Diagnostic reagent for quantitatively measuring procalcitonin in a sample

Abstract

A diagnostic reagent for quantitatively determining procalcitonin in a sample, suitable for simple photometric turbidimetric analysis and with high sensitivity for quantitatively determining procalcitonin in a sample, comprising an aqueous suspension of polymer particles to which an antibody against procalcitonin is covalently bound, wherein the tendency for aggregation / sedimentation is not detectable or is negligible even after prolonged standing, and the specific reactivity of the particles remains substantially unchanged. To provide such a diagnostic reagent, it is proposed according to the present invention that the suspended polymer particles have an average particle size in the range of 150-450 nm, the suspension comprises a proportion of dissolved sugar or sugar alcohol in the range of 25-250 g / l, and the suspension has a pH in the range of 8-10.

Description

[Technical Field]

[0001] The present invention relates to a diagnostic reagent for quantitatively measuring procalcitonin in a sample, which is an aqueous suspension of polymer particles to which an antibody against procalcitonin is covalently bound, and to a method for preparing such a diagnostic reagent. [Background technology]

[0002] Procalcitonin (PCT), a precursor of the hormone calcitonin, is one of the most important markers for diagnosing sepsis. PCT is commonly used to control sepsis, especially postoperatively. This is because the liver and adipocytes produce large amounts of PCT, especially in postoperative bacterial infections. However, in daily clinical practice, PCT is not only used postoperatively, but also in the initial diagnosis and differentiation of sepsis caused by viruses, bacteria, fungi, and protozoa / parasites, as well as in follow-up management, i.e., as an auxiliary tool to confirm the effectiveness of drugs used.

[0003] Sepsis has several diagnostic ranges. Below 0.5 ng / ml, sepsis is unlikely. Above 0.5-2 ng / ml, inflammation is likely. The presence or absence of sepsis must be determined by repeated time-delayed measurements of PCT and other parameters, if necessary. Above 2-10 ng / ml, sepsis possibly involving bacteria appears. Above 10 ng / ml, severe septic shock appears. This classification is used to characterize sepsis and guide the treatments derived from said characterization. This is especially important with regard to the time factor.

[0004] Statistically, every hour that sepsis goes undiagnosed or its diagnosis is delayed is associated with a 7% increase in mortality. This makes sepsis the third leading cause of death in all regions of the world. Given this background, there are very high demands on the sensitivity and reproducibility of sepsis diagnosis. Reagents used for diagnosis must be able to provide reliable results even after long-term storage.

[0005] Various immunoassay-based diagnostic methods are known in the prior art that allow quantitative determination of PCT in a patient's blood. Antibodies against PCT can be bound to polymer particles, which are then reacted with PCT contained in the test sample, followed by analysis.

[0006] CLIA (chemiluminescence immunoassay) methods require the use of chemiluminescent reagent components, allowing analysis based on the chemiluminescence generated, which generally requires continuous resuspension of particles and requires specialized equipment.

[0007] The PETIA (particle-enhanced immunoturbidimetric assay) is a turbidimetric assay based on the decrease in light transmittance of the reaction solution. The more PCT present in the sample, the stronger the cross-linking that occurs between antibody-occupied particles, resulting in a more turbid solution and a decrease in transmittance. Therefore, no additional chemiluminescent reagents are required, and the measurement can be performed with a simple photometer system available in virtually every diagnostic laboratory.

[0008] For the accuracy and diagnostic relevance of turbidimetry, it is absolutely important that the degree of turbidity generated as a result of the reaction with PCT always correlates reliably with the quantification of PCT in the sample. This is not the case, for example, when antibody-loaded particles spontaneously / nonspecifically aggregate or precipitate, or when their specific antigen-binding capacity for PCT changes over time after preparation. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a need for a diagnostic reagent that is suitable for simple photometric turbidimetric analysis and has high sensitivity for quantitatively measuring procalcitonin in a sample, which reagent is an aqueous suspension of polymer particles to which antibodies against procalcitonin are covalently bound, and which shows no or only a slight tendency for aggregation / sedimentation even after prolonged standing, and the specific reactivity of the particles remains substantially unchanged. [Means for solving the problem]

[0010] This issue is The suspended polymer particles have an average particle size in the range of 150 to 450 nm, The suspension comprises a proportion of sugar or sugar alcohol dissolved in the range of 25 to 250 g / l; The suspension has a pH in the range of 8 to 10. This is achieved through invention.

[0011] The combination of features proposed according to the present invention provides a diagnostic reagent for the quantitative determination of PCT with very high sensitivity in the relevant range for very low PCT concentrations in samples. Moreover, this high sensitivity is maintained even during long-term storage of up to 24 months. This high storage stability is particularly noteworthy considering the fact that the polymer particles used according to the present invention have a relatively large particle size of 150 nm or more.

[0012] Large particle sizes of polymer particles are clearly desirable, especially with regard to the required high sensitivity of diagnostic reagents. Relatively large particles have a correspondingly large surface area, and therefore a correspondingly large number of antibodies against PCT can be covalently bound thereto. Thus, there are more potential coupling sites for the PCT molecule, which increases the sensitivity of the diagnostic reagent.

[0013] Large particle sizes are also advantageous in terms of light scattering properties. The "light yield" was found to be significantly better for the polymer particles according to the invention. With the same degree of cross-linking, the particles according to the invention essentially re-amplify the signal. The larger the particle, the stronger the signal. The underlying principle is the effect of Rayleigh scattering, Mie scattering, etc. on colloidal nanoparticles in suspension.

[0014] The present invention surprisingly achieved better storage stability than expected, despite the large particle size. Normally, larger particles have a greater tendency to settle, making resuspension difficult over longer storage periods. However, the proposed combination of sugar or sugar alcohol dissolved in the suspension and the pH of the suspension, surprisingly, makes it possible to obtain a highly storage-stable suspension with consistent reactivity, even with the relatively large particles of the present invention.

[0015] The diagnostic reagent according to the present invention is for quantitatively measuring PCT in a sample. In the context of the present invention, a "sample" is understood to be any material prepared for analytical purposes containing an analytical amount of PCT. In most cases, the sample will be a sample of fresh whole blood, which may have been appropriately prepared for the purpose of performing the analysis. However, the present invention also encompasses other liquid samples containing PCT, such as standard solutions and calibrators.

[0016] The diagnostic reagent of the present invention is suitable for quantitative PCT measurement. "Quantitative measurement" in this context means that inferences can be made about the amount of PCT in a sample from the amount of PCT molecules bound to the polymer particles.

[0017] In the reagent of the present invention, the polymer particles are present in an aqueous suspension. In this context, the term "aqueous suspension" refers to a slurry of polymer particles carrying an antibody against procalcitonin in water, or an aqueous solution in which a sugar, a sugar alcohol, and / or a buffer substance is dissolved. In the context of the present invention, the term "suspension" must be interpreted narrowly, meaning that almost all particles are suspended in the aqueous phase and do not settle. Thus, a suspension in the sense of the present invention means that at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the polymer particles are freely suspended.

[0018] According to the present invention, the average particle size of the polymer particles is in the range of 150 to 450 nm. In some embodiments within the claimed range, the average particle size is preferably greater than 190 nm, greater than 240 nm, or even greater than 300 nm. In other embodiments within the claimed range, the average particle size is preferably less than 410 nm, less than 360 nm, or even less than 300 nm. In the present case, the particle size of the polymer particles is determined by dynamic light scattering at 25°C, for example, using a Malvern Zetasizer Pro. The average particle size refers to a numerical average.

[0019] The polymeric material comprising the polymeric particles is preferably selected from acrylic polymers, dextran-epichlorohydrin copolymers, polymethyl methacrylate, polystyrene, silica (silica gel), and combinations thereof. In some embodiments, the polymeric particles are composed entirely of one polymeric material or a mixture thereof. In other embodiments, the particles are composed of multiple layers of different polymers.

[0020] In certain embodiments, the particles can comprise a core and one or more layers coated on the core. The core and one or more layers coated thereon can be composed of one polymeric material, different polymeric materials, or non-polymeric materials, provided that the polymeric particles consist primarily of polymeric materials.

[0021] Polymer particles in the sense of the present invention are meant when the particles consist of at least 80% by weight, at least 90% by weight, at least 95% by weight, or 100% by weight of polymeric material or a combination of different polymeric materials.

[0022] The overall density of the polymer particles is preferably 0.9 to 1.1 g / cm 3 The range is particularly preferably 1.0±0.5 g / cm 3 The range is.

[0023] On the surface, the polymer particles preferably comprise functional groups that allow for covalent binding of antibodies to the surface of the polymer particles, preferably selected from carboxyl groups (-COOH), primary amine groups (-RNH2), aromatic amine groups (-ArNH2), chloromethyl groups (-CH2Cl), aromatic chloromethyl groups (ArCH2Cl), amide groups (-CONH2), hydrazide groups (-CONHNH2), aldehyde groups (-CHO), hydroxyl groups (-OH), thiol groups (-SH), epoxy groups, and biotin-avidin.

[0024] The aqueous portion of the diagnostic reagent suspension of the present invention comprises a dissolved sugar or sugar alcohol portion at a concentration ranging from 25 to 250 g / L. In certain embodiments, the sugar and / or sugar alcohol concentration ranges from 50 to 200 g / L. In some embodiments, the concentration is 50 to 150 g / L, and in other embodiments, the concentration is 150 to 250 g / L.

[0025] In some embodiments, the sugar or sugar alcohol dissolved in the aqueous portion of the suspension is selected from sucrose, mannitol, sorbitol, xylitol, maltitol, raffinose, rhamnose, and combinations thereof. When a sugar / sugar alcohol combination is used, the above amounts refer to the total sugar / sugar alcohol ratio of that combination.

[0026] The aqueous suspension in which the antibody-loaded polymer particles are suspended has a pH in the range of 8 to 10. In some embodiments, the pH is in the range of 9.0 to 10.0. In certain embodiments, the pH of the suspension is greater than 9. In some embodiments, the pH of the suspension is in the range of 9.1 to 10.0. In certain embodiments, the pH of the suspension is 9.0±0.5. In particular embodiments, the pH of the suspension is 9.5±0.1.

[0027] The diagnostic reagents claimed according to the present invention are characterized by very high storage stability. This is particularly manifested by the fact that the turbidity of the suspension remains stable compared to its initial value even after several months. The suspensions of the present invention are particularly characterized in that the absorbance of the suspension at 660 nm within 90, 120, 150, or 180 days from the time of preparation of the suspension deviates by less than 5% from the initial value on day 0. In some embodiments, the deviation is less than 5% within 12 months or even within 24 months. In some embodiments, the deviation is even less than 3% or even less than 2%. In some embodiments of the present invention, slight deviations in the degree of turbidity within the aforementioned time periods can also be measured at other wavelengths in the range of 340 to 800 nm.

[0028] In certain embodiments of the invention, the antibody covalently attached to the polymer particle is a monoclonal antibody to PCT. In other embodiments, the antibody is a polyclonal antibody to PCT. In yet other embodiments of the invention, the polymer particle carries a covalently attached recombinant antibody to PCT or a covalently attached antibody fragment to PCT.

[0029] The present invention also relates to a method for preparing a diagnostic reagent of the above type, in which polymer particles are first contacted with an antibody against PCT under conditions that result in covalent binding of the antibody to the surface of the polymer particles. The loading of the antibody onto the polymer particles is carried out in aqueous suspension at a specific temperature and a specific pH for a specific period of time.

[0030] The method of the present invention is characterized in that the covalent attachment of the antibody via the functional groups on the surface of the polymer particles occurs at a pH of 3 to 6. In one embodiment, the covalent attachment occurs at a pH of 3 to 5. In one embodiment, the pH of the reaction resulting in the covalent attachment of the antibody via the functional groups on the surface of the polymer particles is 4.0±0.5.

[0031] The pH of the reaction leading to the covalent attachment of the antibody via the functional groups on the surface of the polymer particles is preferably set using inorganic or organic buffer substances such as borate, citrate, phosphate, malate, maleate, succinate, acetic acid / acetate, etc. The pH is preferably set / adjusted using hydrochloric acid / sodium hydroxide solution.

[0032] In some embodiments of the methods of the present invention, covalent attachment of the antibody via functional groups on the surface of the polymer particles occurs at a temperature in the range of 20-29° C. In other embodiments, the reaction occurs in the range of 30-34° C., or even in the range of 35-45° C.

[0033] In some embodiments of the methods of the present invention, the covalent attachment of the antibody occurs at one of the predetermined pH ranges and at one of the predetermined temperature ranges described above for 20 to 80 hours, hi some embodiments, the reaction occurs for more than 30, more than 40, more than 50, or even more than 60 hours.

[0034] At the end of the above-specified period, the pH of the suspension of antibody-loaded polymer particles is lowered to a range of 8 to 10. For this purpose, one or more of the alkalinizing agents or buffer substances mentioned above can be added.

[0035] The pH in the suspension is preferably set using a buffer substance with an amine group, such as EPPS, HEPPS, tricine, tris, glycylglycine, bicine, TAPS, boric acid, ethanolamine, CHES, glycine, and CAPS, etc. The pH is preferably set / adjusted using a hydrochloric acid / sodium hydroxide solution.

[0036] The polymer particles carrying antibodies or antibody fragments obtained using the method according to the present invention exhibit, as mentioned above, very high sensitivity and very high storage stability.

[0037] It should be noted that for the purposes of disclosure, all features that would be apparent to one skilled in the art from the present specification, drawings, and claims, even if they are specifically described only in connection with certain other features, can be combined individually or in any combination with other features or feature groups disclosed herein, unless this is expressly excluded or unless the technical circumstances make such a combination impossible or meaningless. For the sake of brevity and readability, a comprehensive and explicit listing of all possible combinations of features has been omitted herein.

[0038] It should be further noted that it is obvious to those skilled in the art that the following embodiment examples are merely intended to illustrate possible embodiment examples of the present invention. Therefore, those skilled in the art will easily understand that all other embodiments having the features or combinations of features according to the present invention as set forth in the claims also fall within the scope of protection of the present invention. For the sake of brevity and readability, a comprehensive and explicit listing of all possible embodiments is omitted in this specification. [Brief explanation of the drawings]

[0039] [Figure 1] : The effect of incubation time on sensitivity was investigated. [Figure 2] : The effect of pH on sensitivity was investigated. [Figure 3] : Results of storage stability investigation [Figure 4] : Results of storage stability investigation [Figure 5] : The effect of particle size on measurement accuracy [Figure 6] : The effect of particle size on sensitivity and linearity [Figure 7] : Results of investigating the effect of sugar concentration on calibration [Figure 8] : The effect of various combinations of sucrose and pH on sensitivity was investigated. [Figure 9] : The effect of various combinations of sucrose and pH on sensitivity was investigated. [Figure 10] : The effect of various combinations of sucrose and pH on sensitivity was investigated. [Figure 11] : The effect of incubation time on sensitivity was investigated. [Figure 12] : Results of investigating the stability of reactivity at pH 8.1 [Figure 13] : Results of investigating the stability of reactivity at pH 9.0 [Figure 14] : Results of investigating the stability of reactivity at pH 9.5 [Figure 15] : Results of long-term stability investigation [Figure 16] : Results of long-term stability investigation DETAILED DESCRIPTION OF THE INVENTION

[0040] Example of an embodiment Below we present various comparative tests in which different parameters of the method for producing the diagnostic reagent according to the invention were varied. For this purpose, polystyrene polymer particles were loaded with antibodies against PCT under various conditions.

[0041] In one embodiment variation, the polymer particles had carboxyl groups (-COOH) as functional groups, and in another embodiment variation, the polymer particles had chloromethyl groups (-CH2Cl) as functional groups.

[0042] I. Variations of the embodiment having a chloromethyl group 1. Incubation time The polymer particles used had an average particle size of over 350 nm. The particles were coupled to the antibody for 15 to 48 hours, and the results for different batches are shown in Figure 1.

[0043] These results are expressed as absorbance at 660 nm, and the relatively high absorbance value of the batch with an incubation time of 48 hours indicates that the batch with a long incubation time provides significantly higher sensitivity at each PCT concentration in the sample because a significantly higher proportion of the PCT contained in the sample is bound than in the other batches. Therefore, the amount of PCT contained in the sample can be measured in a quantitatively more accurate manner.

[0044] 2. pH In these experiments, the pH was varied from 4 to 9 during the coupling reaction.

[0045] The results shown in Figure 2 indicate that better loading efficiency is achieved at lower pH values, resulting in polymer particles with a higher binding capacity for PCT and therefore higher sensitivity.

[0046] 3.Storage stability To confirm the suspension stability of the diagnostic reagent of the present invention, an aqueous suspension of polymer particles was mixed with a test sample (physiological saline), and the turbidity of the resulting mixture was then measured. The polymer particles used in this manner were stored under different pH conditions for up to 120 days.

[0047] The results shown in Figures 3 and 4 show that the turbidity is significantly lower when the polymer particles are stored at pH 9.0 than when the samples are stored at pH 8.1, especially when the samples are stored for more than 60 days. This clearly indicates that the tendency for aggregation and sedimentation is significantly lower for the batches stored at pH 9.

[0048] As can be seen in Figures 12-14, the reactivity stability in the higher pH range, i.e., pH 9.0 (see Figure 13) to pH 9.5 (see Figure 14), is better than the lower range (see Figure 12: pH 8.1). Particularly high reactivity stability is observed in the pH range above 9 (see Figure 14: pH 9.5) over the 60-day period studied here.

[0049] Figures 15-16 show high long-term stability up to 24 months in one embodiment using a storage buffer at pH 9.0. The small shift in the curve at 12 months for the calibrator reactivity test results can be explained by the detector lamp being replaced after 12 months.

[0050] II. Embodiment Variations with Different Particle Sizes To investigate the effect of particle size, polymer particles with chloromethyl groups and an average particle size greater than 350 nm were compared with polymer particles with chloromethyl groups and an average particle size less than 300 nm.

[0051] Figure 5 shows that the reactivity is significantly stronger for polymer particles with an average particle size greater than 350 nm than for polymer particles with an average particle size less than 300 nm, especially in the concentration range of 0.2 to 2 ng / mL, which is the range of two medically relevant detection ranges for procalcitonin. This significantly improves the measurement accuracy.

[0052] Recovery (precision of concentration) is better for polymer particles with an average particle size greater than 350 nm than for polymer particles with an average particle size less than 300 nm (see Table 1 below).

[0053] [Table 1]

[0054] Figure 6 shows the linearity of the sensitivity (measured as precision at the cutoff) and recovery of diluted samples.

[0055] For polymer particles with an average particle size of less than 300 nm, linear measurement is only possible up to the target value of 35 ng / mL. At higher concentrations, the measurement value becomes constant.

[0056] Polymer particles with an average particle size greater than 350 nm have linear recoveries up to 65 ng / mL. Thus, the correlation coefficients for larger particles are higher than those for smaller particles.

[0057] Sample recovery and precision at the medically relevant cutoff (0.5ng / mL PCT) were also significantly better for larger particles than for smaller particles (see Table 2 below).

[0058] [Table 2]

[0059] III. Variations of the embodiment having different sugar concentrations To investigate the effect of different sugar concentrations, polymer particles with chloromethyl groups and an average particle size greater than 350 nm were analyzed in storage buffers with different sugar concentrations.

[0060] Figure 7 shows that similar calibration curves are obtained when comparing 50 g / L, 75 g / L, and 125 g / L sucrose in storage buffer, although precision (measured as CV% of a 20-fold assay of a sample containing 0.5 ng / mL PCT) is significantly improved (lower CV%) at higher sucrose concentrations (see Table 3 below).

[0061] [Table 3]

[0062] Figures 8, 9, and 10 show data on the combined effect of sucrose and pH. Chloromethyl particles are resuspended in 125 g / L sucrose and three storage buffers at pH 8.1 / 8.5, and 9.0. The slope of the linear regression line of the reagent blank values ​​for the different measurement days corresponds to particle aggregation and should be as close to 0 as possible (Excel slide "Combined Effect suc-pH").

[0063] IV. Carboxyl Group-Containing Embodiment Variations The polymer particles used had an average particle size of less than 310 nm. The particles were coupled to the antibody for 12-24 hours, and the pH of the coupling reaction was 4-6.

[0064] As shown in the results in Figure 11, good reactivity can be achieved using polymer particles with carboxyl groups. However, in comparison, polymer particles containing chloromethyl groups show significantly stronger reactivity.

[0065] Polymer particles containing carboxyl groups also require activation (EDC+NHS) prior to the coupling reaction to render the COOH groups reactive. This activation is complex and does not always work in a reproducible and batch-uniform manner. In this case, activation was actually successful only in one of five attempts. In comparison, polymer particles with chloromethyl groups are much more reliable in coupling (data here from three batches).

Claims

1. 1. A diagnostic reagent for quantitatively measuring procalcitonin in a sample, said reagent being an aqueous suspension of polymer particles to which an antibody against procalcitonin is covalently bound, said diagnostic reagent comprising: The covalent bond of the antibody is formed through functional groups disposed on the surface of the polymer particles, the functional groups being chloromethyl groups (-CH ２ Cl), the suspended polymer particles have an average particle size in the range of from greater than 300 nm to 450 nm; the suspension comprises a proportion of sugar or sugar alcohol dissolved in the range of 25 to 250 g / l; The suspension has a pH in the range of 9.0 to 10.

0. Diagnostic reagents.

2. 2. The diagnostic reagent of claim 1, wherein the polymer of the polymer particles is selected from the group consisting of acrylic polymers, dextran-epichlorohydrin copolymers, polymethyl methacrylate, polystyrene, and silica.

3. 3. The diagnostic reagent according to claim 1, wherein the sugar or sugar alcohol is selected from the group consisting of sucrose, mannitol, sorbitol, xylitol, maltitol, raffinose, rhamnose, and combinations thereof.

4. The diagnostic reagent according to any one of claims 1 to 3, wherein the absorbance of the suspension at a wavelength of 660 nm within 90 days from the time of preparation of the suspension deviates by less than 5% from the initial value on day 0.

5. The diagnostic reagent according to any one of claims 1 to 4, characterized in that the antibody against procalcitonin covalently bound to the polymer particles is a monoclonal, polyclonal or recombinant antibody or antibody fragment.

6. A method for preparing a diagnostic reagent according to any one of claims 1 to 5, characterized in that the covalent attachment of the antibody via the functional groups on the surface of the polymer particles occurs at a pH of 3 to 6.

7. 7. The method for preparing the diagnostic reagent of claim 6, wherein the covalent attachment of the antibody via the functional groups on the surface of the polymer particles occurs at a temperature in the range of 20-29°C, in the range of 30-34°C, or in the range of 35-45°C.

8. 8. The method of claim 6 or 7, wherein the covalent binding of the antibody occurs at a defined pH range and a defined temperature for a period of 20 to 80 hours.

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