Diagnostic reagent for quantitative determination of procalcitonin in a sample

Abstract

The present invention provides a diagnostic reagent which is suitable for nephelometric analysis with a simple photometer and has high sensitivity for the quantitative determination of procalcitonin in a sample, wherein the reagent is an aqueous suspension of polymer particles, wherein procalcitonin antibodies are covalently bound to the polymer particles, wherein even after a longer standing time no or only a very slight agglomeration / settling tendency is detected and the specific reactivity of the particles remains essentially constant, for which the present invention proposes that the average particle size of the suspended polymer particles is 150-450 nm, the suspension contains a proportion of a sugar or sugar alcohol which is dissolved therein in the range from 25-250 g / l and the pH of the suspension is 8-10.

Description

[0001] This invention relates to a diagnostic reagent for the quantitative determination of procalcitonin in a sample, wherein the reagent is an aqueous suspension of polymer particles, and an anticalcitonin antibody is covalently bound to the polymer particles. The invention also relates to a method for preparing this diagnostic reagent.

[0002] Procalcitonin (PCT) is a precursor to the hormone calcitonin and one of the most important biomarkers for diagnosing sepsis. PCT is standardly used, especially for postoperative control of sepsis. This is because, postoperatively, particularly in cases of bacterial infection, the liver and fat cells produce more PCT. However, in routine clinical practice, PCT is not only used postoperatively but also often serves as an adjunct to the initial diagnosis and differentiation of sepsis caused by viruses, bacteria, fungi, and protozoa / parasites, and is subsequently used for control, i.e., to check the effectiveness of the medications used.

[0003] Sepsis has several diagnostically relevant ranges. Below 0.5 ng / ml, sepsis is unlikely. Above 0.5–2 ng / ml, inflammation may be present. The presence of sepsis must be confirmed by repeated time-delayed measurements of PCT and, if necessary, other parameters. Above 2–10 ng / ml, sepsis combined with bacteria may be present. Above 10 ng / ml, severe septic shock is present. This classification is used to characterize sepsis and the measures derived from said characterization. This is particularly important for the time factor.

[0004] Statistically, every hour that sepsis goes undiagnosed or is delayed in diagnosis increases mortality by 7%. For this reason, sepsis is the third leading cause of death in all parts of the world. Given this, extremely high sensitivity and reproducibility are required for the diagnosis of sepsis. Diagnostic reagents must provide reliable results even after prolonged storage.

[0005] Several immunoassay-based diagnostic methods are known in the art, which can be used to quantitatively measure PCT in a patient's blood. PCT antibodies bind to polymer particles and react with PCT contained in the sample to be tested, after which analysis can be performed.

[0006] CLIA (Chemical Immunoassay) requires the use of chemiluminescent reagent components, allowing analysis based on the chemiluminescence emitted. This necessitates a specialized apparatus in which particles must typically be continuously resuspended.

[0007] In PETIA (particulate-enhanced turbidimetric immunoassay), turbidimetric analysis is based on the decrease in light transmittance through the reaction fluid. The more PCT in the sample, the stronger the cross-linking between antibody-bound particles, leading to greater turbidity in the liquid and consequently lower transmittance. Therefore, no additional chemiluminescent reagents are required, and the assay can be performed using a simple photometer system available in almost every diagnostic laboratory.

[0008] For the accuracy and diagnostic relevance of turbidimetric analysis, the degree of turbidity resulting from the PCT reaction is always reliably correlated with the quantification of PCT in the sample. This is not the case, for example, if antibody-loaded particles spontaneously / nonspecifically agglutinate or settle, or if their specific antigen-binding ability against PCT changes over time after preparation.

[0009] Therefore, there is a need for a diagnostic reagent that is suitable for turbidimetric analysis using a simple spectrophotometer and has high sensitivity for the quantitative determination of procalcitonin in a sample, wherein the reagent is an aqueous suspension of polymer particles in which procalcitonin antibodies are covalently bound, wherein no or only very slight agglomeration / sedimentation tendency is detected even after a long period of storage, and the specific reactivity of the particles remains substantially unchanged.

[0010] According to the present invention, this objective is achieved by the following means

[0011] The average particle size of the suspended polymer particles ranges from 150 to 450 nm.

[0012] The suspension contains a certain proportion of sugar or sugar alcohol, dissolved in it in the range of 25-250 g / L, and

[0013] The pH of the suspension is 8-10.

[0014] The proposed combination of features provides a diagnostic reagent for the quantitative determination of PCT, exhibiting very high sensitivity over a range of extremely low PCT concentrations in the sample. Furthermore, this high sensitivity is maintained even during long-term storage of up to 24 months. This high storage stability is particularly noteworthy given the relatively large particle size of the polymer particles used in this invention, which is 150 nm and larger.

[0015] The large particle size of polymer particles is particularly desirable, especially for the high sensitivity required in diagnostic reagents. Relatively larger particles have a correspondingly larger surface area, allowing them to covalently bind a larger number of PCT antibodies. Therefore, PCT molecules have more potential coupling sites, thereby improving the sensitivity of diagnostic reagents.

[0016] Larger particle size is also advantageous in terms of light scattering properties. It has been found that the polymer particles of this invention exhibit significantly better "light yield." Under the same degree of cross-linking, the particles of this invention essentially re-amplify the signal. The larger the particle size, the stronger the signal. The basic principle is the effect of colloidal nanoparticles in suspension, such as Rayleigh scattering and Mie scattering.

[0017] Despite the large particle size, this invention surprisingly achieves better storage stability than expected. Larger particles typically have a greater tendency to settle and are therefore more difficult to resuspend after longer storage periods. However, the combination of the sugar or sugar alcohol dissolved in the suspension and the pH set in the suspension, as proposed in this invention, surprisingly allows for highly storage-stable suspensions with consistent reactivity even with relatively large suspension particles.

[0018] The diagnostic reagents claimed herein are used for the quantitative determination of PCT in samples. In the context of this invention, "sample" should be understood as any material containing an analyte amount of PCT prepared for analytical purposes. In most cases, the sample will likely be a fresh whole blood sample appropriately prepared for analysis. However, this invention also includes other liquid samples containing PCT, such as standard solutions and calibrators.

[0019] The diagnostic reagent of this invention is suitable for the quantitative determination of PCT. As used herein, "quantitative determination" means that the amount of PCT in a sample can be inferred from the amount of PCT molecules bound to polymer particles.

[0020] In the reagents of this invention, the polymer particles are present in an aqueous suspension. Hereinafter, the term "aqueous suspension" refers to a slurry of polymer particles loaded with anticalcitonin antibodies in water or an aqueous solution containing dissolved sugars, sugar alcohols, and / or buffering substances. In the context of this invention, the term "suspension" must be interpreted narrowly and means that virtually all particles are suspended in the aqueous phase and do not settle. Therefore, a suspension in the sense of this invention exists when at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% of the polymer particles are freely suspended.

[0021] According to the present invention, the average particle size of the polymer particles is 150-450 nm. In some embodiments within the scope of the claims, the average particle size is preferably >190 nm, >240 nm, or even >300 nm. In other embodiments within the scope of the claims, the average particle size is preferably <410 nm, <360 nm, or even <300 nm. In the case of the present invention, the particle size of the polymer particles is determined by dynamic light scattering at 25°C, for example using Malvern's Zetasizer Pro. The average particle size refers to the number average value.

[0022] The polymeric material constituting the polymer particles is preferably selected from acrylic polymers, dextran-epimercaprolactone copolymers, polymethyl methacrylate, polystyrene, silica (silicone), and combinations thereof. In some embodiments, the polymer particles consist entirely of a single polymeric material or a mixture thereof. In other embodiments, the particles consist of multiple layers of different polymers.

[0023] In a specific embodiment, the particles may comprise a core and one or more layers coated thereon. The core and the one or more layers coated thereon may consist of a polymeric material, different polymeric materials, or non-polymeric materials, as long as the polymeric particles are primarily composed of polymeric materials.

[0024] Polymer particles in the sense of this invention exist when the particles consist of at least 80 wt%, at least 90 wt%, at least 95 wt%, or 100 wt% of polymeric material or a combination of different polymeric materials.

[0025] The total density of the polymer particles is preferably 0.9-1.1 g / cm³. 3 The preferred value is 1.0 ± 0.5 g / cm³. 3 .

[0026] On the surface, the polymer particles preferably contain functional groups through which antibodies can covalently bind to the surface of the polymer particles. These functional groups are preferably selected from carboxyl (-COOH), primary amino (-RNH2), aromatic amino (-ArNH2), chloromethyl (-CH2Cl), aromatic chloromethyl (-ArCH2Cl), amide (-CONH2), hydrazide (-CONHNH2), aldehyde (-CHO), hydroxyl (-OH), thiol (-SH), epoxy group, and biotin-antibiotin protein.

[0027] The aqueous portion of the diagnostic reagent suspension of the present invention contains dissolved sugar or sugar alcohol fractions in a concentration range of 25-250 g / L. In some embodiments, the concentration of sugar and / or sugar alcohol ranges from 50-200 g / L. In some embodiments, the concentration is 50-150 g / L, while in other embodiments, the concentration is 150-250 g / L.

[0028] 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 combination of sugar / sugar alcohol is used, the above amount refers to the sum of the proportions of the sugar / sugar alcohol in the combination.

[0029] The aqueous suspension in which the antibody-loaded polymer particles are suspended has a pH of 8-10. In some embodiments, the pH is 9.0-10.0. In some embodiments, the pH of the suspension is >9. In some embodiments, the pH of the suspension is 9.1-10.0. In some embodiments, the pH of the suspension is 9.0 ± 0.5. In a particular embodiment, the pH of the suspension is 9.5 ± 0.1.

[0030] The diagnostic reagent claimed in this invention is characterized by very high storage stability. This is, for example, manifested in the fact that the turbidity of the suspension remains stable even after several months compared to the initial value. Specifically, the suspension of this invention is characterized in that, within 90, 120, 150, or even 180 days after preparation, the absorbance of the suspension at 660 nm deviates by less than 5% from the initial value on day zero. In some embodiments, the deviation remains less than 5% even 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 this invention, slight deviations in turbidity within the aforementioned time periods can also be measured at other wavelengths in the 340-800 nm range.

[0031] In one specific embodiment of the invention, the antibody covalently bound to the polymer particles is a PCT monoclonal antibody. In other embodiments, the antibody is a PCT polyclonal antibody. In other embodiments of the invention, the polymer particles are loaded with covalently bound recombinant PCT antibodies or covalently bound PCT antibody fragments.

[0032] This invention also claims a method for preparing diagnostic reagents of the above type. In this method, polymer particles are first contacted with PCT antibodies, which occurs under conditions where the antibody covalently binds to the surface of the polymer particles. Antibody loading onto the polymer particles occurs in an aqueous suspension at a specific time, specific temperature, and specific pH.

[0033] The method of the present invention is characterized in that the antibody covalently binds to functional groups on the surface of the polymer particles at pH 3-6. In some embodiments, covalent binding occurs at pH 3-5. In some embodiments, the pH in the reaction in which the antibody covalently binds to functional groups on the surface of the polymer particles is 4.0 ± 0.5.

[0034] Inorganic or organic buffers such as borate, citrate, phosphate, malate, maleate, succinate, and acetate / acetate are preferably used to set the pH in reactions where antibodies covalently bind to functional groups on the surface of polymer particles. Hydrochloric acid / sodium hydroxide solution is preferably used to set / adjust the pH.

[0035] In some embodiments of the method of the present invention, the antibody covalently binds to functional groups on the surface of the polymer particles in a temperature range of 20-29°C. In other embodiments, the reaction occurs at 30-34°C, or even at 35-45°C.

[0036] In some embodiments of the method of the present invention, the covalent binding of the antibody occurs within a time period of 20-80 hours within one of the pH ranges defined above and one of the temperature ranges defined above. In some embodiments, the reaction occurs within a time period of >30, >40, >50, or even >60 hours.

[0037] At the end of the specified time period, the pH of the suspension of the antibody-loaded polymer particles should be lowered to 8-10. One or more of the aforementioned alkalizing agents or buffering substances may be added for this purpose.

[0038] The pH of the suspension is preferably set using a buffer containing an amine group, such as EPPS, HEPPS, tricine, tris, glycylglycine, bicine, TAPS, boric acid, ethanolamine, CHES, glycine, and CAPS. Hydrochloric acid / sodium hydroxide solution is preferably used to set / adjust the pH.

[0039] As mentioned above, polymer particles loaded with antibodies or antibody fragments obtained using the method of the present invention exhibit extremely high sensitivity and extremely high storage stability.

[0040] For the purposes of the original disclosure, it should be noted that, as disclosed to those skilled in the art from this specification, drawings, and claims, all features, even those described in conjunction with only certain other features, can be combined individually or in any combination with other features or combinations of features disclosed herein, unless this is expressly excluded or the technical conditions make such a combination impossible or meaningless. For the sake of brevity and readability, a comprehensive and explicit description of all possible combinations of features has been omitted herein.

[0041] It should also be noted that, as will be apparent to those skilled in the art, the following examples of embodiments are merely intended to illustrate possible embodiments of the invention. Therefore, those skilled in the art will readily understand that all other embodiments having the features or combinations thereof described in the claims according to the invention also fall within the scope of protection of the invention. For the sake of brevity and readability, a full and explicit description of all possible embodiments has been omitted herein.

[0042] Attached Figure

[0043] Figure 1 Results of the test on the effect of incubation time on sensitivity

[0044] Figure 2 Results of the test on the effect of pH on sensitivity

[0045] Figure 3 Storage stability test results

[0046] Figure 4 Storage stability test results

[0047] Figure 5 Test results on the influence of particle size on measurement accuracy

[0048] Figure 6 Test results on the effect of particle size on sensitivity and linearity

[0049] Figure 7 Test results on the effect of sugar concentration on calibration

[0050] Figure 8 Results of the test on the effect of different combinations of sucrose and pH on sensitivity

[0051] Figure 9 Results of the test on the effect of different combinations of sucrose and pH on sensitivity

[0052] Figure 10 Results of the test on the effect of different combinations of sucrose and pH on sensitivity

[0053] Figure 11 Results of the test on the effect of incubation time on sensitivity

[0054] Figure 12 Results of the test on reactivity stability at pH 8.1

[0055] Figure 13 Results of reactivity stability test at pH 9.0

[0056] Figure 14 Results of the test on reactivity stability at pH 9.5

[0057] Figure 15 Results of long-term stability test

[0058] Figure 16 Results of long-term stability test

[0059] Examples of implementation methods

[0060] Various comparative tests are presented below, in which different parameters of the preparation method of the diagnostic reagent of the present invention were changed. For this purpose, polystyrene polymer particles were loaded with PCT antibodies under different conditions.

[0061] In one embodiment variant, the polymer particles contain a carboxyl group (-COOH) as a functional group, while in another embodiment variant, the polymer particles contain a chloromethyl group (-CH2Cl) as a functional group.

[0062] I. Variations of implementation with chloromethyl groups

[0063] 1. Incubation time

[0064] The polymer particles used had an average particle size >350 nm. The particles were conjugated with the antibody within 15-48 hours; results from different batches are shown below. Figure 1 As shown.

[0065] These results are expressed as absorbance at 660 nm. The relatively high absorbance values ​​of the batch incubated for 48 hours indicate that the longer incubation time provides significantly higher sensitivity at their respective PCT concentrations in the samples, because the binding proportion of PCT in the samples is significantly higher than in other batches. Therefore, the amount of PCT in the samples can be determined more accurately.

[0066] 2. pH

[0067] In these experiments, the pH during the coupling reaction varied in the range of 4–9.

[0068] Figure 2 The results show that better loading efficiency was achieved at lower pH values. In this way, polymer particles with higher binding capacity to PCT and therefore higher sensitivity were obtained.

[0069] 3. Storage stability

[0070] To test the suspension stability of the diagnostic reagent of the present invention, an aqueous suspension of polymer particles was mixed with a teaching sample (physiological saline solution), and the turbidity of the resulting mixture was then measured. The polymer particles thus used were stored under different pH conditions for up to 120 days.

[0071] Figure 3 and Figure 4 The results show that when polymer particles are stored at pH 9.0, the turbidity is significantly lower than that of samples stored at pH 8.1, especially for samples stored for 60 days or longer. This clearly demonstrates that the tendency for aggregation and sedimentation is significantly reduced in batches stored at pH 9.

[0072] like Figures 12 to 14 As shown, in the higher pH range, i.e. at pH 9.0 (see...) Figure 13 To pH 9.5 (see) Figure 14Within the range of ), the stability of reactivity is better than within the lower range (see [reference]). Figure 12 (pH 8.1). Particularly high reactivity stability was observed in the pH > 9 range during the 60-day period studied in this paper (see [reference needed]). Figure 14 (pH 9.5).

[0073] Figure 15 and 16 The embodiment of the pH 9.0 storage buffer demonstrates high long-term stability for up to 24 months. The slight jump in the curve at 12 months in the calibrator reactivity test results can be explained by replacing the lamp on the detector after 12 months.

[0074] II. Variations in implementation methods for different particle sizes

[0075] To investigate the effect of particle size, polymer particles containing chloromethyl groups and with an average particle size >350 nm were compared with polymer particles containing chloromethyl groups and with an average particle size <300 nm.

[0076] Figure 5 The results show that, particularly in the concentration range of 0.2–2 ng / mL (which includes the two medically relevant procalcitonin decision ranges), polymer particles with an average particle size greater than 350 nm are significantly more reactive than polymer particles with an average particle size less than 300 nm. This significantly improves measurement accuracy.

[0077] The recovery rate (concentration accuracy) of polymer particles with an average particle size > 350 nm was also better than that of polymer particles with an average particle size < 300 nm (see Table 1 below).

[0078] Table 1

[0079]

[0080] Figure 6 The results show linearity between the sensitivity determination (as a precision determination at cutoff) and the recovery of diluted samples.

[0081] For polymer particles with an average particle size <300 nm, linear assays can only reach the target value of 35 ng / mL. Higher concentrations cannot be distinguished.

[0082] The linear recovery rate of polymer particles with an average particle size >350 nm was as high as 65 ng / mL. Therefore, the correlation coefficient of larger particles was also higher than that of smaller particles.

[0083] At the medically relevant cutoff value (0.5 ng / mLPCT), the recovery rate and accuracy of larger particles were significantly better than those of smaller particles (see Table 2 below).

[0084] Table 2

[0085]

[0086] III. Variations in implementation methods with different sugar concentrations

[0087] To investigate the effect of different sugar concentrations, polymer particles containing chloromethyl groups and with an average particle size >350 nm were analyzed in storage buffers with different sugar concentrations.

[0088] Figure 7 The comparison of 50 g / L, 75 g / L, and 125 g / L sucrose in the storage buffer produced similar calibration curves. However, the higher the sucrose concentration, the better the accuracy (as measured as CV% of a 20-fold determination of a sample containing 0.5 ng / mLPCT) became (lower CV%) (see Table 3 below).

[0089] Table 3

[0090]

[0091] Figure 8 , 9 Figures 1 and 10 show the combined effect data of sucrose and pH. Chloromethyl particles were resuspended in three storage buffers at 125 g / L sucrose and pH 8.1 / 8.5 and 9.0. The slope of the linear regression line for reagent blank values ​​at different assay days corresponds to particle aggregation and should be as close to 0 as possible (Excel page "Combined Effects suc-pH").

[0092] IV. Variations in the implementation of carboxyl groups

[0093] The polymer particles used have an average particle size of <310 nm. The particles are conjugated with antibodies within 12–24 hours, and the pH of the conjugation reaction is 4–6.

[0094] like Figure 11 The results show that good reactivity can be obtained by using polymer particles containing carboxyl groups. However, polymer particles containing chloromethyl groups exhibit significantly stronger reactivity in comparison.

[0095] Polymer particles containing carboxyl groups must also be activated (EDC+NHS) prior to the coupling reaction to make the COOH groups reactive. This activation is complex and does not always present a reproducible and batch-uniform process. In this case, activation was actually successful only once out of five attempts. In contrast, polymer particles containing chloromethyl groups can be coupled very reliably (data here comes from three batches).

Claims

1. A diagnostic reagent for quantitatively determining procalcitonin in a sample, wherein, The agent is an aqueous suspension of polymer particles, to which anti-procalcitonin antibodies are covalently bound, characterized in that - the average particle size of the suspended polymer particles ranges from > 300 nm to 450 nm, - the suspension contains a proportion of a sugar or sugar alcohol, which is dissolved therein in the range from 25 to 250 g / l, and - the pH of the suspension is > 9.0 to 10, the polymer of the polymer particles is polystyrene, the covalent bond of the antibodies is formed via functional groups located on the surface of the polymer particles, wherein the functional groups are chloromethyl groups (-CH2CI).

2. 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.

3. The diagnostic reagent according to claim 1, wherein The anti-procalcitonin antibodies covalently bound to the polymer particles are monoclonal, polyclonal or recombinant antibodies or antibody fragments.

4. A method for preparing the diagnostic reagent according to any one of claims 1 to 3, characterized in that, The covalent binding of the antibodies takes place at a pH of 3 to 6 via functional groups on the surface of the polymer particles.

5. The method of claim 4, wherein, The covalent binding of the antibodies takes place at a temperature in the range from 20 to 29 °C, from 30 to 34 °C or from 35 to 45 °C via functional groups on the surface of the polymer particles.

6. The method of any one of claims 4 and 5, wherein, The covalent binding of the antibodies takes place at a defined pH range and at a defined temperature over a period of 20 to 80 hours. The covalent binding of the antibodies takes place at a defined pH range and at a defined temperature over a period of 20 to 80 hours.

Images