the present invention relates to those fluorometric biospecific assays in which one biospecific reagent is attached to microparticles as a solid reaction medium. The reaction medium and the components bound thereto are hereinafter referred to as the solid phase. The second biospecific reagent is labeled with a fluorescent label and this reagent is simply referred to hereinafter as the label. Assays can be divided into two main groups of operation: 1) 25 assays with excess solid phase bound. and (2) assays that contain a low amount of solid-phase bound reagent, also called a competitive assay.

The first group is applicable to analyte molecules Ag, which have at least two specific determinants. This assay uses an excess of 104586 relative to Ag.

2 solid phase bound biospecific reagents Ab.

The second reagent for the reaction is the labeled reagent Ab *.

AbAgAb * complexes bind to the solid phase. The signal response of the assay is linear. DNA and RNA assays r5 are also performed according to the same principle.

Another group is suitable for small analyte molecules Ag.

An aliquot of solid phase A bound biospecific reagent Ab is used in this assay.

The second reagent in the reaction is the labeled reagent Ag *, i.e. the labeled analyte molecule is used as the 10 reagent. The Ag and Ag * components bind to the solid phase reagent Ab in proportion to their concentrations and the reaction is termed the competing binding. The signal response of the assay is non-linear.

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In addition to the two main groups set forth above, the method of the present invention is applicable to studying the reaction kinetics of various biomolecules Ab and Ab *, i.e., monitoring the formation of the reaction product over time. In this case, the binding of the labeled molecule Ab * to the solid phase molecule Ab is simply monitored. Ab, Ag, and Ab * symbols used above generally refer to biospecific molecules and are not limited to immunological antibodies and antigens.

25 The problem with conventional assays and assays is their complexity. For example, the measurement of a hormone in the blood by fluorometric immunometric assay usually requires many steps: cell separation from serum, serum sample dosing and * · <30 dilution, incubation with solid phase, free fraction washing, incubation with labeled reagent, additional wash separation, measurement measuring the signal.

Separation of the cells from the serum is necessary because in 35 conventional fluorometric methods the high light absorption of hemoglobin 3 104586 interferes with the measurement. Reagents must be added in two steps and occasionally separation of the free fraction must be performed, otherwise the assay response, i.e. the concentration of AbAgAb *, would be reversed when the analyte concentration exceeds the solid phase binding capacity. 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". The free label also has to be distinguished, because otherwise, due to the signal it sends, the signal transmitted by the bound label would not be determined.

In routine diagnostics, there is a need for simpler and less expensive analytics. The novel fluorescent labeled biospecific assay of the present invention is a single step procedure and does not necessarily require label separation and is suitable for assaying whole blood or other biological suspensions. Furthermore, the present invention makes it possible to measure the bioafinity reaction kinetics simply 20 and in real time.

The process of the invention is characterized by the following steps: - Prepare microparticles which are coated with the biospecific primary reagent that binds the analyte of interest.

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• - The biospecific secondary reagent is labeled with the fluorescent molecule F.

- Sample 30 and biospecific secondary reagent are added to the microparticles, followed by: · binding of the analyte molecule 4 104586 and labeled biospecific reagent to the reagent molecules on the surface of the microparticles by bioaffinity.

- The microparticle suspension is subjected to a light beam having an application volume limited by diffraction, so that this light beam can only be applied to one microparticle at a time. Because of the movement of the suspension, the microparticles drift into and out of the application volume. The dye F on the surface of the microparticle is excited and its emission intensity is measured by a photon detector, the intensity of the electrical signal given by time being proportional to the rate of reaction progression and the current. analyte concentration. By monitoring signal growth, information on kinetic reaction parameters and analyte concentration can be obtained before the reaction reaches an end point of 15, i.e. equilibrium.

The use of homogeneous microparticles as a solid phase for the biospecific assay enables a bioaffinity reaction in a short time. Because the average distance between the analyte molecules and the labeled reagents in the reaction on the microparticles is very small, the reaction equilibrium can be rapidly achieved and results in the formation of a reagent labeled with the reagents on the microparticles.

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close complexes.

It is known that the background signal from scattering and autofluorescence can be removed by using dual photon excitation (FI 90175, FI 951040 and WO 9622531). What's New: However, the method of the present invention has the observation that even the known auto-fluorescence and light scattering of whole blood does not interfere with measurement when twin-photon excitation is used. The high visible light absorption of hemoglobin is also not detrimental because - the wavelength of the laser beam is in the NIR range.

] 3545 bound and free labeled reagent in this method.

The separation of the signals emitted by the 5 diffraction is based on the fact that the dual photon excitation fluorescence excitation and detection used in the method are capable of optically distinguishing the light emission emitted by the microparticle at the laser beam alignment point from the free signal emitted by its labeled reagent.

Also novel in the process of the present invention is that the laser beam used for fluorescence excitation is applied directly through the transparent wall of the reaction cuvette to the reaction suspension. The fluorescence signal can be obtained whenever the microparticle enters and exits the targeting volume due to diffusion or fluid movement. At the same time, signals are obtained whose amplitude is directly proportional to the number of labeled complexes bound to the microparticle in question, i.e., it is possible to monitor the kinetic progress of the reaction up to equilibrium. The signal strength also depends on how the microparticles randomly drift cut the alignment volume. Nonetheless, the progress of the bioaffinity reaction can be tracked with sufficient accuracy based on the intensity of fluorescence they emit when statistically sufficient microparticles are observed.

Due to the above two characteristics, this method allows rapid one-step biospecific determination, for example, of whole blood samples without separation; and without the risk of erroneous analysis results from the hook effect, since the hook effect can be predicted from the rate of rise of the kinetic signal.

Dual photon excitation can occur when a high excitation light target causes the density of photons per unit volume and time unit to be so high that the energy of two photons 35 is absorbed into the same chromophore. In this case also the absorber • «104586.

6 healing energy is the sum of the energy of two photons. The absorption of a single photon in a dye is, according to the concepts of probability calculation, an independent event, and the absorption of multiple photons is a series of single, independent events. The probability of absorption of a single photon can be described by a linear function as long as the excited energy states are not saturated. The absorption of two photons is a non-linear process. In double-photon excitation, excitation of the dye molecule 10 occurs only when both photons are simultaneously absorbed. The absorption probability of two photons is the product of the absorption probabilities of the individual photons. The emission of two photons is thus a quadratic process.

15 The characteristics of an optical system for microparticle excitation can be described by the system's response to a point light source. The point light source produces the intensity distribution characteristic of the optical system at the focus point (point-20 response) due to diffraction. Normalized, this intensity distribution is the probability distribution of how photons emitted from a point light source arrive in the focus area. With dual photon technology, the tuning probability distribution is first. .. normalized product of intensity distributions of photonic and second photon. The probability distribution thus obtained in the 3-dimensional space, especially in the depth direction, is clearly more limited to the probability distribution of a single photon. Dual photon excitation thus only excites the fluorescence that is generated by the focus of the light beam; 30 points in a clearly delimited 3-D environment.

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When the toner is excited by dual photon excitation, the light scattering around the target point and optical components is significantly reduced compared to normal excitation. Further, dual photon tuning reduces the occurrence of background fluorescence in the sample at the outer side of the targeting point, 104586, in the sample environment, and in optics. Because the excited beam of light must be focused to the lowest point possible, dual photon excitation is best suited for examination of small sample volumes or structures, which is also an issue in the method of the present invention.

Another advantage of dual photon tuning is that visible or near-infrared (NIR) light can be used, for example, in ultraviolet or blue region excitation. Likewise, excitation in the visible region can be performed by a beam of light in the NIR region. Because the light source wavelength is significantly longer than the dye emission wavelength, the scattering and possible autofluorescence of the light source at and around the wavelength can be effectively suppressed by using low pass filters 15 (at least 10 orders of magnitude) and prevented from reaching the fluorescence emission detector. The red light absorption by the red cells in the NIR region is very low and we have also found that, for example, the excitation light at 1064 nm does not excite the fluorescence of hematoprophyrins and consequently the red fluorescence background fluorescence is insignificant even though they are present.

• j In most cases, dual photon tuning requires the use of high-power ultra-short pulse laser devices.

In our experiments, we have found that dual-photon excitation and short-lived fluorescent labels can achieve very good signal-to-background ratios and sensitivity. As examples of short-lived double fluorescent dyes suitable for double-tone excitation, coumarin and rhodamine derivatives may be mentioned.

According to the present invention, the measurement of the microparticles is performed directly from the reaction cell provided with an optical window, and the measurement can be kinetic, i.e. measurement; complex formation on the surface of the microparticle 35 is monitored. In this case, the microparticles drift to the target point of the laser beam 8 104586 due to e.g. diffusion-based motion. In order to speed up the measurement, the suspension in the cuvette may also be otherwise moved or the cuvette may be moved or rotated at a suitable speed.

5 The microparticles drift randomly to the targeting point and the number of microparticles drifting to the targeting point depends on the speed of movement and the concentration of microparticles. A sufficient number of microparticles are identified and analyzed to obtain statistically accurate 10 results. The results of the measurements shall be related to the measurements carried out on standard samples, on the basis of which the final results shall be calculated.

It is also characteristic of the present invention that the microphotometric measuring system disclosed herein is capable of optically separating light emission from the surface of the microparticles from light emission from a solution surrounding the microparticles. This resolution is based on the fact that when the reaction solution is suitably diluted, only one microparticle fits into the target region of the photon detector at a time, and the background signal from free labeled reagents or red cells in solution is minimal. Optical separation provides sufficient separation of free and bound fraction without chemical or physical separation, but if necessary, separation can be effected by any known separation method, such as washing, filtration or centrifugation.

The method of the present invention and the apparatus required thereto can be practically implemented in a variety of ways. However, it is essential in the invention to observe that by directing a laser beam directly on the reaction suspension, the progress of the reaction can be monitored, and that red cells, like other cells, do not produce a measurable background signal: whether these cells are at or outside the targeting point. The fluorescence detector can only be activated for the duration of the laser pulse used for dual photon excitation, whereby the photon emitted during the intervals of the excitation pulses will not be measured. Similarly, the photon detector can only be activated at the moment the microparticle is in the focus volume. Information on the drift of the microparticle 5 to the targeting volume is obtained, for example, from the scattering generated by the laser beam wavelength, which is best measured by a confocal scattering detector. The scattering signal produced by blood cells is an order of magnitude lower, since their refractive index is significantly lower than that of micro-10 particles. Cell-mediated scattering and background fluorescence can be further reduced by puncturing cell membranes with isotonic solution media, whereby the cellular fluid and hemoglogin (hematoporphyrin) are diluted in the assay suspension.

The method of using dual photon tuning according to the present invention can also be made multiparametric as disclosed in WO 9622531. Microparticles are then used, which are divided into several different classes coated with primary reagents that bind different analytes. Herein, it is of particular importance to the manufacturing cost of the measuring device that the excitation light used to identify the particle and the double photon excitation of the analyte quantification stamp F .. can be performed with the same laser beam having a red or NIR wavelength.

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.

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Claims (2)

10 104586 1. A method for real-time monitoring and measuring the kinetics of a bioaffinity reaction between a biomolecule and its specific fluorescent labeled reagent molecule in biological fluids or suspensions using microparticles as a solid phase of a bioaf reagent-coated microparticles, and the fluorescence of microparticle-binding labels is induced by double photon excitation, characterized in that the biological fluid or suspension to which the laser beam is applied is a reaction suspension in which the microparticles are randomly drifted into the laser beam targeting volume. Method according to Claim 1, characterized in that the information about the drift of the microparticle to the target volume is obtained by scattering at the wavelength of the laser beam used for double photon excitation, which is measured by a confocal scattering detector. , «:: • I · i a M i 11 104586