A beta-amyloid protein detection kit with wide linear range adapting to various sample uses

Abstract

This invention discloses a β-trace protein detection kit with a wide linear range adaptable to various sample applications, belonging to the field of bioassay technology. The kit, comprising reagent R1 with 2-4 mg / L retinol-binding protein and 0.3-0.6 mg / L rabbit polyclonal antibody against thyroxine T4, exhibits a linear range of 0.05-35.00 mg / L. It is suitable for detecting cerebrospinal fluid, blood, urine, and nasal swab solutions, covering over 95% of sample concentrations, reducing retesting costs and improving detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically to a β-trace protein detection kit with a wide linear range adaptable to various sample applications. Background Technology

[0002] β-trace protein (BTP), also known as lipid carrier prostaglandin D synthase (L-PGDS / PGDS), is a lipid carrier protein widely found in bodily fluids such as cerebrospinal fluid, blood, seminal plasma, amniotic fluid, and intraocular fluid. Due to varying degrees of glycosylation, its molecular weight ranges from 23 to 29 kDa, and it exists in multiple glycoform isomers in different bodily fluids and disease states.

[0003] Over the past 30 years, the value of detecting BTP concentrations in cerebrospinal fluid (nasal leak), cerebrospinal fluid, blood, and urine for various diseases has been continuously confirmed. In recent years, numerous studies have explored its value in different diseases by using different methods and reagent kits to detect BTP concentrations in different body fluids. In other words, there are many emerging research projects in the field of in vitro diagnostics regarding BTP detection methods and reagent kits.

[0004] Literature documents describe BTP detection methods including ELISA, lateral chromatography, latex turbidimetry, electrochemical aptamer methods, and the obsolete electrophoresis method. Those skilled in the art, considering the numerous BTP isoforms, can infer that polyclonal antibodies are the preferred method for BTP detection using immunoassays, as some monoclonal antibody-based methods are fundamentally flawed. Furthermore, a review of the literature indicates a high correlation between BTP concentration and disease progression; therefore, reproducible and accurately quantifiable methodologies are preferred.

[0005] The currently accepted and most accurate BTP detection method is the latex turbidity assay based on polyclonal antibodies. Currently, only Siemens' BTP detection kit has obtained NMPA medical device registration certification. The basic performance of the Siemens BTP detection kit is as follows: for an initial dilution of 1:100, the measurement range is 0.22 to 14.0 mg / L, with an accuracy CV of 2.3%-6.5%. At a 1:1 dilution, the detection limit is 2.5 µg / L (i.e., 0.0025 mg / L). In other words, the machine automatically dilutes the initial sample 100 times, with a linear range of 0.22 to 14.0 mg / L. If the initial sample concentration is below 0.22 mg / L, the machine reduces the dilution factor; if the initial concentration is above 14.0 mg / L, the machine increases the dilution factor.

[0006] BTP concentrations vary significantly in different body fluids. For example, cerebrospinal fluid has the highest concentration, with a median of 9 mg / L and high values ​​reaching 15-30 mg / L. Blood concentrations are usually lower, with a 95% confidence interval of less than 0.7 mg / L. In kidney disease, concentrations can rise significantly to 5-15 mg / L. Nasal swab solutions used for nasal leak detection typically have a concentration below 0.5 mg / L, which usually rises to 1-6 mg / L when nasal leak occurs. However, nasal swab solutions often require manual dilution of 2-5 times for testing due to their high viscosity, which can clog the initial detection needle after triggering an alarm.

[0007] The performance of Siemens BTP test kits shows that when testing blood samples, the linear range is ideal, and results can be obtained with a single initial test. However, when testing cerebrospinal fluid, there is a high probability of encountering an alarm when the initial sample concentration is >14.0 mg / L, requiring the instrument to increase the dilution factor and retest, consuming an additional test and time. When testing nasal swab solutions, the BTP concentration is often below 0.22 mg / L, requiring the instrument to decrease the dilution factor and retest, also consuming an additional test and time. Since each Siemens BTP test kit costs over $20, operators are willing to risk needle clogging by attempting to use the undiluted nasal swab solution directly on the machine to minimize testing and reduce costs. Only when needle clogging does occur is the nasal swab solution manually diluted for testing.

[0008] It is evident that a BTP detection kit with a wide linear range (high sensitivity, low detection limit, and high detection limit) can be adapted to various sample applications, obtain test results in a single test as much as possible, reduce the retest rate, improve detection efficiency, and reduce detection costs, which is urgently needed for both clinical testing and scientific research.

[0009] Therefore, providing a β-trace protein detection kit with a wide linear range adaptable to various sample applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention provides a β-trace protein detection kit with a wide linear range adaptable to various sample applications.

[0011] There is no international standard for BTP protein, and high-value sample addition and recovery experiments are required for accuracy verification. Our researchers, during these experiments, found significant differences in recovery rates (the deviation between measured and theoretically calculated concentrations) when adding cerebrospinal fluid samples with normal blood-brain barrier (containing high concentrations of free BTP) to different types of samples at the NMPA-recommended fixed ratio (usually 1:9). This was particularly evident when adding BTP to samples taken with vitamin A (retinol), those with hypothyroidism, those with high T4 levels (hyperthyroidism), those with jaundice (high bilirubin), and routine blood samples. The recovery rate was significantly lower in samples with high T4 levels and those taken with vitamin A (retinol) compared to those with hypothyroidism. While there was no significant difference between jaundice and routine samples, the recovery rate was slightly lower than in samples with hypothyroidism. This indicates that the sensitivity of BTP detection was significantly inhibited in these two types of samples.

[0012] For example, if cerebrospinal fluid with a BTP concentration of 20 mg / L is added at a ratio of 1:9 to a blood sample of a hyperthyroid patient with high thyroxine levels and a BTP concentration of only 0.3 mg / L, the theoretical recovery value should be 20 mg / L. 10% + 0.3 90% = 2.27 mg / L, but the actual measured concentration of BTP was only 1.25 mg / L, resulting in a recovery rate of only 1.25 / 2.27 = 55.1%, meaning the measured value was far lower than the theoretically calculated value. For samples taking vitamin A (retinol), the recovery rate was generally below 70%, while for samples with low thyroid hormone levels, the recovery rate could reach over 90%. For jaundice and routine samples, the recovery rate was 80-85%. Research revealed that BTP has a high affinity for various lipophilic compounds. BTP selectively binds to retinol, bilirubin, biliverdin, thyroid hormones, etc., acting as a medium for the extracellular transport of these compounds. Our researchers hypothesize that relatively large molecules like retinol and thyroid hormones, when binding to BTP, mask some of the BTP antigenic epitopes, thus affecting the binding of antibodies in the BTP detection reagent and reducing detection sensitivity (turbidity signal is suppressed), resulting in a low recovery rate. Small molecule compounds such as bilirubin have a limited impact, so jaundice samples are not significantly different from regular samples. However, because they contain a small amount of thyroxine, which slightly inhibits BTP detection, the recovery rate is lower than that of low thyroxine samples.

[0013] Those skilled in the art can deduce that different blood samples, due to varying concentrations of retinol and thyroxine, will have different levels of inhibition on BTP test results; similarly, cerebrospinal fluid samples, with varying degrees of blood-brain barrier rupture and different amounts of retinol and thyroxine entering the cerebrospinal fluid, will also lead to different degrees of inhibition on cerebrospinal fluid BTP test results.

[0014] To achieve high-sensitivity, multi-sample-use BTP detection, it is necessary to address the issue that different samples contain varying amounts of macromolecules that can bind to BTP, resulting in different levels of inhibition of BTP detection.

[0015] Invention principle:

[0016] Retinol-binding protein (RBP) is an readily available and inexpensive protein that can compete with BTP for retinol binding. Excessive RBP may render some retinol-bound BTP free, facilitating BTP binding to the detection antibody. Anti-thyroxine polyclonal antibodies can mask the binding site of thyroxine to BTP, rendering thyroxine-bound BTP free. Furthermore, when anti-thyroxine antibodies and BTP detection antibodies are homologous, they can act as passive blockers, reducing the interference of RF (rheumatoid factor) and HA (heterophile antibodies) on BTP detection. Therefore, adding appropriate amounts of RBP protein and thyroxine polyclonal antibodies to reaction buffer R1 can significantly improve the high-value recovery rate of the aforementioned special samples.

[0017] Furthermore, when the detection reagent has sufficient sensitivity, samples that are easily viscous, such as nasal swab solutions or seminal plasma, as well as small-volume samples like intraocular fluid, can all be uniformly diluted 4-fold (50µl of nasal swab solution added to 150µl of dedicated diluent) before testing. The test result is then multiplied by 4 to obtain the final result, which significantly reduces the difficulty and cost of detection and research. The reason for choosing 4-fold is that, with conventional 2-fold manual dilution, extremely viscous samples occasionally still trigger alarms and cause needle blockage. 4-fold dilution solves this problem, as excessively large dilution factors challenge the reagent's sensitivity.

[0018] To achieve the above objectives, the present invention adopts the following technical solution: A β-trace protein detection kit with a wide linear range adaptable to various sample uses includes reagent R1; said reagent R1 comprises the following components at the following concentrations: 100mM pH6.6 BES-NaOH buffer, NaCl 1500mM, Tween-20 10ml / L, polyethylene glycol 8000 10g / L, rabbit anti-blocking agent 5ml / L, retinol-binding protein 2-4 mg / L (preferably 2 mg / L), and rabbit polyclonal antibody against thyroxine T4 0.3-0.6 mg / L (preferably 0.3 mg / L).

[0019] Furthermore, the kit also includes reagent R2; reagent R2 comprises the following components at the following concentrations: 100mM PBS buffer pH 7.5, Tween-20 6ml / L, BSA 1g / L, sucrose 30g / L, trehalose 10g / L, 180nm latex microspheres 6.25g / L, 230nm latex microspheres 2.5g / L, 300nm latex microspheres 1.56g / L, and rabbit anti-human BTP polyclonal antibody 26.25ml / L.

[0020] Furthermore, reagents R1 and R2 also include preservative PC300 0.5 ml / L.

[0021] Furthermore, the kit also includes calibrators; the calibrators are BTP recombinant antigens at concentrations of 2 mg / L, 1.2 mg / L, 0.6 mg / L, 0.3 mg / L, 0.15 mg / L, 0.05 mg / L, and 0.025 mg / L.

[0022] Furthermore, the kit also includes a viscous sample diluent containing a final concentration of 0.2% V / V Tween-20.

[0023] As can be seen from the above technical solution, compared with the prior art, this invention discloses a β-trace protein detection kit with a wide linear range adaptable to various sample uses. This BTP detection kit has a linear range of 0.05-35.00 mg / L, suitable for the detection of cerebrospinal fluid, blood, urine, and nasal swab solutions, covering more than 95% of sample concentrations, reducing retesting costs and improving detection efficiency. It also includes a diluent and a standardized dilution method, allowing for the study of various small-volume or viscous body fluids such as seminal plasma and intraocular fluid. In summary, compared with existing Siemens detection reagents, it represents a significant improvement in detection cost and efficiency. Attached Figure Description

[0024] 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.

[0025] Figure 1 The linear dilution validation plot and regression equation are shown for Comparative Example 1.

[0026] Figure 2 The following is a linear dilution validation plot and regression equation for Example 1.

[0027] Figure 3The following is a linear dilution verification plot and regression equation for Example 2.

[0028] Figure 4 The following is a comparison plot and regression equation for linear dilution validation in Example 2.

[0029] Figure 5 This is a comparison diagram of the present invention and clinical samples of Siemens BTP reagent. Detailed Implementation

[0030] 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.

[0031] R1 Basic Recipe: 100mM pH6.6 BES-NaOH buffer, 1500mM NaCl; 10ml / L Tween-20; 10g / L polyethylene glycol 8000; 0.5ml / L preservative PC300; 5ml / L rabbit antiblocker (Feipeng Biotechnology E-015G).

[0032] R2 basic formulation and preparation process: R2 basic formulation: 100mM PBS buffer pH 7.5, Tween-20 6ml / L, preservative PC300 0.5ml / L, BSA 1g / L, sucrose 30g / L, trehalose 10g / L, 180nm latex microspheres 6.25g / L, 230nm latex microspheres 2.5g / L, 300nm latex microspheres 1.56g / L, rabbit anti-human BTP polyclonal antibody 26.25ml / L.

[0033] Activation buffer: 100mM MES-NaOH buffer, pH 5.5, preservative PC300 0.2ml / L.

[0034] Coating buffer: 100mM PBS buffer, pH 7.5, preservative PC300 0.2ml / L.

[0035] Blocking and storage buffer: 100mM PBS buffer pH 7.5, Tween-20 6ml / L, preservative PC300 0.5ml / L, BSA 1g / L, sucrose 30g / L, trehalose 10g / L.

[0036] R2 preparation process: A three-latex system was used, consisting of 180nm latex microspheres (Polymicroshperes CB0180E), 230nm latex microspheres (Polymicroshperes CB0230D), and 300nm latex microspheres (Polymicroshperes CB0297C).

[0037] (1) Latex cleaning: Using the pre-prepared activation buffer, pH 5.5, centrifuge and change the latex microspheres twice; take 10 ml of each of the 10% mass concentration of 180 nm, 230 nm and 300 nm latex microspheres, i.e., 1 g of each of the three types of latex, centrifuge at 22000 rpm for 15 min, remove the supernatant, reconstitute with 10 ml of the above activation buffer, centrifuge again, remove the supernatant and reconstitute, and complete the cleaning for later use.

[0038] (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 activation buffer, and add 1ml of each to 10ml of the three types of microspheres that have been washed. Stir magnetically at room temperature for 10min.

[0039] (3) Cleaning after activation: Centrifuge the three types of latex activated in step (2) at 22000 rpm for 15 min, remove the supernatant, add 20 ml of coating buffer to reconstitute for later use. At this time, the latex concentration is 5%.

[0040] (4) Antibody conjugation: Take three portions of rabbit anti-human BTP polyclonal antibody (Huirui Xinyuan, antibody catalog number: 301020, name β-Trace Rabbit pAb) and add them to the three activated latex solutions reconstituted with the coating buffer. Add 3 ml of BTP antibody to the 180 nm latex; add 2 ml of BTP antibody to the 230 nm latex; and add 1.6 ml of BTP antibody to the 300 nm latex. Stir magnetically at room temperature for 3 hours to complete antibody conjugation.

[0041] (5) After centrifuging the three types of latex at 22,000 rpm for 15 min, remove all supernatant and add blocking and storage buffer respectively. Add 80 ml of blocking and storage buffer to the 180 nm latex (final latex concentration of 1.25% and final antibody concentration of 37.5 ml / L), add 100 ml of blocking and storage buffer to the 230 nm latex (latex concentration of 1% and antibody concentration of 20 ml / L), and add 160 ml of blocking and storage buffer to the 300 nm latex (final latex concentration of 0.625% and final antibody concentration of 10 ml / L). Reconstitute and mix overnight. Mix 50ml of 180nm latex, 25ml of 230nm latex, and 25ml of 300nm latex after reconstitution and mixing to obtain product R2 (the concentration of 180nm latex in product R2 is 6.25g / L, the concentration of 230nm latex is 2.5g / L, the concentration of 300nm latex is 1.56g / L, and the final concentration of BTP antibody is 26.25ml / L).

[0042] Formula for viscous sample diluent: Take 60 ml of sample diluent type II (BYS92430, product number BYS92430, Bairong Diagnostics Products (Shanghai) Co., Ltd.), add 120 µl of Tween-20 (i.e., final concentration 0.2% V / V Tween-20), mix well for 1 hour, and prepare a viscous sample diluent.

[0043] Basic formulation and preparation process of calibrators: Diluent: Sample diluent type II (BYS92430, Bairong Diagnostics Products (Shanghai) Co., Ltd.).

[0044] Preparation process: Preparation of high-value stock solution from natural samples: Cerebrospinal fluid (CSF) samples from several patients with normal blood-brain barrier levels remaining in the department's laboratory were collected and mixed to form a CSF matrix. This matrix was then mixed with the aforementioned diluent 1+4 (2 ml of CSF matrix mixed with 8 ml of diluent), and stirred for 2 hours. The solution was then assigned a value using Siemens BTP reagent, and the average value was obtained from 5 tests. This concentration is generally 2.5-4 mg / L. Based on the assigned value, it was diluted to 2 mg / L using sample diluent II and named the high-value stock solution for BTP calibrator. For example, if the assigned concentration is 3.2 mg / L, 10 ml of the assigned matrix was taken and 6 ml of diluent was added to obtain 16 ml of high-value stock solution with a final concentration of 2 mg / L.

[0045] Preparation of Master Cal: The above high-value mother liquor was diluted to 1.2 mg / L, 0.6 mg / L, 0.3 mg / L, 0.15 mg / L, 0.05 mg / L, and 0.025 mg / L, respectively, and together with the 2 mg / L high-value mother liquor, Master Cal was prepared.

[0046] Preparation of product calibrators: Dilute the BTP recombinant antigen (Huirui Xinyuan, catalog number: P2010Z, name: Human β-TraceProtein (BTP)) with diluent to a target concentration of 3-5 mg / L.

[0047] Using the diluent as the zero point and Master Cal as the calibrator, the prepared reagent was calibrated on a Hitachi 3500 specific protein biochemical analyzer. Using a method similar to high-value stock solution, the recombinant antigen solution was assigned a value to prepare a 2 mg / L product calibrator high value.

[0048] The high value of the recombinant antigen was diluted with diluent to 1.2 mg / L, 0.6 mg / L, 0.3 mg / L, 0.15 mg / L, 0.05 mg / L, and 0.025 mg / L, respectively, and confirmed on the above calibration curve. A deviation of less than ±3% was considered acceptable. Adding 2 mg / L to the concentration yielded the product calibrator.

[0049] Reagent performance verification and acceptance criteria: The general instrument parameters are as follows: calibrator 5µl, samples (quality control and clinical samples) are pre-diluted 1:20 for testing (e.g. on Hitachi 3500, the instrument automatically dilutes the sample by adding 6µl of the original solution to 114µl of diluent (onboard purified water), and after automatic dilution, 5µl of the sample (i.e., 5 / 20=0.25µl of the original solution sample) participates in R1 and R2 reactions), R1 150µl, R2 75µl, transmission wavelength 700nm, scattering detector 2, and the two-point endpoint method is used to calculate the signal value.

[0050] Scattering calibrators and values: 0.00 mg / L (diluent), 0.50 mg / L (0.025 mg / L product calibrator), 1.00 mg / L (0.05 mg / L product calibrator), 3.00 mg / L (0.15 mg / L product calibrator), 6.00 mg / L (0.3 mg / L product calibrator), scattering spline curve 2, five-point calibration.

[0051] Transmission calibrators and values: 3.00 mg / L (0.15 mg / L product calibrator), 6.00 mg / L (0.3 mg / L product calibrator), 12.00 mg / L (0.6 mg / L product calibrator), 24.00 mg / L (1.2 mg / L product calibrator), and 40.00 mg / L (2.0 mg / L product calibrator), transmission spline curve 2, five-point calibration.

[0052] The limits for scattering technology are 0.05-6.00, and the limits for transmission technology are 4.00-35.00. Scattering is preferred, that is, for results of 0.05-6 mg / L, output scattering results, and for results of 6.01-35 mg / L, output transmission results. For results below 0.05 mg / L, reduce the dilution factor and re-examine, and for results above 35 mg / L, increase the dilution factor and re-examine.

[0053] Experimental validation of accuracy recovery for different types of samples: High-value mixed samples of cerebrospinal fluid with Siemens-assigned concentrations greater than 20 mg / L.

[0054] Hyperthyroidism (high thyroid hormone T4) samples, numbered blood samples 1, 2, 3; Vitamin A (high retinol) samples, numbered blood samples 4, 5, 6; Jaundice samples, numbered blood samples 7, 8, 9; Hypothyroidism (low thyroid hormone T4) samples, numbered blood samples 10, 11, 12; Control samples, numbered blood samples 13, 14, 15.

[0055] The nasal swab samples were diluted four times and numbered nasal swab sample 1, 2, and 3. Sample 2 was a blood-contaminated sample.

[0056] Urine samples, numbered 1, 2, and 3. Sample 1 was a hematuria sample.

[0057] Cerebrospinal fluid samples, numbered CSF 1, 2, and 3. Among them, CSF sample 1 was a blood-brain barrier rupture sample.

[0058] All the above samples were initially assigned values ​​using Siemens reagents; the accuracy was considered acceptable if the high-value spiking recovery rate (the ratio of the measured concentration to the theoretically calculated concentration) was within 85%-115%.

[0059] Reagent linearity range verification: A high-value cerebrospinal fluid sample with a Siemens value close to 35 mg / L was selected. Following the NMPA linearity validation method, the sample was diluted to at least six gradients (100%, 66.7%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%) using diluent. The theoretical estimated value (calculated using a linear equation of the measured value and the dilution factor) was compared with the measured value using Excel. A linear correlation coefficient R-squared greater than 0.99, within the range of 3.01-35.00, with a relative deviation ≤10%, and an absolute deviation ≤0.30 mg / L within the range of 0.05-3.00, was considered linearly acceptable.

[0060] Precision and detection limit validation: Using low-value samples, serially dilute to three levels (e.g., a 0.12 mg / L sample, diluted to 0.06 mg / L, 0.03 mg / L, a total of three levels), test 10 times, calculate the precision CV, and estimate the lower limit of detection (LOD) when the CV is less than or equal to 20%. If the LOD is less than the lower limit of linearity (LDL) of 0.05 mg / L, the detection limit is considered acceptable.

[0061] Select low values ​​(diluted nasal swab samples), medium values ​​(serum samples from kidney disease patients), and high values ​​(cerebrospinal fluid samples), and test 10 times. Calculate the precision CV. A CV < 8% is considered to be of acceptable precision.

[0062] Comparative Example 1 R1 is the basic R1; R2 is the finished product R2 mentioned above; the calibrator is the product calibrator; the general parameters are used; and the experimental instrument is a Hitachi 3500 specific protein biochemical analyzer.

[0063] The calibration data are shown in Table 1.

[0064] Table 1

[0065] Different sample recovery experiments were conducted, and the results are shown in Table 2.

[0066] Table 2

[0067] As shown in Table 2, the recovery rates of the blood samples from the control group (Comparative Example 1), the blood samples from hyperthyroidism, the blood samples from jaundice, the blood samples from samples taken with vitamin A, and the urine sample 1 (hematuria) were all below 85%, and were therefore unqualified. The cerebrospinal fluid sample 1 with a ruptured blood-brain barrier and the nasal swab sample 2 with blood contamination had a significant impact, with recovery rates significantly lower than other similar samples, and the recovery rates were on the verge of being qualified.

[0068] Reagent linearity range verification, results are shown in [link to documentation]. Figure 1 And Table 3.

[0069] Table 3

[0070] We can see the linearity correlation coefficient R. 2 =0.995 is greater than 0.99, the linear deviation is <10% in the range of 3.01-35.00 mg / L, and the absolute linear deviation is less than 0.30 mg / L in the range of 0.05-3.00 mg / L, so the linearity verification is qualified.

[0071] The reagent precision and detection limit were validated, and the results are shown in Tables 4 and 5.

[0072] Table 4

[0073] As shown in Table 4, the detection limit is below 0.03 mg / L and less than the lower limit of linearity of 0.05 mg / L, which is acceptable.

[0074] Table 5

[0075] As shown in Table 5, the precision CV of various samples is less than 8%, which is acceptable.

[0076] Overall, the basic formulation's R1, the finished product's R2, and the accompanying product calibrators all meet basic performance standards, but they cannot adequately address sample diversity.

[0077] Example 1 R1: Improved R1, with each liter of R1 containing 2.0 mg of retinol-binding protein (RBP, Guilin Yingmeite, catalog number KAY0024.C) and 0.30 mg of thyroxine T4 rabbit polyclonal antibody (Huirui Xinyuan: catalog number 301249, name T4Rabbit pAb).

[0078] The calibrators are product calibrators with general parameters, and the experimental instrument is a Hitachi 3500 specific protein biochemical analyzer.

[0079] The calibration data are shown in Table 6.

[0080] Table 6

[0081] Different sample recovery experiments were conducted, and the results are shown in Table 7.

[0082] Table 7

[0083] As shown in Table 7, compared with Comparative Example 1, after adding sufficient RBP antigen and rabbit polyclonal antibody against thyroxine T4, the recovery rate of all samples reached over 90%, which is qualified.

[0084] Reagent linearity range verification, results are shown in [link to documentation]. Figure 2 See Table 8.

[0085] Table 8

[0086] As can be seen, adding sufficient amounts of RBP antigen and rabbit polyclonal antibody against thyroxine T4 had no effect on the linearity test, and the linearity correlation coefficient R was [value missing]. 2 =0.9996 is greater than 0.99, the linear deviation is <10% in the range of 3.01-35.00 mg / L, and the absolute linear deviation is less than 0.30 mg / L in the range of 0.05-3.00 mg / L, so the linearity verification is qualified.

[0087] The reagent precision and detection limit were validated, and the results are shown in Tables 9 and 10.

[0088] Table 9

[0089] Table 10

[0090] As shown in Tables 9 and 10, adding sufficient amounts of RBP antigen and rabbit polyclonal antibody against thyroxine T4 has no effect on the detection limit and precision of the reagents, and both are qualified.

[0091] Example 2 Improved R1 2: Over-addition of RBP and T4 rabbit polyclonal antibody, with 4.0 mg of retinol-binding protein (RBP, Guilin Yingmeite, catalog number KAY0024.C) and 0.60 mg of thyroxine T4 rabbit polyclonal antibody (Huirui Xinyuan: catalog number 301249, name T4 Rabbit pAb) added per liter of R1.

[0092] The calibrators are product calibrators with general parameters, and the experimental instrument is a Hitachi 3500 specific protein biochemical analyzer.

[0093] The calibration data are shown in Table 11.

[0094] Table 11

[0095] Different sample recovery experiments were conducted, and the results are shown in Table 12.

[0096] Table 12

[0097] As shown in Table 12, compared to Example 1, after adding excessive amounts of RBP antigen and rabbit polyclonal antibody against thyroxine T4, the recovery rate of all samples reached over 93%, which is qualified. However, doubling the addition yielded lower benefits. The reason for this is that RBP antigen and T4 antibody mainly compete with BTP interferon, resulting in diminishing marginal benefits from the increased dosage.

[0098] Reagent linearity range verification, results are shown in [link to documentation]. Figure 3 And Table 13.

[0099] Table 13

[0100] As can be seen, adding excessive amounts of RBP antigen and rabbit polyclonal antibody against thyroxine T4 had no effect on the linearity test, and the linearity correlation coefficient R was [value missing]. 2=0.9996 is greater than 0.99, the linear deviation is <10% in the range of 3.01-35.00 mg / L, and the absolute linear deviation is less than 0.30 mg / L in the range of 0.05-3.00 mg / L, so the linearity verification is qualified.

[0101] The reagent precision and detection limit were validated, and the results are shown in Tables 14 and 15.

[0102] Table 14

[0103] Table 15

[0104] As shown in Tables 14 and 15, adding excessive amounts of RBP antigen and rabbit polyclonal antibody against thyroxine T4 has a slight impact on the detection limit and precision of the reagents. The sensitivity of the calibration data decreases slightly, and the detection CV increases slightly, but it is still within the acceptable range.

[0105] Taking into account cost, sensitivity, and marginal benefit of recovery rate, the RBP antigen and rabbit polyclonal antibody against thyroxine T4 are sufficient within the scope of this invention. Further increasing the dosage will have little effect and will also bring negative impacts such as increased sensitivity and cost.

[0106] Comparative Example 2 R1: Improved from basic R1 3, with insufficient addition of anti-interference agents, including 1.0 mg of retinol-binding protein (RBP, Guilin Yingmeite, catalog number KAY0024.C) and 0.15 mg of thyroxine T4 rabbit polyclonal antibody (Huirui Xinyuan: catalog number 301249, name T4 Rabbit pAb) per liter of R1.

[0107] The calibrators are product calibrators with general parameters, and the experimental instrument is a Hitachi 3500 specific protein biochemical analyzer.

[0108] The calibration data are shown in Table 16.

[0109] Table 16

[0110] Different sample recovery experiments were conducted, and the results are shown in Table 17.

[0111] Table 17

[0112] As shown in Table 17, compared to Comparative Example 1, the recovery rates of all samples improved after adding a small amount of RBP antigen and rabbit polyclonal antibody against thyroxine T4. The recovery rates of jaundice samples, routine control samples, and urine sample 1 (hematuria) improved from unacceptable to acceptable. However, the recovery rates of severely affected hyperthyroidism samples and samples treated with vitamin A remained below 85%, which is unacceptable. This indicates that sufficient amounts of additives are needed to ensure compatibility with various sample types.

[0113] Reagent linearity range verification, results are shown in [link to documentation]. Figure 4 See Table 18.

[0114] Table 18

[0115] As can be seen, adding a small amount of RBP antigen and rabbit polyclonal antibody against thyroxine T4 had no effect on the linearity test, and the linearity correlation coefficient R was [value missing]. 2 =0.9997 is greater than 0.99, the linear deviation is <10% in the range of 3.01-35.00 mg / L, and the absolute linear deviation is less than 0.30 mg / L in the range of 0.05-3.00 mg / L, so the linearity verification is qualified.

[0116] The reagent precision and detection limit were validated, and the results are shown in Tables 19 and 20.

[0117] Table 19

[0118] Table 20

[0119] As shown in Tables 19 and 20, adding a small amount (insufficient amount) of RBP antigen and rabbit polyclonal antibody against thyroxine T4 has no effect on the detection limit and precision of the reagent, and is therefore acceptable.

[0120] Considering Comparative Example 1 (without RBP antigen and T4 rabbit polyclonal antibody), Example 1 (with sufficient addition), Example 2 (with excessive addition), and Comparative Example 2 (with insufficient addition), based on the impact on recovery rate, cost, and marginal benefit of addition, adding 2.0 mg of retinol-binding protein and 0.30 mg of thyroxine T4 rabbit polyclonal antibody per liter of R1 is the optimal solution. Those skilled in the art can conclude from the data analysis of Example 2 above that when BTP detection encounters stronger interference, the problem can be solved by appropriately increasing the dosage of RBP and T4 rabbit polyclonal antibody within the acceptable range of cost and reagent performance, based on the optimal solution. The optimal solution does not choose the dosage close to the 85% recovery limit, but rather ensures that the test samples all have a recovery rate greater than 90%, also considering the complexity of clinical samples and leaving a certain margin.

[0121] Example 3 Parallel testing of nasal swab solutions with Siemens samples demonstrates their efficiency and cost advantages.

[0122] Using R1 from Example 1, finished product R2, and product calibrators, the experimental instrument was a Hitachi 3500 specific protein biochemical analyzer.

[0123] Remaining nasal swab solutions from the Siemens test on the same day were selected and uniformly diluted 4-fold (50µl nasal swab solution added to 150µl viscous sample diluent) using a viscous sample diluent. The solutions were then tested. The test results were multiplied by 4 and compared with the Siemens results. It is crucial to compare only the nasal swab samples from the same day. A correlation analysis was performed on a total of 40 results from multiple days, along with an analysis of testing efficiency and cost. The results are shown in Table 21 and... Figure 5 .

[0124] Table 21

[0125] NA: The test was unsuccessful due to needle blockage or detection limit being below the detection limit, or this step is not involved.

[0126] From the data and Figure 5 As can be seen, when this invention and the Siemens BTP test kit are used to test nasal swab solutions, R 2 >0.95 indicates a good correlation. The test results are reliable.

[0127] Testing cost (number of test samples consumed for the same sample): 40 nasal swab solutions. Siemens used incremental testing due to samples being below the detection limit, requiring needle blockage and dilution for retesting, consuming a total of 60 test samples. This invention used retesting for 2 cases due to samples being below the detection limit, consuming a total of 42 test samples. This invention reduces the number of tests by 30% compared to Siemens.

[0128] Detection efficiency: Siemens only provides results for 23 cases in one test, 14 cases require two tests to obtain results, and 3 cases require two dilutions, totaling 3 tests to obtain results. In contrast, this invention provides results for 38 cases in one test, and only 2 cases require two tests to obtain results.

[0129] This invention is significantly superior to the Siemens BTP detection kit in terms of detection efficiency and cost.

[0130] 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 β-trace protein detection kit with a wide linear range adaptable to various sample applications, characterized in that, Includes reagent R1; said reagent R1 comprises the following components at the following concentrations: 100mM pH6.6 BES-NaOH buffer, NaCl 1500mM, Tween-20 10ml / L, polyethylene glycol 8000 10g / L, rabbit anti-blocking agent 5ml / L, retinol-binding protein 2-4 mg / L, and rabbit polyclonal antibody against thyroxine T4 0.3-0.6 mg / L.

2. The β-trace protein detection kit with a wide linear range adaptable to various sample applications according to claim 1, characterized in that, It also includes reagent R2; said reagent R2 comprises the following components at the following concentrations: 100mM PBS buffer pH 7.5, Tween-20 6ml / L, BSA 1g / L, sucrose 30g / L, trehalose 10g / L, 180nm latex microspheres 6.25g / L, 230nm latex microspheres 2.5g / L, 300nm latex microspheres 1.56g / L, and rabbit anti-human BTP polyclonal antibody 26.25ml / L.

3. The β-trace protein detection kit with a wide linear range adaptable to various sample applications according to claim 2, characterized in that, The reagents R1 and R2 also include preservative PC300 0.5 ml / L.

4. The β-trace protein detection kit with a wide linear range adaptable to various sample applications according to claim 3, characterized in that, It also includes calibrators; the calibrators are BTP recombinant antigens at concentrations of 2 mg / L, 1.2 mg / L, 0.6 mg / L, 0.3 mg / L, 0.15 mg / L, 0.05 mg / L, and 0.025 mg / L.

5. The β-trace protein detection kit with a wide linear range adaptable to various sample applications according to claim 4, characterized in that, It also includes a viscous sample diluent; the viscous sample diluent contains a final concentration of 0.2% V / V Tween-20.

Images