Method for simultaneously improving sensitivity and cross contamination resistance of latex turbidimetric kit and application

Abstract

The invention discloses a method for simultaneously improving the sensitivity and cross contamination resistance of a latex turbidimetric kit and application, and belongs to the technical field of biology. According to the method for simultaneously improving the sensitivity and the cross contamination resistance of the latex turbidimetric kit, latex microspheres used in a sensitization latex solution are low-mass-density latex microspheres, and the sensitization latex solution contains prednisone. According to the method disclosed by the invention, the sensitivity is improved by 39%-46%; meanwhile, after the reagent is subjected to cross contamination, the onboard service time is prolonged by 3-10 times, the scrapping time is prolonged from 2-3 days to 7-30 days for stabilization, and the basic use requirement is met.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method and application for simultaneously improving the sensitivity and anti-cross-contamination ability of a latex turbidimetric reagent kit. Background Technology

[0002] Latex immunoturbidimetry has been the mainstream method in the field of biochemistry and specific protein detection due to the widespread availability of equipment (various biochemical transmission, specific protein scattering, and transmission-scattering fusion analyzers), fast detection speed and quantification (quantitative results are usually available in 8-10 minutes, and even in POCT in 1-2 minutes), and high detection throughput (from 50T / hour for small mini machines to 2000T / hour for large equipment). It is also constantly being developed.

[0003] The main performance characteristics of latex immunoturbidimetric assay kits include sensitivity, linearity and antigen excess safety zone, specificity, and reagent stability. Many companies and research institutions have invested resources in upgrading and optimizing these aspects.

[0004] For example, regarding reagent sensitivity: CN201811426490.6, "A method to improve the detection sensitivity of latex turbidimetric reagents," explores the improvement of reagent kit sensitivity by introducing an amino acid scaffold to change the steric hindrance of the two Fab structures of the antibody. CN202010813669.8, "A method for improving the sensitivity of latex turbidimetric assays using polystyrene microspheres," explores the improvement of the number and length of carboxyl chains on the surface of polystyrene microspheres. CN201410401284.5, "Anti-cyclic citrulline peptide antibody detection kit," explores the improvement of detection sensitivity by increasing the intensity of scattered and transmitted light through high refractive index and chemical inertness. CN202510645838.4 A method for improving the sensitivity of latex immunoturbidimetric reagents; CN102759631B A latex-enhanced immunoturbidimetric kit for quantitative detection of procalcitonin (PCT); CN108872616B An immunoturbidimetric kit for the detection of single-size latex particles based on NGAL, which introduces a biotin-streptavidin binding system to improve the detection sensitivity of latex turbidimetric reagents.

[0005] Numerous companies and research institutions have conducted various studies to improve the detection sensitivity of latex turbidimetric reagent kits, thereby achieving better detection results or enabling the detection of more items. However, research on controlling the reaction rate in different sections of the reaction curve to control reagent sensitivity and the reagent's resistance to cross-contamination is rare.

[0006] Therefore, providing a method and application that simultaneously improves the sensitivity and anti-cross-contamination ability of latex turbidimetric reagent kits is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method and application for simultaneously improving the sensitivity and anti-cross-contamination ability of a latex turbidimetric reagent kit.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Currently, it is rare to control the reaction rate in different segments of the reaction curve to control reagent sensitivity. Taking Hitachi 7180 and 3500 as examples, a typical latex turbidimetric reaction curve is shown below. Figure 1 The horizontal axis represents the reaction time, counted at measurement time points 1 to 34, and the vertical axis represents the reaction signal value (absorbance ABS for transmission and light intensity INT for scattering). The sample is added to the buffer and incubated for measurement time points 1 to 16. Typically, antibody-labeled sensitized latex is added between measurement points 16 and 17. From measurement point 17 onwards, antigen-antibody latex immune complexes gradually form, increasing turbidity and consequently the signal value. However, the immune reaction usually does not reach its endpoint within 5 minutes between measurement points 17 and 34.

[0010] The parameters are two-point endpoint method, with 18-34 reading points; 18-34 reading points means that the first measurement point is the 18th measurement point after adding the sensitized latex, and the second measurement point is the last 34 measurement points.

[0011] When the immune response does not reach its endpoint, the reaction rate and the shape of the reaction curve will affect the calculation of the δ signal value. For reading points 18-34, the calculation method is: δ signal value = (signal value at reading point 34 + signal value at reading point 33 - signal value at reading point 18 - signal value at reading point 17) / 2. Figure 2 Taking Table 1 as an example, for latex immunoturbidimetric reactions with a deviation of only 1.7% between the two final signal values ​​(16800 VS 16500), the final calculated δ signal values ​​showed a reversal, with the difference widening to 6.3% (6350 VS 6750). Reactions with a higher initial reaction rate (more curved reaction curve) had lower δ signal values ​​even if the final signal value of 16800 was higher.

[0012] Table 1

[0013] Based on the above fundamental principles, our company's technical personnel believe that for any method using the two-point endpoint method, where the latex turbidimetric reagent is used at the first measurement point after the addition of the sensitized latex, slowing down the initial reaction rate after the addition of the sensitized latex, or increasing the reaction rate in the middle and final stages, can lead to an increase in the final δ signal value through the mathematical formula for calculating the δ signal value. When the reagent and instrument are constant, an increase in the δ signal value indicates an increase in reagent sensitivity.

[0014] Therefore, by controlling the reaction rate in different sections of the reaction curve, the sensitivity of this type of reagent can be significantly altered by changing the δ signal value.

[0015] The ability of a reagent to resist cross-contamination is a branch of reagent stability.

[0016] Taking latex immunoturbidimetric reagents for antigen detection with labeled antibodies as an example, during the production process, the reagent may be accidentally introduced into the test antigen or its analogue during opening and use. This introduces the antigen into the antibody-sensitized latex solution. During reagent transportation and storage, as well as onboard storage, the antigen-antibody reaction gradually produces antigen-antibody latex immune complex precipitation, which leads to reagent instability. This problem is commonly referred to as cross-contamination in the industry (conversely, for reagents for antibody detection with labeled antigens, cross-contamination is caused by the introduction of the test antibody).

[0017] Due to the storage and transportation of the above reagents, the airborne environment is usually 2-8 degrees Celsius, and the antigen-antibody reaction is slow. After cross-contamination occurs, it usually takes several days or even months to detect the problem. By the time it is detected, it is too late, which will cause the reagents to be scrapped and the measurement values ​​of airborne reagents to be biased.

[0018] To reduce such cross-contamination, the production process is usually carried out in a clean room, where antigen and antibody sites, materials, operators, and equipment are strictly physically isolated. Under strict management, major losses caused by cross-contamination during the production process are generally rare.

[0019] However, cross-contamination is a common problem for hospitals and testing institutions. It comes from the fact that operators carry contamination from gloves when opening the test box, from aerosols in the testing environment, and from contamination from instruments and reagent needles that act as antigens and antibodies to each other. This leads to all sorts of bizarre cross-contaminations, and there are many uncontrollable factors that make it difficult to avoid.

[0020] To prevent bacterial contamination, preservatives and antibiotics are typically added to reagent kits. However, there seems to be no good way to prevent cross-contamination, since materials that can resist cross-contamination, such as immunosuppressants, will suppress the immune response and theoretically reduce the sensitivity of antigen-antibody immune response kits.

[0021] Latex immunoturbidimetric reactions are typically performed at 37°C, a significant range compared to storage temperatures of 2-8°C. Our technical staff hypothesizes that appropriate amounts of temperature-sensitive, heat-degradable immunosuppressants (such as prednisone) can inhibit cross-contamination from small amounts of contaminants during low-temperature storage or onboard incubation. Furthermore, when a sensitized latex solution containing such inhibitors is added to a reaction vessel and incubated at 37°C, these inhibitors, due to their temperature sensitivity, are rapidly decomposed. If the dosage is carefully controlled, this could potentially inhibit the immune response at its inception, but the decomposition would have a limited impact on subsequent immune responses (a slight reduction in the final signal value). Theoretically, this slightly reduces sensitivity while simultaneously improving the reagent's resistance to cross-contamination.

[0022] However, during the experiment, we unexpectedly discovered that, due to the aforementioned δ signal value calculation formula, for latex immunoturbidimetric reagents using the two-point endpoint method with the initial measurement point located after the addition of R2 (such as the Hitachi 3500 specific protein biochemical analyzer, two-point endpoint method, 18-34 reading point mode), as described above regarding the effect of reaction rate on sensitivity, appropriately suppressing the initial reaction rate unexpectedly resulted in an increase in the δ signal value (sensitivity). This improved resistance to cross-contamination while slightly increasing sensitivity by approximately 10-20%.

[0023] Furthermore, reviewing our company's patent CN201710986970.7, "A Method for Improving Antigen Excess and Linear Range in Latex Immunoturbidimetric Method," we can increase the mass density of latex microspheres through secondary convergent polymerization, which can reduce the reaction rate of high-concentration samples. Conversely, reducing the mass density of latex microspheres (such as hollow microspheres or other low-mass-density microspheres) can theoretically increase the overall reaction rate of the reagent. For latex immunoturbidimetric reagents using the two-point endpoint method, with the initial measurement point located after the addition of R2, a higher δ signal value can be obtained per unit time, thereby improving sensitivity.

[0024] Hollow latex microspheres, such as those shown in CN 202511258123.X "Hollow Porous Monodisperse Carboxylated Polystyrene Microspheres and Preparation Methods and Their Application in Quantitative Detection Kits for MxA", have been commercially available. They are mainly used to solve the stability problems of reagent agglomeration and sedimentation in the preparation of large-particle-size latex and also improve the sensitivity of the kit. However, the latex in these kits is only 0.4-0.6 μm (400-600 nm), which does not cover the 200-500 nm microspheres that are more widely used in detection reagents, and the mechanism of improving sensitivity has not been systematically analyzed.

[0025] Furthermore, it can be inferred that when the mass density is too low, such as below 0.85 g / ml, the latex microspheres will exhibit a phenomenon similar to oil-water separation more quickly during storage and onboard transport, that is, the latex microspheres gradually float to the top of the reagent.

[0026] Therefore, the mass density of hollow and low-density microspheres needs to be controlled within a certain range to be effectively used in reagents. The seed method for preparing low-density microspheres effectively solves this problem, and the process is relatively mature. The mass density of carboxylated polystyrene is typically around 1.03 g / ml. Introducing a core (seed) of moderate size and low mass density, such as polypropylene (PP) with a mass density of only 0.89 g / ml, and copolymerizing it to produce carboxylated polystyrene-polypropylene microspheres can reduce the final microsphere mass density to 0.92-0.96 g / ml. When the polypropylene (PP) seed particle size is 100-350 nm, the final particle size can be controlled within 200-450 nm. These carboxylated polystyrene-polypropylene microspheres can improve the overall reaction rate of the reagent and are relatively less prone to sedimentation or oil-water separation.

[0027] Our technical staff deduced that when using sensitized latex reagents prepared with low-density latex microspheres, combined with an appropriate amount of temperature-sensitive immunosuppressant, and employing a specific two-point endpoint method, with the initial measurement point located in the latex immunoturbidimetric reagent after the addition of the sensitized latex, the combined effects of the two methods can result in a reagent with a lower initial reaction rate and a higher final reaction rate, thereby significantly improving the δ signal value, i.e., reagent sensitivity. This improves the reagent's resistance to cross-contamination while simultaneously achieving a certain degree of increase in reagent sensitivity.

[0028] A method for simultaneously improving the sensitivity and cross-contamination resistance of a latex turbidimetric reagent kit, wherein the latex turbidimetric reagent kit contains reagent R1 and reagent R2; wherein reagent R1 is a reaction buffer and reagent R2 is a sensitized latex solution; The latex microspheres used in the sensitized latex solution are low mass density latex microspheres, and the sensitized latex solution contains a certain concentration of the temperature-sensitive immunosuppressant prednisone. The mass density (mass density of a single microsphere, not the density of the latex solution) of the low mass density latex microspheres is 0.92-0.96 g / ml, preferably 0.94 g / ml; The dosage of the temperature-sensitive immunosuppressant prednisone in 1L of sensitized latex solution is 0.20-2.00mg, and the preferred dosage for different projects is 0.8-1.4mg depending on the actual situation.

[0029] Furthermore, the low mass density latex microspheres are hollow latex microspheres prepared by the seed method or other methods; the seed is a polymer with a mass density of less than 1.03 g / ml; the polymer is polyethylene (PE) or polypropylene (PP); preferably, polypropylene PP is prepared into carboxylated polystyrene-polypropylene microspheres.

[0030] Furthermore, a method for simultaneously improving the sensitivity and anti-cross-contamination ability of a latex turbidimetric reagent kit involves, on a fully automated biochemical or specific protein analyzer, mixing the sample with reagent R1 and incubating for 1 to 5 minutes, then adding reagent R2 to sensitize the latex solution for antigen-antibody latex reaction, and calculating the δ signal value using a two-point endpoint method; and after the sensitized latex solution is added to the mixture of reagent R1 and sample, the first light signal is acquired (the first photometric point is after R2 is added), and the second light signal is acquired 2-5 minutes after the immune reaction.

[0031] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method and application for simultaneously improving the sensitivity and anti-cross-contamination ability of latex turbidimetric reagent kits. Compared with latex turbidimetric reagents prepared by conventional latex particles, this method improves the sensitivity by 39%-46%; at the same time, it extends the onboard use time of the reagent by 3-10 times after cross-contamination, from 2-3 days to 7-30 days of stability, meeting the basic usage requirements.

[0032] Note that due to the hollowing phenomenon that occurs in non-secondary convergent polymerization latex microspheres, CN 202511258123.X also utilizes this phenomenon to prepare porous hollow microspheres. The final product density is usually slightly lower than the theoretically calculated value. Due to limited conditions in our laboratory, it is difficult to calculate the accurate mass density of the final latex by drying and weighing. The values ​​of 0.92-0.96 g / ml and the preferred 0.94 g / ml are estimated values, but a detailed preparation process is provided later for implementing this invention. Those skilled in the art will also understand that a core seed material with a density of 0.89 g / ml and a peripheral material with a density of 1.03 g / ml, in different proportions, can yield a latex with a mass density between 0.89-1.03 g / ml. When the particle size of the final product remains constant, the larger the core, the lower the mass density of the final product. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0034] Figure 1 This is a typical latex turbidity reaction curve.

[0035] Figure 2 For latex immunoturbidimetric reactions with a deviation of only 1.7% between the two final signal values ​​(16800 vs 16500), the final δ signal value was calculated.

[0036] Figure 3The reaction curves of conventional latex reagent at a concentration of 30 ng / ml and low mass density latex with 1.4 mg prednisone for the FER project.

[0037] Figure 4 The reaction curves of a conventional latex reagent at a concentration of 3.5 ng / ml and a low mass density latex with 0.8 mg prednisone are shown for the PCT project. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Different projects involve latex microspheres with different particle sizes, resulting in variations in core particle size and final mass density during seed-based preparation. Similarly, different projects use different antibodies, leading to variations in the dosage of immunosuppressants. The following examples will progressively illustrate the application of the principles of this invention in different projects.

[0040] Common seed-based microsphere preparation processes and calculation methods: Taking the preparation of 1L of 10% mass concentration 260nm carboxylated polystyrene-polypropylene low mass density microspheres (density approximately 0.94 g / ml) using 150nm polypropylene (PP) as an example: (1) Preparation of external aqueous phase: 877 ml of purified water, stirred at room temperature at 300 rpm, first add 15 g of polyethylene glycol PEG4000, then add 2 g of sodium dodecylbenzene sulfonate (SDBS), after the above two surfactants are dissolved, add 70 g of polypropylene (150 nm) powder, stir at 300 rpm for at least 5 minutes to form an emulsion, then add 120 ml of styrene (Note: first wash with a separatory funnel and 5% NaOH 3 times to remove the anticoagulant styrene) and continue stirring at 300 rpm for 5 minutes; then add 12 ml of acrylic acid (at least analytical grade), and finally add 1.2 ml of crosslinking agent DVB (divinylbenzene), mix at room temperature at 300 rpm for at least 30 minutes to form microdroplets of polypropylene, styrene, acrylic acid and DVB encapsulated by PEG and SDBS.

[0041] (2) Preheat the 2L reactor to 65℃ in a water bath for 2 minutes. Use a 10cm diameter Teflon blade stirrer to stir at 350rpm. Pour the well-mixed external aqueous phase into the reactor. Equilibrate the temperature at 65℃ for 10 minutes. Then, introduce argon gas for 5 minutes to remove the air in the upper layer of the reactor.

[0042] (3) Take a 100ml beaker, add 40ml of purified water, weigh 1.6g of potassium persulfate (KPS) initiator and add it to the purified water. Gently shake the beaker in a 65℃ water bath to dissolve the KPS. Open the sample loading port of the reactor and use a 5ml pipette to add 40ml of the KPS-dissolved aqueous solution into the reactor within 1 minute. Cover the sample loading port and purge with argon gas for 2 minutes. Stir at 65℃ and 350rpm for 6 hours to carry out the polymerization reaction.

[0043] (4) After the reaction is complete, filter the latex solution after the polymerization reaction using silk cloth, discard the large polymer particles, and obtain the carboxylated polystyrene-polypropylene latex solution with a mass concentration of about 10% (about 100g / L).

[0044] (5) After the above latex has cooled to room temperature, filter it through an 800nm ​​PP filter membrane, and then wash and change the liquid using an ultrafiltration membrane or centrifugation to obtain latex microspheres that can be used for antibody conjugation.

[0045] Formulas for preparing latex of different particle sizes from seeds of different sizes; let the final particle size be D and the seed kernel particle size be d.

[0046] To prepare a latex with a final particle size of D=260nm from a core of d=150nm, assuming the microspheres are regular spheres with a volume V = (1 / 6)πD³, and the volume of carboxylated polystyrene = final volume - core volume = (1 / 6)π(D³-d³), then the amount of polymer raw material used is proportional to D³-d³.

[0047] Based on the above formula, it can be deduced that when using 250nm core PP to prepare 360nm carboxylated polystyrene-polypropylene latex, D³-d³=31031000, compared to D³-d³=14201000 when using 150nm core PP to prepare 260nm latex. The required amount of styrene is 120ml. 31031000 / 14201000=262ml. For easy operation with a graduated cylinder, round to the nearest 0 or 5. If the particle size is large and sensitivity is improved, round up to 265ml.

[0048] The styrene-acrylic acid ratio is a key factor in the synthesis. The conventional styrene:acrylic acid:DVB ratio is 100:10:1, as in the example above (120ml:12ml:1.2ml).

[0049] When preparing 360nm carboxylated polystyrene-polypropylene latex (mass density approximately 0.94 g / ml) using a 250nm PP core, the amount of 250nm polypropylene remains constant at 70g, and the amount of styrene is 265ml. Maintaining the styrene-to-acrylic ratio, the calculated yields 26.5ml of acrylic acid, 2.65ml of DVB, and 777ml of purified water in the external aqueous phase. Note: A styrene-water mixture of 800ml water + 100ml styrene yields approximately 860ml, with a volume yield of 0.6%. Similarly, the acrylic acid volume yield is 0.9. Therefore, the volume of purified water in the external aqueous phase is approximately equal to 1000ml minus the volume of styrene. 0.6, acrylic acid volume (Calculated using 0.9 and 40 ml of water for dissolving the initiator). Other process flows are the same as before.

[0050] The process of preparing carboxylated polystyrene-polypropylene (or polyethylene) latex microspheres using polyethylene (PE) and polypropylene (PP) as core seeds is consistent.

[0051] Those skilled in the art can deduce the required amounts of each material for preparing carboxylated polystyrene-polypropylene latex microspheres of different particle sizes using the above formulas, and prepare them according to the above process. In subsequent embodiments, the preparation formulas and processes will not be described in detail.

[0052] R2 sensitizing latex formulation, preparation process and formulation: FER R2 preparation and formulation (common process): To facilitate demonstration of the effects of this invention, the following formula for FER reagent R2 was obtained by referring to and adjusting the patent application No. 202111453716.3, "A High-Specific Transmittance Scattering Integrated Method Ferritin Latex Turbidimetric Detection Kit": 50mM PBS pH 7.5; Tween-20 16ml / L; 205nm carboxylate 1.0g / L; 360nm carboxylate 2.0g / L; protectants (sucrose 30g / L, trehalose 10g / L, fructose 10g / L); FER rabbit polyclonal antibody 21.6ml / L (Agilent Dako catalog number A0133); preservative 0.5ml / L PC300.

[0053] The antibody labeling process is as follows: After R2 is produced according to the conventional chemical coupling method, its sensitivity is processed in accordance with the document 2018114264906, "A method to improve the detection sensitivity of latex turbidimetric reagent". Prepare 100 ml of 300 mM urea solution and set aside at room temperature.

[0054] Prepare the scaffold dissolution solution: 100mM pH 7.5 PBS buffer containing 0.1% v / v Tween-20, ready for use at room temperature.

[0055] Prepare a 50mM potassium persulfate solution: Dissolve 2.7032g of potassium persulfate in 200ml of stent dissolving solution and store at 4℃ for later use.

[0056] Prepare the blocking and washing solution: 50mM pH7.5 PBS buffer containing 16ml / L Tween-20, 30g / L sucrose, 10g / L trehalose, 10g / L fructose, and 0.05% v / v preservative PC300. Store at room temperature.

[0057] A quantity of Agilent Dako FER polyclonal antibody (catalog number A0133, antibody concentration 2.5 mg / ml) is available for use.

[0058] The latex microspheres described below are conventional carboxylated polystyrene latexes used in Comparative Examples 1 and 2, or carboxylated polystyrene-polypropylene (polyethylene) latexes prepared by the above-mentioned seed method used in Examples 1 and 2.

[0059] (1) Latex cleaning: Use 100mM MES-NaOH buffer, pH 5.5, centrifuge and change the latex microspheres twice; take 10ml of the above conventional carboxylated latex or carboxylated polystyrene-polypropylene (polyethylene) latex prepared by seed method with a mass concentration of 10%, i.e., 1g of each of the two latexes, centrifuge at 22000rpm for 15min, remove the supernatant, reconstitute with 10ml of the above MES-NaOH buffer, centrifuge again, remove the supernatant and reconstitute, and complete the cleaning for use.

[0060] (2) Latex activation: Taking 10ml of the above-washed latex as an example, weigh 50mg NHS (N-hydroxysuccinimide) and 30mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5ml of the above-washed MES-NaOH buffer, add 1ml of each to 10ml of the two types of microspheres that have been washed, and mix them magnetically at room temperature for 10min.

[0061] (3) Cleaning after activation: Centrifuge the two latexes activated in step (2) at 22000 rpm for 15 min, remove the supernatant, and add 20 ml of 100 mM pH 7.5 PBS buffer to reconstitute for later use.

[0062] (4) Antibody conjugation: Take two portions of Agilent Dako FER antibody and add them to the two activated latex solutions reconstituted with PBS buffer. Add 5.6 ml of FER antibody to the 205 nm latex and 8 ml of FER antibody to the 360 ​​nm latex. Stir magnetically at room temperature for 3 hours to complete antibody conjugation (final volume 25 ml for 205 nm and 27 ml for 360 nm).

[0063] (5) R2 modification: 1) Take 4 ml of each of the two types of latex that have been antibody-conjugated, and add 200 μl of 300 mM urea solution to each, so that the urea concentration is 15 mM.

[0064] 2) Centrifuge both types of latex at 22000 rpm for 15 min, and discard 2 ml of supernatant, i.e., “replace 50% of the liquid”.

[0065] 3) Dissolve 1 mg of an amino acid scaffold with the X sequence EDED (synthesized by Sangon Biotech (Shanghai) Co., Ltd.) in 50 ml of scaffold dissolution solution. The calculated antibody mass for each experimental group is approximately 205 nm. Latex: 5.6 2.5 4 / 25 = 2.24 mg, 360 nm latex: 8 2.5 4 / 27 = 2.96mg.

[0066] Add 2 ml of solution containing amino acid scaffold to each of the two latexes, and reconstitute. The amount of antibody and scaffold used can be calculated as follows: 1 mg of antibody corresponds to 0.0179 mg of latex at 205 nm and 0.0135 mg of amino acid scaffold in latex at 360 nm. 4) Add 0.6 ml of 50 mM potassium persulfate solution, which means that the molar amount of potassium persulfate is about 50% of the amount of urea in step 1).

[0067] 5) Incubate at 37 degrees Celsius in a constant temperature shaker, gently rotating at 120 rpm for 30 minutes to mix and incubate.

[0068] (6) Sealing and cleaning.

[0069] After centrifuging both latexes at 22,000 rpm for 15 minutes, all supernatant was discarded. 49.3 ml of blocking and washing buffer was added to the 360 ​​nm latex to reconstitute it (final latex concentration approximately 3.0 g / L), and 53.3 ml of the 205 nm latex was added to reconstitute it (final latex concentration approximately 3.0 g / L). The two latexes were mixed at a volume ratio of 2:1 and stirred at room temperature for 2 hours (final concentration: 2.0 g / L for the 360 ​​nm carboxylated latex and 1.0 g / L for the 205 nm carboxylated latex). The antibody concentration was approximately 21.6 ml / L, which is the final product R2.

[0070] PCT R2 preparation and formulation (common process): To facilitate demonstration of the effects of this invention, please refer to our company's patent, application number: 202111453821.7, "A High-Specific Transmissivity-Scattering Integrated Method for Calcitonin Progeny Latex Turbidimetric Detection Kit," which simplifies the PCT reagent R2 formulation: 50mM PBS pH7.5; Tween-20 16ml / L; 450nm latex 1.4g / L; 260nm latex 0.7g / L; protectant (sucrose 30g / L, trehalose 10g / L, fructose 10g / L); PCT rabbit polyclonal antibody 28ml / L (Chengdu Huirui Xinyuan Biotechnology); preservative 0.5ml / L PC300; antibody labeling process: After production using the conventional chemical coupling method, sensitivity processing was performed in accordance with 2018114264906, "A Method for Improving the Sensitivity of Latex Turbidity Reagent Detection". Prepare 100 ml of 300 mM urea solution and set aside at room temperature.

[0071] Prepare the scaffold dissolution solution: 100mM pH 7.5 PBS buffer containing 0.1% v / v Tween-20, ready for use at room temperature.

[0072] Prepare a 50mM potassium persulfate solution: Dissolve 2.7032g of potassium persulfate in 200ml of stent dissolving solution and store at 4℃ for later use.

[0073] Prepare the blocking and washing solution: 50mM pH7.6 PBS buffer containing 16ml / L Tween-20, 30g / L sucrose, 10g / L trehalose, 10g / L fructose, and 0.5ml / L preservative PC300. Store at room temperature.

[0074] Several units of Huirui Xinyuan PCT polyclonal antibody (6mg / ml) are available for use.

[0075] The latex microspheres described below are conventional carboxylated polystyrene latexes used in Comparative Examples 1 and 2, or carboxylated polystyrene-polypropylene (polyethylene) latexes prepared by the above-mentioned seed method used in Examples 1 and 2.

[0076] (1) Latex cleaning: Use 100mM MES-NaOH buffer, pH 5.5, centrifuge and change the latex microspheres twice; take 10ml of the above latex with a 10% mass concentration, i.e., 1g of microspheres with a mass concentration of 260nm and 450nm, centrifuge at 22000rpm for 15min, remove the supernatant, reconstitute with 10ml of the above buffer, centrifuge again, remove the supernatant and reconstitute, and complete the cleaning for use.

[0077] (2) Latex activation: Taking 10ml of the above-washed latex as an example, weigh 50mg NHS (N-hydroxysuccinimide) and 30mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5ml of the above-mentioned cleaning buffer, and add 1ml of each to 10ml of the three types of cleaned microspheres. Stir magnetically at room temperature for 10min.

[0078] (3) Cleaning after activation: Centrifuge the three latexes activated in step (2) at 22000 rpm for 15 min, remove the supernatant, and add 20 ml of 100 mM / L pH7.5 PBS buffer to reconstitute them for later use.

[0079] (4) Antibody conjugation: Take two portions of Huirui Xinyuan PCT antibody and add them to the three activated latex solutions reconstituted with PBS buffer. Add 12.5 ml of PCT antibody to the 450 nm latex and 15 ml of PCT antibody to the 260 nm latex. Stir magnetically at room temperature for 3 hours to complete antibody conjugation (final volume 31 ml for 450 nm and 33 ml for 260 nm).

[0080] (5) R2 modification: 1) Take 4 ml of each of the two types of latex that have been antibody-conjugated, and add 200 μl of 300 mM urea solution to each, so that the urea concentration is 15 mM.

[0081] 2) Centrifuge both types of latex at 22000 rpm for 15 min, and discard 2 ml of supernatant, i.e., “replace 50% of the liquid”.

[0082] 3) Dissolve 1 mg of an amino acid scaffold with the X sequence EDED (synthesized by Sangon Biotech (Shanghai) Co., Ltd.) in 12 ml of scaffold dissolution solution. The calculated antibody mass for each experiment is approximately 450 nm latex: 12.5. 6 4 / 31 = 9.7mg, 260nm latex: 15 6 4 / 33 = 10.9mg.

[0083] Add 2 ml of solution containing amino acid scaffold to each of the two latexes and reconstitute. The amount of antibody and scaffold used can be calculated as follows: 1 mg of antibody corresponds to 0.0172 mg of latex at 450 nm and 0.0153 mg of amino acid scaffold at 260 nm.

[0084] 4) Add 0.6 ml of 50 mM potassium persulfate solution, which means that the molar amount of potassium persulfate is about 50% of the amount of urea in step 1).

[0085] 5) Incubate at 37 degrees Celsius in a constant temperature shaker, gently rotating at 120 rpm for 30 minutes to mix and incubate.

[0086] (6) Sealing and cleaning: After centrifuging the two latexes at 22,000 rpm for 15 minutes, all supernatant was discarded. Blocking and washing buffer was added to redissolve the two latexes: 61.4 ml of 450 nm latex (final latex concentration approximately 2.1 g / L) and 57.7 ml of 260 nm latex (final latex concentration approximately 2.1 g / L). The two latexes were then mixed at a volume ratio of 2:1 and stirred at room temperature for 2 hours (final concentration of 1.4 g / L for 450 nm latex and 0.7 g / L for 260 nm latex). The antibody concentration was approximately 28 ml / L, which is the final product R2.

[0087] Methods for adding the immunosuppressant prednisone: After completing the preparation of R2 as described above, 0.2-2.0 mg of prednisone was added per L of R2 for use in each embodiment.

[0088] Pharmaceutical grade prednisolone acetate 5mg per tablet, take 4 tablets and dissolve them in 200ml of purified water, add 60µl of preservative PC300 for later use, to obtain a 0.1mg / ml prednisolone acetate solution.

[0089] Add 2 ml of prednisolone acetate solution (0.1 mg / ml) and 18 ml of water to 1 L of R2 to obtain the anti-cross-contamination R2 containing a final concentration of 0.2 mg prednisolone per L of R2. Similarly, add 8 ml of prednisolone acetate solution (0.1 mg / ml) and 12 ml of water to 1 L of R2, add 14 ml of prednisolone acetate solution (0.1 mg / ml) and 6 ml of water to 1 L of R2, and add 20 ml of prednisolone acetate solution (0.1 mg / ml) to 1 L of R2 to obtain anti-cross-contamination R2 concentrations of 0.8 mg, 1.4 mg, and 2.0 mg prednisolone per L of R2, respectively. Mix well and set aside.

[0090] R2 has no ability to resist cross-contamination. Simply add 20ml of water per L of R2 and mix well before use.

[0091] R1 formula: Please refer to our company's patent, application number: 202111453716.3, "A High-Specific Transmittance Scattering Integrated Method Ferritin Latex Turbidimetric Detection Kit", FER reagent R1: 100mM Hepes-NaOH buffer pH 7.0, NaCl 1500mM, polyethylene glycol 12000 2g / L, Tween-20 16ml / L, deferoxamine 11 g / L, rabbit polyclonal antibody blocking agent 30ml / L.

[0092] Please refer to our company's patent, application number: 202111453821.7, "A High-Specific Transmission-Scattering Integrated Method for Calcitonin Progeny Latex Turbidimetric Detection Kit", PCT reagent R1: 100mM Hepes-NaOH buffer pH 7.0, NaCl 1600mM, polyethylene glycol 12000 3g / L, Tween-20 16ml / L, TiterMax® classic water-soluble adjuvant 12ml / L, polyclonal antibody blocking agent 30ml / L.

[0093] Prepare sufficient quantities of the above-mentioned R1 for use as reagents for the FER and PCT projects in subsequent examples.

[0094] Sensitivity evaluation method: Taking the Hitachi 3500 specific protein biochemical analyzer as an example, a two-point endpoint method was used. Reading points 18-34 were used for calibration and testing, with the same reaction buffer R1, parameters, and calibrators. The delta signal values ​​of low-concentration calibration data from different experimental groups were compared, and two levels of low concentration were measured 10 times, with the mean, STDEV, and CV calculated. The CV was compared. A higher delta signal value indicates higher sensitivity, and a lower CV also indicates higher sensitivity. For quantitative detection reagents, a CV < 10% is considered acceptable, < 5% does not affect clinical use, and ≤ 2% is excellent.

[0095] Evaluation method for resistance to cross-contamination: The method simulates the impact of cross-contamination at the hospital client level, i.e., onboard stability. Specifically: For onboard reagents with open caps, the bias of the two-level quality control values ​​measured within 30 days is assessed. A bias ≥8.33% triggers an alert, and >12.5% ​​is considered unacceptable. The onboard stability period is compared between uncontaminated reagents, uncontaminated reagents with anti-interference protocols, reagents with artificially added contaminants, and reagents with artificially added contaminants but with anti-interference protocols, considering the number of days it takes for the reagent to reach the alert or fail. The earlier the alert or failure is reached, the worse the result. A stability period exceeding 7 days is considered acceptable, 14 days does not affect clinical use, 21 days or more is considered excellent, and 30 days is considered outstanding. Failure in any one of the two-level quality controls results in a failure.

[0096] Comparative Example 1: Conventional FER latex immunoturbidimetric kit and procalcitonin PCT latex immunoturbidimetric kit, without the use of low-density latex and without the addition of immunosuppressants. FER Kit: Latex microspheres, using conventional carboxylated polystyrene microspheres, such as Polymicroshperes CB0200E (approximately 205 nm) and CB0376C (approximately 360 nm) latex, with a 205 nm:360 nm mixing ratio of 1:2, and Agilent Dako's A0133 ferritin FER rabbit polyclonal antibody, were prepared according to the above common process. The instrument parameters were as follows: 2µl sample, 160µl reaction buffer (R1), 80µl sensitized latex (R2), 600nm transmission wavelength, scattering detector 2, transmission technology threshold of 400 to 4000 ng / ml, scattering technology threshold of -999 to 800 ng / ml, with priority given to scattering in the fusion region. A two-point endpoint method was used. The sensitivity of the instrument was determined and calibrated on a Hitachi 3500 specific protein biochemical analyzer at readings 18-34. The δ signal value at a concentration of 30 ng / ml was compared. The quality control materials at 10 ng / ml and 30 ng / ml were tested 10 times, and the mean, STDEV, and CV were calculated.

[0097] PCT kit: Latex microspheres, using conventional carboxylated polystyrene microspheres, such as Polymicroshperes CB0270D (approximately 260 nm) and CB0430B (approximately 450 nm), with a 260 nm:450 nm mixing ratio of 1:2, and PCT rabbit polyclonal antibody (catalog number 301089) from Chengdu Huirui Xinyuan Co., Ltd., were prepared according to the above-mentioned common process. The instrument parameters were as follows: 2µl sample, 150µl reaction buffer (R1), 75µl sensitized latex, 700nm transmission wavelength, scattering detector 2, transmission technology threshold of 12 to 400 ng / ml, scattering technology threshold of -999 to 17 ng / ml, with priority given to scattering in the fusion region. A two-point endpoint method was used. Calibration and sensitivity assessment were performed on a Hitachi 3500 specific protein biochemical analyzer at readings 18-34. The δ signal value at a concentration of 3.5 ng / ml was compared. The 1 ng / ml and 5 ng / ml quality control materials were tested 10 times, and the mean, STDEV, and CV were calculated.

[0098] Cross-contamination simulation: Simulate the situation of trace contamination carried by hospital client reagents or aerosols by adding 10µl of the test antigen to 10ml of sensitized latex R2.

[0099] To prevent cross-contamination of FER reagent, 10 µl of antigen at a concentration of 1000 ng / mL was added to 10 ml of sensitized latex R2, which is equivalent to adding 10 ng of FER antigen. After 30 days on-board, the quality control materials at 200 ng / ml and 1000 ng / ml were measured at a higher concentration before and lower concentration afterward, and the changes in the quality control materials were observed.

[0100] To prevent cross-contamination of PCT reagent, 10 ml of sensitized latex R2 was added with 100 ng / ml of antigen, i.e., 1 ng of PCT antigen was added. After 30 days on-board, the quality control material was measured at 8 ng / ml and 40 ng / ml, with the concentration increased at the beginning and decreased at the end, and the changes in the quality control material were observed.

[0101] The uncontaminated reagent does not contain any contaminating antigens and is tested in the same way as the cross-contaminated reagent.

[0102] Comparative Example 1: FER project test data are shown in Tables 2-5.

[0103] Table 2

[0104] Table 3

[0105] Table 4

[0106] Table 5

[0107] Comparative Example 1: PCT project test data are shown in Tables 6-9.

[0108] Table 6

[0109] Table 7

[0110] Table 8

[0111] Table 9

[0112] Tables 2-9 show that reagents prepared from conventional carboxylated polystyrene latex have acceptable sensitivity and achieve 30 days of onboard stability under uncontaminated conditions. However, with prolonged onboard operation with the lid open, the measurement deviation gradually increases, possibly due to reagent oxidation or trace aerosol contamination. In cases of cross-contamination, for the FER project, stability was acceptable for the first two days, but from the third day onwards, the 1000 ng / ml quality control deviation exceeded 12.5%, resulting in failure. By the 14th day, reagent sedimentation was visibly observed, rendering the reagent unusable. For the PCT project, possibly due to the larger latex particle size and weaker resistance to cross-contamination, warnings were triggered at the 8 ng / ml quality control level on the second day, and both levels of quality control failed on the third day. On the 7th day of onboard operation, reagent sedimentation was observed, rendering the reagent unusable.

[0113] Comparative Example 2: Conventional FER latex immunoturbidimetric kit and procalcitonin PCT latex immunoturbidimetric kit, which were used without low mass density latex and only had different concentrations of immunosuppressants added.

[0114] FER Kit: Latex microspheres, using conventional carboxylated polystyrene microspheres, such as Polymicroshperes CB0200E (approximately 205 nm) and CB0376C (approximately 360 nm) latex, with a 205 nm:360 nm mixing ratio of 1:2, and Agilent Dako's A0133 ferritin FER rabbit polyclonal antibody, were prepared according to the above common process. Furthermore, 0.2 mg, 0.8 mg, 1.4 mg, and 2.0 mg of prednisone were added per L of sensitized latex to compare the effects of different immunosuppressants on sensitivity and anti-contamination ability. The instrument parameters were as follows: 2 µl sample, 160 µl reaction buffer (R1), 80 µl sensitized latex, 600 nm transmission wavelength, scattering detector 2, transmission technology thresholds of 400 to 4000 ng / ml, scattering technology thresholds of -999 to 800 ng / ml, with priority given to scattering in the fusion region. A two-point endpoint method was used, with 18-34 readings on a Hitachi 3500 specific protein biochemical analyzer. All experimental groups used the same R1, the same calibrators, and calibration and sensitivity assessment methods. The δ signal value at a concentration of 30 ng / ml was compared. Ten tests were performed on quality controls at 10 ng / ml and 30 ng / ml, and the mean, STDEV, and CV were calculated.

[0115] PCT kit: Latex microspheres, using conventional carboxylated polystyrene microspheres, such as Polymicroshperes CB0270D (approximately 260 nm) and CB0430B (approximately 450 nm), with a 260 nm:450 nm mixing ratio of 1:2, and PCT rabbit polyclonal antibody (catalog number 301089) from Chengdu Huirui Xinyuan Co., Ltd., were prepared according to the above-mentioned common process. Furthermore, 0.2 mg, 0.8 mg, 1.4 mg, and 2.0 mg of prednisone were added per L of sensitized latex to compare the effects of different immunosuppressants on sensitivity and anti-contamination ability. The instrument parameters were as follows: 2 µl sample, 150 µl reaction buffer (R1), 75 µl sensitized latex, 700 nm transmission wavelength, scattering detector 2, transmission technology limits of 12 to 400 ng / ml, scattering technology limits of -999 to 17 ng / ml, with priority given to scattering in the fusion region. A two-point endpoint method was used, with 18-34 readings on a Hitachi 3500 specific protein biochemical analyzer. All experimental groups used the same R1, the same calibrators, and calibration and sensitivity assessment. The δ signal value at a concentration of 3.5 ng / ml was compared. Ten tests were performed on quality controls at 1 ng / ml and 5 ng / ml, and the mean, STDEV, and CV were calculated.

[0116] To prevent cross-contamination of FER reagent, 10 µl of antigen at a concentration of 1000 ng / mL was added to 10 ml of sensitized latex R2, which is equivalent to adding 10 ng of FER antigen. After 30 days on-board, the quality control materials at 200 ng / ml and 1000 ng / ml were measured at a higher concentration and then lower concentration, and the changes in the quality control materials were observed.

[0117] To prevent cross-contamination of PCT reagent, 10 µl of 100 ng / ml antigen was added to 10 ml of sensitized latex R2, which is equivalent to adding 1 ng of PCT antigen. After 30 days on-board, the quality control material was measured at 8 ng / ml and 40 ng / ml, with the concentration increased at the beginning and decreased at the end, and the changes in the quality control material were observed.

[0118] The uncontaminated reagent does not contain any contaminating antigens and is tested in the same way as the cross-contaminated reagent.

[0119] The FER test data results are shown in Tables 10-22.

[0120] Table 10

[0121] Table 11

[0122] Table 12

[0123] Table 13

[0124] Table 14

[0125] Table 15

[0126] Table 16

[0127] Table 17

[0128] Table 18

[0129] Table 19

[0130] Table 20

[0131] Table 21

[0132] Table 22

[0133] Table 22 summarizes the analysis of Tables 2 to 21. The results show that for latex turbidimetric reagents prepared from conventional carboxylated polystyrene latex particles, when different concentrations of the temperature-sensitive immunosuppressant prednisone were added to the sensitized latex, the reagent sensitivity initially increased and then decreased as the prednisone concentration increased from 0.2 to 2.0 mg. Compared to reagents without prednisone, the sensitivity increased by approximately 11.9-19.88%. A similar pattern was observed in the precision detection of low concentrations; the CV at 10 ng / ml improved from acceptable to approximately 5% or less, which is not considered unusable. This may be because an appropriate amount of prednisone can slow down the reaction rate in the initial stage of the reaction, and at a constant temperature of 37 degrees Celsius, it decomposes rapidly, leading to increased sensitivity through the δ signal value calculation formula. For the FER test, the sensitivity gradually increased with increasing prednisone concentration from 0.2 mg to 1.4 mg. However, when the concentration of prednisone is too high, such as 2.0 mg, it cannot be completely and timely decomposed. Excessive prednisone lowers the overall signal value, resulting in a decrease in the δ signal value. Therefore, the concentration of prednisone needs to be appropriate, and the optimal concentration needs to be determined by combining the ability to resist cross-contamination.

[0134] The airborne stability data of conventional latex reagents with different prednisone concentrations are shown in Tables 23-26.

[0135] Table 23

[0136] Table 24

[0137] Table 25

[0138] Table 26

[0139] The results in Tables 23-26 show that as the concentration of prednisone increases, the immunosuppressive capacity and the ability to resist cross-contamination increase, and the number of days of airborne stability continues to increase. Both 1.4 mg and 2.0 mg can achieve 30 days of stability. Combined with the analysis of previous sensitivity data, the optimal prednisone concentration for the FER project is 1.4 mg per liter of sensitized latex.

[0140] The test data results for the PCT project are shown in Tables 27-37.

[0141] Table 27

[0142] Table 28

[0143] Table 29

[0144] Table 30

[0145] Table 31

[0146] Table 32

[0147] Table 33

[0148] Table 34

[0149] Table 35

[0150] Table 36

[0151] Table 37

[0152] Table 37 summarizes the analysis of Tables 27 to 36. It shows that for the PCT project using latex turbidimetric reagents prepared from conventional carboxylated polystyrene latex particles, when different concentrations of the temperature-sensitive immunosuppressant prednisone were added to the sensitized latex, the reagent sensitivity initially increased and then decreased as the prednisone concentration increased from 0.2 to 2.0 mg. Compared to reagents without prednisone, the sensitivity increased by approximately 8.76-19.05% (the 3.5 ng / ml transmission sensitivity was not measured because it was difficult to measure). A similar pattern was observed in the precision detection of low concentrations, with the CV at 1 ng / ml improving from acceptable to approximately 5% or less, which does not affect usability. The reason for this may be that an appropriate amount of prednisone can slow down the reaction rate in the initial stage of the reaction, and then rapidly decompose at a constant temperature of 37 degrees Celsius, leading to increased sensitivity through the δ signal value calculation formula. Slightly different from the FER project, for the PCT project, the sensitivity gradually increased as the prednisone concentration increased from 0.2 mg to 0.8 mg. However, when the prednisone concentration is too high, such as ≥1.4 mg, it may not be completely and timely decomposed. Excessive prednisone lowers the overall signal value, resulting in a decrease in the δ signal value. Therefore, the prednisone concentration needs to be appropriate, and the optimal concentration needs to be determined by combining the ability to resist cross-contamination. The optimal prednisone concentration varies significantly among different projects.

[0153] The airborne stability data of conventional latex for the PCT project with different prednisone concentrations are shown in Tables 38-41.

[0154] Table 38

[0155] Table 39

[0156] Table 40

[0157] Table 41

[0158] As can be seen, the pattern is exactly the same for the PCT project and the FER project. With the increase of prednisone concentration, that is, the immunosuppressive ability increases, the ability to resist cross-contamination increases, and the number of days of qualified airborne stability continues to increase. At 1.4mg and 2.0mg, both can achieve 30 days of stability. Combined with the analysis of previous sensitivity data, for the PCT project, the optimal prednisone concentration is 0.8mg per liter of sensitized latex, which has the highest sensitivity and qualified stability for 20 days.

[0159] Example 1: Low mass density latex (approximately 0.94 g / ml) prepared by the seed method was used with FER latex immunoturbidimetric kit and procalcitonin PCT latex immunoturbidimetric kit containing optimal concentrations of immunosuppressants.

[0160] FER Kit: Latex microspheres, using polypropylene (PP) as the core seed, are prepared using a conventional seeding process to prepare carboxylated polystyrene-polypropylene latex microspheres. For example, using 100nm polypropylene will produce a particle size of approximately 205nm (mass density of approximately 0.94g / ml), and using 250nm polypropylene will produce a particle size of approximately 360nm (mass density of approximately 0.94g / ml). The 205nm:360nm mixing ratio is 1:2. Agilent Dako's A0133 ferritin FER rabbit polyclonal antibody is used. The latex turbidimetric kit is prepared according to the above common process. Furthermore, 1.4 mg of prednisone was added per L of sensitized latex; the instrument parameters were as follows: 2 µl sample, 160 µl reaction buffer (R1), 80 µl sensitized latex, 600 nm transmission wavelength, scattering detector 2, transmission technology limit of 400 to 4000 ng / ml, scattering technology limit of -999 to 800 ng / ml, scattering of the fusion region preferred, a two-point endpoint method was used, and 18-34 readings were performed on a Hitachi 3500 specific protein biochemical analyzer. The above different experimental groups used the same R1, the same calibrator, and the same calibration and test sensitivity judgment. The δ signal value at a concentration of 30 ng / ml was compared. The 10 ng / ml and 30 ng / ml quality control materials were detected 10 times, and the mean, STDEV and CV were calculated.

[0161] PCT kit: Latex microspheres, using polypropylene (PP) as the core seed, are prepared using a conventional seeding method to prepare carboxylated polystyrene-polypropylene latex microspheres. For example, using 150nm polypropylene will produce a particle size of approximately 260nm (mass density of approximately 0.94g / ml), and using 350nm polypropylene will produce a particle size of approximately 450nm (mass density of approximately 0.94g / ml). The 260nm:450nm mixing ratio is 1:2. PCT rabbit polyclonal antibody (catalog number 301089) from Chengdu Huirui Xinyuan Co., Ltd. is used to prepare the latex turbidimetric kit according to the above common process. Furthermore, 0.8 mg of prednisone was added per L of sensitized latex; the instrument parameters were as follows: 2 µl sample, 150 µl reaction buffer (R1), 75 µl sensitized latex, 700 nm transmission wavelength, scattering detector 2, transmission technology thresholds of 12 to 400 ng / ml, scattering technology thresholds of -999 to 17 ng / ml, scattering of the fusion region preferred, and a two-point endpoint method was used. Readings 18-34 were performed on a Hitachi 3500 specific protein biochemical analyzer. The same R1 and the same calibrators were used for different experimental groups. Calibration and sensitivity testing were performed, and the δ signal value at a concentration of 3.5 ng / ml was compared. The 1 ng / ml and 5 ng / ml quality control materials were tested 10 times, and the mean, STDEV, and CV were calculated.

[0162] To prevent cross-contamination of FER reagent, 10 µl of antigen at a concentration of 1000 ng / mL was added to 10 ml of sensitized latex R2, which is equivalent to adding 10 ng of FER antigen. After 30 days on-board, the quality control materials at 200 ng / ml and 1000 ng / ml were measured at a higher concentration before and lower concentration afterward, and the changes in the quality control materials were observed.

[0163] To prevent cross-contamination of PCT reagent, 10 µl of 100 ng / ml antigen was added to 10 ml of sensitized latex R2, which is equivalent to adding 1 ng of PCT antigen. After 30 days on-board, the quality control material was measured at 8 ng / ml and 40 ng / ml, with the concentration increased at the beginning and decreased at the end, and the changes in the quality control material were observed.

[0164] The uncontaminated reagent does not contain any contaminating antigens and is tested in the same way as the cross-contaminated reagent.

[0165] The FER test data results are shown in Tables 42-44.

[0166] Table 42

[0167] Table 43

[0168] Table 44

[0169] As can be seen, for the FER project, the low-density latex prepared with polypropylene (PP) as the core seed, at a relatively optimal prednisone concentration, combined with the two beneficial factors, resulted in a sensitivity improvement of approximately 45% at 30 ng / ml compared to Comparative Example 1. CV tests at 10 ng / ml and 30 ng / ml showed levels that were at or near excellent.

[0170] The reason can be found by comparing the reaction signal values ​​of Comparative Example 1 and Preferred Example 1 at 30 ng / ml (the scattering value is higher, so the scattering is used for analysis).

[0171] The results are shown in Table 45 and Figure 3 .

[0172] Table 45

[0173] Comparing the response curves at measurement points 17-34, it can be clearly seen that the better low-density latex in Example 1, combined with a better prednisone concentration, resulted in a lower initial reaction rate due to prednisone, while the low-density latex resulted in a higher overall reaction rate. The combined effect of these two factors achieved the aforementioned sensitivity gain.

[0174] The airborne stability data for FER low-density latex and optimal concentration of prednisone are shown in Table 46.

[0175] Table 46

[0176] Stability data shows that with the addition of 1.4mg of prednisone, airborne stability can reach an excellent level of 30 days even under slight contamination.

[0177] In the PCT project, low-density latex microspheres were prepared using polypropylene (PP) cores as seeds, combined with a prednisolone dosage of 0.8 mg per liter of sensitized latex. The test data are shown in Tables 47-49.

[0178] Table 47

[0179] Table 48

[0180] Table 49

[0181] As can be seen, for the PCT project, similar to the FER project, the low-density latex prepared with polypropylene (PP) as the core seed, at a relatively optimal prednisone concentration, the two beneficial factors combined, resulting in a sensitivity improvement of approximately 40% at 3.5 ng / ml compared to the control in Example 1. CV tests at 1 ng / ml and 5 ng / ml have reached or are close to excellent levels.

[0182] The reason can be found by comparing the reaction signal values ​​of Example 1 control and the better Example 3 at 3.5 ng / ml (the concentration of 3.5 ng / ml for PCT is low, below the limit of transmission detection capability, and can only be detected by scattering, so the analysis is based on scattering data).

[0183] The results are shown in Table 50 and Figure 4 .

[0184] Table 50

[0185] Comparing the response curves at metering points 17-34, it can be clearly seen that, similar to FER, in the PCT test, Example 3's superior low mass density latex combined with a superior prednisone concentration results in a lower initial reaction rate due to prednisone, while the low mass density latex leads to a higher overall reaction rate. The combined effect of these two factors achieves the aforementioned sensitivity gain.

[0186] Airborne stability data for low-density latex and optimal concentration of prednisone in the PCT project are shown in Table 51.

[0187] Table 51

[0188] Stability data shows that with the addition of 0.8mg prednisone, airborne stability can reach a relatively good level for 20 days even under slight contamination.

[0189] Example 2: Taking the FER project as an example, the impact of optimal prednisolone concentration on density and seed material was evaluated. FER kit: Latex microspheres, using polypropylene (PP) of different diameters and polyethylene (PE) as core seeds, carboxylated polystyrene-polypropylene latex microspheres were prepared using a conventional seeding method. The 205nm:360nm mixing ratio was 1:2, so the preparation method for 205nm remained unchanged. Using 100nm polypropylene resulted in particle sizes of approximately 205nm. Only the preparation method for 360nm was changed. Single-factor comparison showed that using 200nm and 300nm polypropylene as core seeds resulted in carboxylated polystyrene-polypropylene latex microspheres with a particle size of approximately 360nm (200nm and 300nm). PP core, 360nm latex microspheres were prepared with a mass density of approximately 0.96 g / ml and 0.92 g / ml, respectively. The mass density of PP is approximately 0.89 g / ml. The larger the core, the lower the mass density of the final product, and vice versa. The mass density of PP is approximately 0.89 g / ml. 300nm polyethylene (mass density approximately 0.93 g / ml) was used to prepare 360nm carboxy-polystyrene-polyethylene latex microspheres (mass density approximately 0.96 g / ml). The 205nm:360nm mixing ratio was 1:2. Agilent Dako's A0133 ferritin FER rabbit polyclonal antibody was used to prepare a latex turbidimetric kit according to the above common process. Furthermore, 1.4 mg of prednisone was added per L of sensitized latex; the instrument parameters were as follows: 2 µl sample, 160 µl reaction buffer (R1), 80 µl sensitized latex, 600 nm transmission wavelength, scattering detector 2, transmission technology threshold of 400 to 4000 ng / ml, scattering technology threshold of -999 to 800 ng / ml, scattering of the fusion region was preferred, and a two-point endpoint method was used. Readings 18-34 were performed on a Hitachi 3500 specific protein biochemical analyzer. The same R1 and the same calibrators were used for different experimental groups. Calibration and sensitivity testing were performed, and the δ signal value at a concentration of 30 ng / ml was compared. The 10 ng / ml and 30 ng / ml quality control materials were tested 10 times, and the mean, STDEV, and CV were calculated.

[0190] The FER test data results are shown in Tables 52-57.

[0191] Table 52

[0192] Table 53

[0193] Table 54

[0194] Table 55

[0195] Table 56

[0196] Table 57

[0197] In summary, when using a polypropylene (PP) core, the final latex density at 360nm decreases with increasing core particle size (from approximately 0.96 g / ml at 200nm to approximately 0.92 g / ml at 300nm). The delta signal value at 30 ng / ml gradually increases, while the coefficient of variation (CV) at 10 ng / ml and 30 ng / ml shows an excellent decrease of less than 2%. However, the reagent prepared from latex using a 300nm PP core begins to show oil-water separation after 4-5 months of storage, with latex starting to aggregate on the top layer. This is considered borderline and does not affect clinical use (the reagent needs to be mixed every 2-3 months). Polyethylene, with its relatively high density of 0.93, requires a 300nm core to prepare 360nm microspheres with a density of 0.96 g / ml, resulting in the lowest sensitivity gain; therefore, it is not the preferred choice.

[0198] Low-density latex prepared with a 250nm core as the seed will only show oil-water separation after being left for more than 8 months. Considering the overall sensitivity gain and ease of use, the 250nm polypropylene core is preferred as the seed for preparing 360nm carboxylated polystyrene-polypropylene latex.

[0199] This embodiment uses 360nm latex, which accounts for 2 / 3 of the FER, as an example to analyze the seed core material and seed particle size. Those skilled in the art should be able to deduce the preparation methods and effects of latex with other particle sizes from this embodiment, which will not be listed here.

[0200] Those skilled in the art will know from the reaction curves of the two preferred formulations of the kit in Example 1 that, for the selection of the photometric point, as long as the sample is first mixed with R1 and incubated for 1 to 5 minutes, and then R2 is added to sensitize the latex for antigen-antibody latex reaction, the δ signal value is calculated using the two-point endpoint method, and the first light signal is collected after the sensitized latex R2 is added to the mixture of R1 and the sample reaction solution (the first photometric point is after R2 is added), and the second light signal is collected after the immune reaction has proceeded for 2-5 minutes, the latex turbidimetric kit using the content of this invention can achieve both reagent anti-interference ability and a certain degree of improvement in detection sensitivity.

[0201] Taking the Hitachi 3500 as an example, using the content of this invention, according to the δ signal value calculation formula, the optimal reading point is generally 18-34 or 19-34 (considering the time required for R2 to be added to the reaction solution and mixed, the measurement point is delayed by one point). This can obtain a higher δ signal value and sensitivity gain compared to other reading point methods. Simply changing the reading point to 18-24 (the immune response takes about 2 minutes) can also be considered an extension of 18-34, except that the obtained δ signal value is less than that of 18-34.

[0202] Similarly, as long as the first measurement point is delayed and the reaction rate is still within the range of lower reaction rate compared to conventional reagents, such as when the reading point becomes 20-34, the corresponding effect can still be obtained. However, the δ signal value will be lower and the sensitivity gain will be lower than 18-34.

[0203] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously improving the sensitivity and cross-contamination resistance of a latex turbidimetric reagent kit, characterized in that, The latex turbidimetric reagent kit contains reagent R1 and reagent R2; reagent R1 is a reaction buffer solution and reagent R2 is a sensitized latex solution. The latex microspheres used in the sensitizing latex solution are low-mass-density latex microspheres, and the sensitizing latex solution contains prednisone; The mass density of the low-mass-density latex microspheres is 0.92-0.96 g / ml; The dosage of the temperature-sensitive immunosuppressant prednisone in 1L of sensitized latex solution is 0.20-2.00 mg.

2. The method for simultaneously improving the sensitivity and anti-cross-contamination ability of a latex turbidimetric reagent kit according to claim 1, wherein the mass density of the low-mass-density latex microspheres is 0.94 g / ml; The amount of prednisone used in 1L of sensitized latex solution is 0.8-1.4 mg.

3. The method for simultaneously improving the sensitivity and cross-contamination resistance of a latex turbidimetric reagent kit according to claim 1, characterized in that, The low mass density latex microspheres are hollow latex microspheres prepared by seed method or other methods; the seed is a polymer with a mass density of less than 1.03 g / ml; the polymer is polyethylene or polypropylene.

4. The method for simultaneously improving the sensitivity and cross-contamination resistance of a latex turbidimetric reagent kit according to claim 1, characterized in that, On a fully automated biochemical or specific protein analyzer, the sample and reagent R1 are first mixed and incubated for 1 to 5 minutes, and then reagent R2 is added to sensitize the latex solution for antigen-antibody latex reaction. The δ signal value is calculated using the two-point endpoint method. After the sensitized latex solution is added to the mixture of reagent R1 and sample, the first light signal is collected, and the second light signal is collected after the immune reaction has been carried out for 2-5 minutes.

5. The application of the method according to any one of claims 1-4 in simultaneously improving the sensitivity and cross-contamination resistance of latex turbidimetric reagent kits.

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