DIAGNOSTIC REAGENT FOR QUANTITATIVE DETERMINATION OF PROCALCITONIN IN A SAMPLE

Abstract

A diagnostic reagent that is suitable for turbidimetric analysis with a simple photometer and has high sensitivity for quantitative determination of procalcitonin in a sample is provided. The reagent is an aqueous suspension of polymer particles with antibodies against procalcitonin covalently bound to said polymer particles, in which no or only an extremely slight tendency to agglutination/sedimentation is detectable even after longer standing times and the specific reactivity of the particles remains largely unchanged. The suspended polymer particles have an average particle size in the range from 150 to 450 nm, the suspension includes a proportion of sugar or sugar alcohol dissolved therein in the range from 25 to 250 g/l and the suspension has a pH in the range from 8 to 10.

Claims

1. A diagnostic reagent for quantitative determination of procalcitonin in a sample, wherein the reagent is an aqueous suspension of polymer particles with antibodies against procalcitonin covalently bound to said polymer particles, characterised in that the suspended polymer particles have an average particle size in the range from >300 nm to 450 nm, the suspension comprises a proportion of sugar or sugar alcohol dissolved therein in the range from 25 to 250 g/l, and the suspension has a pH in the range from 8.5 to 10.

2. The diagnostic reagent according to claim 1, wherein the polymer of the polymer particles is selected from acrylic polymer, dextran-epichlorohydrin copolymer, polymethyl methacrylate, polystyrene and silica.

3. The diagnostic reagent according to claim 1, wherein that the covalent bond of the antibodies is formed via functional groups disposed on the surface of the polymer particles, wherein the functional groups are selected from a carboxyl group (—COOH), primary amine group (—RNH.sub.2), aromatic amine group (—ArNH.sub.2), chloromethyl group (—CH.sub.2CI), an aromatic chloromethyl group (—ArCH.sub.2CI), amide group (—CONH.sub.2), hydrazide group (—CONHNH.sub.2), aldehyde group (—CHO), hydroxyl group (—OH), thiol group (—SH), epoxy group and biotin-avidin.

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

5. The diagnostic reagent according to claim 1, wherein the absorbance of the suspension at 660 nm within 90, 120, 150 or 180 days or even within 12 months or 24 months from the time of preparation of the suspension deviates by less than 5%, less than 3% or even less than 2% from the initial value on day 0.

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

7. The diagnostic reagent according to claim 1, wherein the suspension has a pH in the range from 9.0 to 10.0.

8. A method for preparing the diagnostic reagent according to claim 1, comprising covalent binding the antibodies via the functional groups on the surface of the polymer particles at a pH of 3 to 6.

9. The method according to claim 8, wherein the covalent binding of the antibodies via the functional groups on the surface of the polymer particles takes place at a temperature in the range from 20 to 29° C.

10. The method according to claim 8, wherein the covalent binding of the antibodies takes place in a defined pH range and at a defined temperature over a period of 20 to 80 hours.

11. Diagnostic reagent for quantitative determination of procalcitonin in a sample, wherein it is obtained using the method according to claim 8.

Description

FIGURES

[0042] FIG. 1: Results of the examination of the effect of incubation time on sensitivity

[0043] FIG. 2: Results of the examination of the effect of pH on sensitivity

[0044] FIG. 3: Results of the examination of the storage stability

[0045] FIG. 4: Results of the examination of the storage stability

[0046] FIG. 5: Results of the examination of the effect of particle size on the measurement precision

[0047] FIG. 6: Results of the examination of the effect of particle size on sensitivity and linearity

[0048] FIG. 7: Results of the examination of the effect of the sugar concentration on calibration

[0049] FIG. 8: Results of the examination of the effect of different combinations of sucrose and pH on sensitivity

[0050] FIG. 9: Results of the examination of the effect of different combinations of sucrose and pH on sensitivity

[0051] FIG. 10: Results of the examination of the effect of different combinations of sucrose and pH on sensitivity

[0052] FIG. 11: Results of the examination of the effect of incubation time on sensitivity

[0053] FIG. 12 Results of the examination of the stability of reactivity at a pH of 8.1

[0054] FIG. 13 Results of the examination of the stability of reactivity at a pH of 9.0

[0055] FIG. 14 Results of the examination of the stability of reactivity at a pH of 9.5

[0056] FIG. 15 Results of the examination of long-term stability

[0057] FIG. 16 Results of the examination of long-term stability

EXAMPLES FOR EMBODIMENTS

[0058] In the following, a variety of comparative tests are presented, in which different parameters of the method for producing the diagnostic reagent according to the invention are changed. For this purpose, polymer particles of polystyrene were loaded with antibodies against PCT under different conditions.

[0059] In one embodiment variant, the polymer particles comprised carboxyl groups (—COOH) as the functional group and, in another embodiment variant, the polymer particles comprised chloromethyl groups (—CH.sub.2Cl) as the functional group.

I. Embodiment Variant with Chloromethyl Groups

1. Incubation Time

[0060] The polymer particles used had an average particle size of >350 nm. The particles were coupled to the antibodies over a period of 15 to 48 hours, and the results of the different batches are shown in FIG. 1.

[0061] These results are expressed in terms of absorbance at 660 nm, and the comparatively high absorbance values of the batch in which the incubation time was 48 hours demonstrate that the batches incubated for a longer period of time provide significantly higher sensitivity at the respective concentration of PCT in the sample, because clearly higher proportions of the PCT contained in the sample are bound than in the other batches. The amount of PCT contained in the sample can thus be determined in a quantitatively more accurate manner.

2. pH

[0062] In these experiments, the pH during the coupling reaction was varied in the range from 4 to 9.

[0063] The results shown in FIG. 2 demonstrate that better loading efficiency is achieved at lower pH values. In this way, polymer particles are obtained which have a higher binding capacity to PCT and thus a higher sensitivity.

3. Storage Stability

[0064] To check the suspension stability of the diagnostic reagent according to the invention, an aqueous suspension of the polymer particles was mixed with a teaching sample (physiological saline solution) and the turbidity of the resulting mixture was then determined. The thus used polymer particles were stored under different pH conditions for a period of up to 120 days.

[0065] The results shown in FIGS. 3 and 4 demonstrate that there was significantly less turbidity when the polymer particles had been stored at a pH of 9.0 than in the samples stored at a pH of 8.1, in particular for the samples stored for 60 days and more. This clearly demonstrates that the tendency to agglutination and sedimentation is significantly lower in the batches stored at pH 9.

[0066] As can be seen in FIGS. 12 to 14, the stability of reactivity in the higher pH range, namely in the range from pH 9.0 (see FIG. 13) to pH 9.5 (see FIG. 14), is better than in the lower range (see FIG. 12: pH 8.1). In the period of 60 days studied here, a particularly high stability of reactivity can be observed at a pH in the range >9 (see FIG. 14: pH 9.5).

[0067] FIGS. 15 and 16 demonstrate the high long-term stability of up to 24 months in an embodiment with a storage buffer having a pH of 9.0. The small jump in the curve at 12 months in the results of the examination of the reactivity of the calibrators can be explained by the lamp on the detector being changed after 12 months.

II. Embodiment Variants with Different Particle Sizes

[0068] To investigate the effect of particle size, polymer particles comprising chloromethyl groups and having an average particle size of >350 nm were compared with polymer particles comprising chloromethyl groups and having an average particle size of <300 nm.

[0069] FIG. 5 shows that, especially in the range of concentrations 0.2-2 ng/mL, in which the two medically relevant decision ranges of procalcitonin are located, the reactivity is much stronger for the polymer particles having an average particle size of >350 nm than for the polymer particles having an average particle size of <300 nm. This significantly improves the measurement precision.

[0070] The recovery (accuracy of concentration) is also better with the polymer particles having an average particle size of >350 nm than with the polymer particles having an average particle size of <300 nm (see Table 1 below).

TABLE-US-00001 TABLE 1 Target value PCT Range Recovery Recovery [ng/mL] [ng/mL] <300 nm >350 nm Control Level 1 0.80 0.60-1.00  1.14-1.12 0.75-0.74 Control Level 2 10.85 8.68-13.03 10.58-10.57 10.46-10.24

[0071] FIG. 6 shows the results of the measurement of the sensitivity (measured as precision at the cut-off) and the linearity of the recovery of a diluted sample.

[0072] With the polymer particles having an average particle size of <300 nm, linear measurement is possible only to the target value 35 ng/mL. Higher concentrations are no longer differentiated.

[0073] The polymer particles having an average particle size of >350 nm have linear recovery up to 65 ng/mL. The correlation coefficient of the larger particles is accordingly also higher than that of the smaller particles.

[0074] The recovery and precision of a sample at the medically relevant cut-off (0.5 ng/mL PCT) are also significantly better for the larger particles than for the smaller ones (see Table 2 below).

TABLE-US-00002 TABLE 2 Measured concentration PCT [ng/mL] <300 nm >350 nm Mean [ng/mL] (target 1.01 0.60 value = 0.5 ng/mL) Std. deviation 0.0773 0.0294 CV % 7.65 4.86

III. Embodiment Variants with Different Sugar Concentrations

[0075] To investigate the effect of different sugar concentrations, polymer particles comprising chloromethyl groups and having an average particle size of >350 nm were analysed in storage buffers having different sugar concentrations.

[0076] FIG. 7 shows that a comparison of 50 g/L vs. 75 g/L vs. 125 g/L sucrose in the storage buffer yields similar calibration curves. However, the precision (measured as CV % of 20-fold determination of a sample containing 0.5 ng/mL PCT) becomes significantly better (CV % lower) the higher the sucrose concentration (see Table 3 below).

TABLE-US-00003 TABLE 3 Sucrose Sucrose Sucrose 50 g/L 75 g/L 125 g/L Precision [CV %] 13.97 12.53 6.53

[0077] FIGS. 8, 9 and 10 show data of the combined effect of sucrose+pH. The chloromethyl particles are resuspended in three storage buffers with sucrose 125 g/L and pH 8.1/8.5 and 9.0. The slope of the linear regression line of the reagent blank values on the different measurement days corresponds to the aggregation of the particles and should be as close to 0 as possible (Excel slide “Combined effect suc-pH”).

IV. Embodiment Variant with Carboxyl Groups

[0078] The polymer particles used had an average particle size of <310 nm. The particles were coupled to the antibodies over a period of 12 to 24 hours and the pH in the coupling reaction was 4 to 6.

[0079] As the results shown in FIG. 11 indicate, good reactivity can be achieved with polymer particles comprising a carboxyl group. By comparison, however, the polymer particles comprising chloromethyl groups exhibit a significantly stronger reactivity.

[0080] Polymer particles comprising a carboxyl group also have to be activated (EDC+NHS) prior to the coupling reaction in order to make the COOH groups reactive. This activation is complex and does not always work in a reproducible and batch-homogeneous manner. In the present case, only one activation out of five attempts was actually successful. By comparison, polymer particles comprising chloromethyl groups couple very reliably (here the data from three batches).